Physiol Rev 90: 291–366, 2010;
doi:10.1152/physrev.00021.2009.
Inwardly Rectifying Potassium Channels: Their Structure,
Function, and Physiological Roles
HIROSHI HIBINO, ATSUSHI INANOBE, KAZUHARU FURUTANI, SHINGO MURAKAMI, IAN FINDLAY,
AND YOSHIHISA KURACHI
Department of Pharmacology, Graduate School of Medicine and The Center for Advanced Medical Engineering
and Informatics, Osaka University, Osaka, Japan; and Laboratoire de Physiologie des Cellules Cardiaques et
Vasculaires (EA4433), Université François-Rabelais, Tours, France
I. Overview of Inwardly Rectifying K⫹ Channels
A. Introduction
B. Molecular structure and basic stoichiometry
C. Kir channel function and localization
D. Structure
E. Pharmacology
II. Classical Kir Channels (Kir2.x)
A. Historical view and molecular diversity
B. Pore function and structure bases
C. Intracellular localization
D. Physiological functions in cells and organs
E. Pharmacology
F. Diseases
III. G Protein-Gated Kir Channels (Kir3.x)
A. Historical view and molecular diversity
B. Pore function and subunit structure
C. Channel localization
D. Physiological functions in cells and organs
E. Pharmacology
F. Diseases
IV. KATP Channels (Kir6.x/SURx)
A. Historical view and molecular diversity
B. Pore structure and function
C. Intracellular localization
D. Physiological functions in cells and organs
E. Pharmacology
F. Diseases
V. K⫹ Transport Channels (Kir1.1, Kir4.x, Kir5.x, Kir7.x)
A. Kir1.1
B. Kir4.x and Kir5.1
C. Kir7.1
VI. Conclusion
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Hibino H, Inanobe A, Furutani K, Murakami S, Findlay I, Kurachi Y. Inwardly Rectifying Potassium Channels: Their
Structure, Function, and Physiological Roles. Physiol Rev 90: 291–366, 2010; doi:10.1152/physrev.00021.2009.—Inwardly
rectifying K⫹ (Kir) channels allow K⫹ to move more easily into rather than out of the cell. They have diverse physiological
functions depending on their type and their location. There are seven Kir channel subfamilies that can be classified into
four functional groups: classical Kir channels (Kir2.x) are constitutively active, G protein-gated Kir channels (Kir3.x) are
regulated by G protein-coupled receptors, ATP-sensitive K⫹ channels (Kir6.x) are tightly linked to cellular metabolism,
and K⫹ transport channels (Kir1.x, Kir4.x, Kir5.x, and Kir7.x). Inward rectification results from pore block by intracellular
substances such as Mg2⫹ and polyamines. Kir channel activity can be modulated by ions, phospholipids, and binding
proteins. The basic building block of a Kir channel is made up of two transmembrane helices with cytoplasmic NH2 and
COOH termini and an extracellular loop which folds back to form the pore-lining ion selectivity filter. In vivo, functional
Kir channels are composed of four such subunits which are either homo- or heterotetramers. Gene targeting and genetic
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HIBINO ET AL.
analysis have linked Kir channel dysfunction to diverse pathologies. The crystal structure of different Kir channels is opening
the way to understanding the structure-function relationships of this simple but diverse ion channel family.
I. OVERVIEW OF INWARDLY RECTIFYING
Kⴙ CHANNELS
A. Introduction
Inwardly rectifying K⫹ (Kir) currents were first identified in skeletal muscle (363). Instead of outward rectification predicted by the Nernst equation, they showed
greater flow into rather than out of the cell (Fig. 1A). Kir
currents were therefore originally described as “anomalous” rectifier K⫹ currents. This characteristic was
clearly different from the voltage-gated K⫹ (Kv) channel
current in squid giant axon (273). Their relationship
with membrane voltage did not follow Hodgkin-Huxley
kinetics (233); instead, their behavior seemed to depend more on the electrochemical gradient for K⫹
[membrane potential (Em) minus equilibrium potential
of K⫹ (EK)] (Fig. 1A). These defining characteristics of
Kir currents result not from a bending of the rules of
biological chemistry by the Kir channel but from asymmetric open channel pore block by intracellular divalent cations and other molecules.
Therefore, under physiological conditions, Kir channels generate a large K⫹ conductance at potentials negative to EK but permit less current flow at potentials positive to EK (237, 537, 575, 679). Cells that express a large
Kir conductance are expected to show the resting membrane potential (Eres) close to EK and no spontaneous
electrical activity. This, and their essential voltage independence, permits Kir channels to play key roles in the
maintenance of Eres and in regulation of the action potential duration in electrically excitable cells such as cardiac
muscle (Fig. 1B) (237, 537, 679).
Kir channels have been found in a wide variety of
cells: cardiac myocytes (41, 406, 514, 575, 667), neurons
(68, 201, 423, 581, 759, 838), blood cells (445, 517), osteoclasts (732), endothelial cells (727), glial cells (400, 563),
epithelial cells (217, 247, 475, 480), and oocytes (235–237).
G protein-gated K⫹ (KG) channels (405, 678), which are
activated via pertussis toxin (PTX)-sensitive G proteins
(65, 411, 412, 414, 580, 584, 620), also show inward rectification (see sect. III). In addition, ATP-sensitive K⫹ (KATP)
channels, which were originally defined as being opened
by a decrease in intracellular ATP (ATPi) (577) also belong to the Kir channel family (see sect. IV) (299, 300, 317).
Accordingly, Kir channels not only orchestrate the passive and active electrical properties of cells, but they are
also involved in G protein-coupled receptor (GPCR) signaling, and they may link cellular metabolic state and
membrane excitability in vivo.
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FIG. 1. Functional properties of Kir channels. A: the dependence of
inward rectification and conductance of Kir channels on [K⫹]o. I-V relationships of the starfish egg cell membrane at four different extracellular [K⫹]o (10,
25, 50, and 100 mM) in Na⫹-free media. Continuous and broken lines indicate
instantaneous and steady-state currents, respectively. [From Hagiwara et al.
(236), copyright 1976. Originally published in The Journal of General Physiology.] B: control of excitability in cardiac cells by Kir channels. Schematic
representations of action potentials in ventricular myocytes (a and c) and in the
sinoatrial node (b) under various conditions are shown. In ventricular myocytes, inhibition of classical Kir and KATP channels causes Eres to depolarize
and Em may oscillate (a). On the other hand, activation of KATP channels
hyperpolarizes Em, shortens action potential duration, and may suppress action
potential generation (c). Cells in the sinoatrial node express a number of KG
channels; their activation may result in hyperpolarization of Em and/or bradycardia (b).
In recent years, the molecular make up, basic architecture, physiology, and pathological relevance of different Kir
channels have been described. Here we will discuss these
topics, with a particular focus on the molecular and functional characters.
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STRUCTURE AND PHYSIOLOGICAL FUNCTION OF Kir CHANNELS
B. Molecular Structure and Basic Stoichiometry
In 1993, two Kir channel cDNAs were isolated by
expression-cloning techniques. The ATP-dependent Kir
channel ROMK1/Kir1.1 (272) and the classical Kir channel
IRK1/Kir2.1 (394) were isolated, respectively, from the
outer medulla of rat kidney and a mouse macrophage cell
line. Their primary structures possessed a common motif
of two putative membrane-spanning domains (TM1 and
TM2) linked by an extracellular pore-forming region (H5)
and cytoplasmic amino (NH2)- and carboxy (COOH)terminal domains (Fig. 2A). We now recognize this as the
basic building block that is common to all types of Kir
channel. The H5 region serves as the “ion-selectivity filter”
(249) that shares with other K⫹-selective ion channels the
signature sequence T-X-G-Y(F)-G (46). Kir channel structures lack the S4 voltage sensor region that is conserved
in voltage-gated Na⫹, Ca2⫹, and K⫹ channels. As a result,
Kir channels are insensitive to membrane voltage and,
293
when particular mechanisms regulating channel activity
(e.g., ATP closing KATP channels and G␤␥ activating KG
channels) are absent, would be active at all Em. Their
defining characteristic, inward rectification, turns out not
to be an intrinsic function of the channel protein but a
result of the block of outward K⫹ flux by intracellular
substances such as Mg2⫹ and polyamines (see sect. IC1).
The primary structure of two transmembrane strands is
insufficient to form a complete ion channel, and functional Kir channels are made up of four such subunits in
a tetrameric complex (see Fig. 4B) (208, 866). This stoichiometry was confirmed by velocity sedimentation with
sucrose density gradients, size exclusion column chromatography, and chemical cross-linking (305, 639).
To date, 15 Kir subunit genes have been identified
and classified into seven subfamilies (Kir1.x to Kir7.x)
(Fig. 2B). These subfamilies can be categorized into four
functional groups: 1) classical Kir channels (Kir2.x), 2) G
protein-gated Kir channels (Kir3.x), 3) ATP-sensitive K⫹
channels (Kir6.x), and 4) K⫹-transport channels (Kir1.x,
Kir4.x, Kir5.x, and Kir7.x) (Fig. 2B).
The simplicity and strong homology of the basic Kir
channel subunit allow for both homomeric and heteromeric combinations to form functional Kir channels. Heteromerization generally occurs between members of the
same subfamily, for example, Kir2.1 can associate with
any one of other Kir2.x subfamily members, namely,
Kir2.2, Kir2.3, or Kir2.4 (626, 695) (see sect. II), and Kir3.1
forms heteromeric complexes with either Kir3.2, Kir3.3,
or Kir3.4 (see sect. III). An exception is where Kir4.1
assembles with Kir5.1 (see sect. VB). Heteromeric assemblies confer distinct properties to their particular channels; they can determine their location on a cell as well as
extend the functional range of Kir channels in different
cell types.
C. Kir Channel Function and Localization
FIG. 2. Basic structure and Kir channel phylogenetic tree. A: primary structure of the Kir channel subunit (left). Each Kir subunit contains two transmembrane (TM1 and TM2) regions, a pore-forming (H5)
loop, and cytosolic NH2 and COOH termini. As a comparison, the structure of voltage-gated K⫹ (Kv) channel subuit, which possesses six transmembrane (TM1-TM6) regions, is shown on the right. B: amino acid
sequence alignment and phylogenetic analysis of the 15 known subunits
of human Kir channels. These subunits can be classified into four
functional groups.
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The physiological activity and functions of Kir channels depend on regulation of pore opening, ion flux, and
channel localization in the cell. Major factors that regulate
pore opening and ion flux include ions, polyamines, nucleotides, lipids, and a variety of intracellular proteins
(Fig. 3A). Many of these interact directly with crucial
elements of the Kir channel to modulate properties such
as ion flux and channel pore opening kinetics. The localization of the channels in particular regions of a cell, such
as apical or basolateral membranes in epithelial cells and
pre- or postsynaptic sites in neurons, and that in membrane microdomains where they may be in close proximity with other transport molecules are also important
contributors to the functional roles of Kir channels in
different cells and tissues (Fig. 3B).
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FIG. 3. Regulators of Kir channel function. A: the
functions of Kir channels can be regulated by small substances (top panel) and by proteins (bottom panels). The
small substances are ions such as H⫹, Mg2⫹, and Na⫹;
polyamines; phosphorylation; and membrane-bound phospholipids. Protein-protein interaction involves sulfonylurea receptors (SUR), G proteins liberated from G proteincoupled receptors (GPCR), and anchoring proteins. B: the
localization of Kir channels on a cell may determine their
particular function. The channels may be distributed homogeneously in nonpolarized cells or in a specific pattern
in membranes of polarized cells such as epithelia and
neurons (top panel). Specific localization patterns play a
role in unidirectional transport of K⫹ and in the organization of signal transduction. Particular channels and other
transport mechanism may be gathered in microdomains
such as detergent-resistant membrane microdomains
(DRMs) and caveolae (bottom panel). Such colocalization
may be necessary for the physiological coupling of iontransport mechanisms.
1. Regulation of the Kir channel pore
A) INTRACELLULAR Mg
AND POLYAMINES. Inward rectification of K⫹ flux through Kir channels results from interaction between two intracellular substances, Mg2⫹ and polyamines, and the lining of the channel pore (Fig. 3A) (see
sects. ID and II). They physically block K⫹ permeation by
binding to residues localized in the transmembrane and
cytoplasmic regions of the channels (471, 508).
Early studies led to the conclusion that rectification
arises from a combination of intracellular Mg2⫹ (Mg2⫹
i )mediated blockage and an intrinsic activation gating process
(310, 406, 507, 508). For instance, in the current of cardiac
classical Kir channels (IK1), Mg2⫹
caused instantaneous ini
2⫹
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ward rectification, which was followed by a time-dependent
further enhancement of rectification by the intrinsic gating
mechanism (310). But the intrinsic gating property was lost
in inside-out patches and restored by submicromolar concentrations of polyamines such as spermine and spermidine
applied to the intracellular side of the Kir channels (160, 172,
471, 856). Because polyamines exist in cells at submillimolar
concentrations, the putative activation gating behavior is
now assumed to result from their slow blocking and unblocking of the Kir channel. Thus, on depolarization, what
was previously called “deactivation” corresponds to polyamines causing a time-dependent decrease of outward current. On hyperpolarization, the inward Kir current first in-
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STRUCTURE AND PHYSIOLOGICAL FUNCTION OF Kir CHANNELS
creases time-independently due to fast Mg2⫹ unblocking and
then increases in a time-dependent fashion, which was referred to as “activation,” is due to slow polyamine unblocking (472).
Not all types of Kir channels show the same degree of
inward rectification. There are “strong” (Kir2.x and Kir3.x),
“intermediate” (Kir4.x), and “weak” (Kir1.1 and Kir6.x) rectifiers. A negatively charged residue Asp at position 172 in
the TM2 helix in a strong rectifier of Kir2.1 seems to be a
critical determinant of inward rectification for various Kir
channels. The weakly rectifying Kir1.1 has an uncharged
residue Asn (N171) in this position, and switching Asn for
Asp (N171D) in Kir1.1 was found to increase the affinity for
Mg2⫹ and cause strong rectification (483, 742, 831, 866). This
position is now known as the “D/N site.” Studies of mutated
Kir channels have also revealed that Kir channel subunits
possess more than one binding site for Mg2⫹ and polyamines, and further details will be described in section ID
and for each Kir channel subfamily. The competitive blockage of Kir channels by Mg2⫹ and polyamine is crucial for the
control of the magnitude of outward current (see an example of Kir2.2 in Ref. 861).
⫹
B) EXTRACELLULAR K CONCENTRATIONS. The conductance
of all Kir channels except for Kir7.1 (140, 393) increases as
extracellular K⫹ concentration ([K⫹]o) augments, which depends on the square root of [K⫹]o (237, 394, 473, 493, 614,
679). This property implies that ion permeations through Kir
channels do not follow the Goldman-Hodgkin-Katz permeability theory describing independent movement of ions in
the pore but rather agree with the multi-ion pore model
(266). In support of this idea, the degree of Cs⫹-induced
block of the Kir current in starfish egg is modified by change
of [K⫹]o (93, 94). Hagiwara and co-workers (93, 94) proposed that Kir channel pore would have at least two binding
sites for K⫹. Moreover, Kir2.1 conductance in the absence of
Mg2⫹ and polyamines also exhibits the square-root dependence on [K⫹]o, strongly suggesting that this dependence
seems to be a property of the open-channel pore (473).
Consistently, crystal structure of KcsA K⫹ channel reveals
that the selective filter contains two K⫹ ions in the condition
of 150 mM [K⫹]o (143).
C) PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE. A membraneanchored phospholipid, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], is essential to sustain the normal
function of the majority of Kir channels (263, 264, 287,
760) (Fig. 3A). In membrane patches excised from their
parent cells, Kir channel activity gradually declines. This
“run-down” activity can be restored by the application of
ATP to the intracellular surface of the membrane which
replenishes PtdIns(4,5)P2 via the action of lipid kinases
(263). Mutation analyses suggest that PtdIns(4,5)P2 is associated with positively charged residues in the COOH
termini (287, 474). The structural basis of PtdIns(4,5)P2
regulation can be estimated using the high-resolution
structures of Kir subunits (see sects. ID and II).
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D) OTHER SMALL SUBSTANCES. Intracellular and/or extracellular pH can regulate Kir channels such as Kir1.1,
Kir2.3, and channels which contain the Kir4.1 subunit
(Fig. 3A); generally acidic shifts of pH reduce channel
activity (see sects. II and V, A and B). KG channels that
contain either Kir3.2 or Kir3.4 can be activated by intracellular Na⫹ (Na⫹
i ) (270, 619) (see sect. III). KATP channels,
which are made up of pore-forming Kir6.x and auxiliary
sulfonylurea receptor (SURx) proteins, are inhibited by
ATPi and activated by intracellular nucleotide diphosphates (NDPsi) (see sect. IV). ATP controls pore function
through direct interaction with cytoplasmic regions of
Kir6.2 (15, 111, 344, 450, 632, 649, 790 –792). On the other
hand, NDPsi bind to SURx which then affects the pore
function of Kir6.x to increase channel opening (28, 114,
221, 512, 720, 796) (see sect. IV).
E) PHOSPHORYLATION. Phosphorylation of Kir channel subunits by protein kinases such as protein kinases A and C
(PKA and PKC, respectively) can modulate their activity
(Fig. 3A). A serum-glucocorticoid-regulated kinase (SGK)
phosphorylates Kir1.1 and promotes its surface expression
(877) (see sect. VA3). Phosphorylation of Ser residues in
Kir1.1 by PKC results in suppression of channel activity
(456) (see sect. VA3). PKA phosphorylates both Kir6.1 and
SUR2B subunits of the smooth muscle type KATP channel
and enhances its activity (637, 713) (see sect. IVB).
F) PROTEIN-PROTEIN INTERACTION. Protein-protein interactions are involved in control of Kir channel pore function.
They include interaction between ␤␥ subunits of G protein (G␤␥) and Kir3.x, association of SUR with Kir6.x, and
binding of anchoring proteins to diverse Kir channels
(Fig. 3A). KG channels are activated by direct association
between cytoplasmic regions of Kir3.x and G␤␥ subunits
released from Gi/o-coupled receptors (78, 246, 288, 289,
305, 326, 392, 402, 734, 863) (see sect. III). Functional KATP
channels require the association of SURx subunits with
Kir6.x subunits to form a hetero-octameric structure (299,
300, 317, 853). Cytoplasmic regions of SURx interact with
Kir6.x to modify channel activity (30, 649) (see sect. IV).
Anchoring proteins such as PSD-95, SAP97, and sorting
nexin 27 play key roles in determining the localization of
some Kir channels in the cell surface (9, 279, 484, 595)
(see sects. II, III, and V).
2. Regulation of localization of Kir channels
A) MEMBRANE MACRODOMAINS. Many cells are polarized.
Epithelial cells have apical and basolateral membrane domains; neurons are divided into soma, dendrites, axon, and
axonal terminal; and astrocytes harbor somatic, perivascular
(end feet), and perisynaptic membrane domains (Fig. 3B)
(568, 725). These membrane “macrodomains” may express
different Kir channels. The postsynaptic membrane bears
KG channels made up of Kir3.1/3.2 (307, 453, 623) (see sect.
III). Renal epithelial cells have Kir1.1 upon their apical mem-
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HIBINO ET AL.
branes (384, 846) and Kir4.1/5.1 on their basolateral membranes (476, 765, 793) (see sect. VB).
This manner of organizing the expression of channels
on the cell surface may serve to couple Kir channels to
selective apparatus to regulate particular cell functions
(197). Thus, in the postsynaptic membrane, Kir3.1/3.2 is
associated with Gi/o-coupled receptors such as ␥-aminobutyric acid type B (GABAB) to produce ligand-induced inhibitory postsynaptic current (390, 535, 778) (see sect. III). In the
apical membrane of renal epithelia, Kir1.1 is functionally
associated with the Na⫹-K⫹-2Cl⫺ cotransporter to balance
salt and water homeostasis (247) (see sect. VA).
A number of mechanisms are involved in the control
of localization of membrane proteins to specific macrodomains. They include protein-protein interaction and protein-lipid interaction. Scaffolding proteins containing
PSD-95/Dlg/ZO-1 (PDZ) and src-homology-3 (SH3) domains play important roles (197). Sphingolipids are required for axonal delivery of glycosylphosphatidylinositol
(GPI)-anchored proteins in neurons (433). Cholesterol is
necessary for apical sorting of some membrane proteins
in epithelia (365). For certain Kir channels, PDZ domaincontaining proteins (PDZ proteins) are involved in their
localization in polarized cells (99, 279, 302, 441, 442, 555)
(see sects. IIC, IIIC, and VB3).
B) MEMBRANE MICRODOMAINS. Membrane microdomains
may also contain various functional proteins (730, 731) (Fig.
3B). Microdomains include caveolae, which are flask-shaped
membrane invaginations (13). There are also “detergentresistant microdomains of cell membranes” (DRMs) or “lipidrafts,” which are enriched with several types of lipids such as
cholesterol and sphingolipids and isolated biochemically as
nonionic detergent-insoluble components (69, 730, 731) (Fig.
3B). Caveolae contain abundant lipids, and they are now
considered as one type of DRMs (730). Distinct microdomains may concentrate particular sets of molecules. For
example, caveolae accumulate particular GPCRs, G proteins, and adenylyl cyclases (707). Noncaveolar DRMs also
anchor particular receptors and cytosolic proteins such as
neurotrophins and mitogen-activated protein (MAP) kinases
(731). In the case of Kir channels, some DRMs harbor Kir4.1
in astroglial cells and Kir3.1/3.2 in neurons (127, 259). Such
localization would synchronize channel activities with other
functional proteins and control various signaling and physiological activities of the cells. Kir4.1 may occur in close
vicinity with the water channel AQP4 to mediate K⫹-driven
water transport in astroglial cells (259) (see sect. VB3). Accordingly, the specific localization of Kir channels in membrane macro- and microdomains will contribute to cell and
organ function.
D. Structure
X-ray crystallography has provided details of the
three-dimensional structure of Kir channels down to the
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atomic level (306, 404, 573, 574, 611). A generic view of
the Kir subunit and the tetrameric Kir channel are shown
in Figure 4, A and B (573). The NH2 and COOH termini of
Kir subunits are exposed to the cytoplasm and associate
with each other to form a cytoplasmic domain that is
linked to but distinct from the transmembrane domain
(Fig. 4A). The transmembrane domain is mainly responsible for ion selectivity and gating (Fig. 4C). The cytoplasmic domain is thought to act as a gating regulator.
1. Transmembrane domain
The architecture of the transmembrane domain of
Kir channels has been determined by solving the crystal
structures of a bacterial homolog KirBac1.1 (404) and a
chimera between bacterial KirBac3.1 and mouse Kir3.1
(573) (Fig. 4C). The domain is composed of outer (TM1)
and inner (TM2) membrane spanning helices, with two
short additional helical elements, the slide and the pore
helices. The channel pore is delimited by one TM2 helix
from each of the four Kir subunits. The arrangement of
the helices to form the transmembrane domain shares
significant similarity with that of other K⫹ channel families including a bacterial channel KcsA (143, 539, 894), a
bacterial calcium-activated channel MthK (336, 337), a
bacterial voltage-gated channel KvAP (338), and a mammalian voltage-gated channel Kv1.2 (466, 467).
The ion conduction pore (Fig. 4C) can be functionally
divided into three distinct zones that consist of the selectivity filter, a water-filled central cavity, and the internal
face of the pore made up of the internal bases of the four
inner (TM2) helices. The K⫹ channel signature sequence
[T-X-G-Y(F)-G] is the selectivity filter. This makes a narrow region in the ion conduction pathway which separates the central cavity from the extracellular solution.
The central cavity is a 10-Å spherical water-filled space
about halfway through the membrane. The bases of four
TM2 helices (one from each subunit) come together to
make another narrow region at the cytoplasmic face of
the channel.
There are two distinct gating mechanisms in Kir
channels: slow gating and fast gating. In single-channel
recordings, slow gating corresponds to channel openings
occurring as bursts separated by long closed periods, and
fast gating corresponds to rapid opening and closing
within the burst. Mutations introduced around the bundle
crossing area of the inner TM2 helix tend to modulate the
behavior of bursts (785, 792, 872). On the other hand,
mutations around the selectivity filter cause alterations in
fast gating (85, 87, 157, 232, 630, 872).
Each TM2 helix can bend around a hinge point (Gly)
about halfway along their length. This bending would vary
the cross-sectional area of the narrow region at the cytoplasmic face of the transmembrane domain. Analyses of
the mechanisms underlying the activation of the consti-
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297
FIG. 4. Molecular architecture of Kir channels. A: a schematic representation of the structure of a generic Kir channel. The Kir channel is divided
into transmembrane and cytoplasmic domains. The NH2 and COOH termini are cytosolic and together contribute to the formation of the cytoplasmic
domain. B: tetrameric assembly of Kir channels. The molecular architecture of a tetrameric Kir channel (protein database ID 2QKS: Kir3.1-KirBac3.1
chimera) is represented as a cartoon model. The front subunit has been omitted for clarity. The organization of the tetramer of NH2 and COOH
termini leads to an extended pore for ion permeation. C: the transmembrane domain. An enlarged view of the transmembrane domain indicates
several important secondary structures for Kir channel function. The transmembrane domain comprises three helices: TM1, pore, and TM2. At the
membrane-cytoplasm interface, there is also an amphiphilic slide helix. The residue that is largely responsible for the interaction with polyamines
and Mg2⫹ and thus inward rectification is indicated by the yellow/red spheres (D131 in Kir3.1 which was mutated in the original coordinate). D: the
cytoplasmic domain. The opening of Kir channels requires PtdIns(4,5)P2. Those amino acid residues (blue) associated with the interaction with
PtdIns(4,5)P2 are distributed on the surface of the cytoplasmic domain towards the plasma membrane. The center of the cytoplasmic domain is a
water-filled cavity that contributes to the ion permeation pathway. Some residues associated with inward rectification (red) map along this cavity.
Kir3.x channels are activated by direct interaction with a G protein ␤␥ (G␤␥) subunit and channels containing Kir3.2 and Kir3.4 subunits can also
be activated by Na⫹. The amino acids responsible for these stimuli are indicated in green (G␤␥) and pink (Na⫹). C and D have been derived from B.
tutively closed KG/Kir3.x channels and comparisons with
the constitutively open classical IK1/Kir2.x channels has
provided clear evidence for mobility of the TM2 helices
contributing to Kir channel gating.
KG/Kir3.x channels are closed in the absence of an
external agonist activating a GPCR to release G␤␥. G␤␥
causes specific conformation changes in KG channels that
allow channel opening. Jin et al. (340) demonstrated that
the highly conserved G175 residue in the middle of the
TM2 helix was crucially involved in G␤␥-induced activation of Kir3.4. They also found that replacement of certain
residues in the TM2 helix below G175 with Pro, which
would increase the flexibility of the TM2 helix, formed
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constitutively active channels (340). These results, together with homology modeling, imply that this Gly may
serve as a hinge in the TM2 helix and allow the inner part
of the helix to swing away from the permeation pathway
upon G␤␥ stimulation. This idea is similar to the gating
mechanism of the KcsA channel proposed by Jiang et al.
(337) and consistent with the result that substitution of
Pro for S171 in Kir3.1 disrupted G␤␥-induced activation
gating because of loss of the rigidity of the below-hinge
part of the TM2 helix (674).
In addition to the importance of Gly as a hinge,
another residue in the TM2 helix seems to play a role in
activation gating. In Kir3.1 and Kir3.4, the replacement of
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a bulky hydrophobic residue Phe (F181 in Kir3.1; F187 in
Kir3.4) with Ala or Ser, but not with Met, at the bundlecrossing region converted channels from agonist activation to constitutive activity (97, 674). The corresponding
residue in KirBac1.1 (F146) is thought to act as a barrier
to ion permeation (404) which supports the idea that G␤␥
stimulation alters the conformation of this region.
Comparisons between Kir3.x and Kir2.x channels have
also been instructive. In the constitutively inactive channel
Kir3.2, the mutation V188G, which is situated below Phe
(F181) in the bundle-crossing region of TM2, converted activation gating to constitutive activity (872). Homology modeling suggested that, in the closed state, V188 might mediate
an interaction between adjacent TM2 helixes and the V118G
mutation destabilized the closed conformation by disrupting
this interaction (872). The corresponding residue (I176) in
the constitutively active Kir2.1 faces the pore cavity and thus
provides no interaction between the TM2 helixes in this
position (534). These structural differences imply that, in
Kir3.2, G␤␥ stimulation would induce a clockwise rotation
of TM2 to open the channel when it is viewed from the
outside of the cell, and this would bring the Kir3.2 channel
pore into a configuration equivalent to that of Kir2.1 (872).
This model is also supported by the following observation.
An overexpression of G␤␥ can weaken both the inward
rectification and Ba2⫹ and Cs⫹ block of KG channels (277).
An excess of G␤␥ may induce sufficient rotation of TM2
helices to alter the coordination of the binding sites for
polyamines, Mg2⫹, Ba2⫹, and Cs⫹ between Kir subunits,
because the residues critically involved in block of the pore
by these substances are localized near the Gly hinge in TM2
helices (see sect. IIB) (339).
Not only TM2 but also TM1 are critically involved in
gating machinery of Kir channels. In Kir3.2, mutation of N94
of TM1 to His results in formation of constitutively active
channel, suggesting that this residue is involved in the gating
process (872). Homology modeling of Kir1.1 indicates that
K80 on TM1, which corresponds to N94 in Kir3.2, forms with
A177 of TM2 a hydrogen (H⫹) bond in the bundle-crossing
region (644). Further analyses of Kir1.1 demonstrate that the
H⫹ bonding plays key roles in control of gating property by
intracellular substances such as H⫹ and PtdIns(4,5)P2; mutation of K80 that would disrupt the bonding lowers intracellular pH (pHi) sensitivity and fastens recovery from acidification-induced channel inhibition and reactivation of rundown channel by PtdIns(4,5)P2. The profile of pHi- and
PtdIns(4,5)P2-dependent modulation of Kir4.1 that is predicted to harbor H⫹ bonding resembles the characteristic of
Kir1.1, whereas Kir2.1 that is expected to have no H⫹ bonding mimics the phenotype of the mutated Kir1.1. Therefore,
the H⫹-bond linking between TM1 and TM2 at the bundlecrossing region is a crucial determinant for channel gating in
all the Kir channels (644) (also see sect. VA2).
While the pore lining helices in the KcsA structure
are arranged in a fourfold symmetry, those in KirBac1.1
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are misaligned (404, 894). Furthermore, only three K⫹
ions could be modeled in place in the selectivity filter of
KirBac1.1, and the temperature factor of one of these K⫹
ions is much smaller than for the other two. This is quite
different from the character of the selectivity filter of
KcsA and KirBac3.1 where four K⫹ ions are accommodated with similar temperature factors (573). The unequal
ion occupancy among the four K⫹ binding sites in the
selectivity filter of KirBac1.1 suggests that this structure is
in the closed state. These structural alterations around
the selectivity filter may also serve to gate Kir channels,
and they may correspond to fast gating.
The slide helix at the cytoplasmic end of TM1 is a
unique structural element of the Kir channel family compared with other K⫹ channels (143, 336, 338, 404, 466)
(Fig. 4C). It is situated outside of the inner (TM2) and the
outer (TM1) helices and connects the TM1 domain and
the NH2-terminal part of the cytoplasmic domain. Since
the slide helix is amphiphilic, it may lie at the interface
between the inner leaflet of membrane and the cytoplasm.
Loss-of-function mutations in the slide helix of Kir1.1 and
Kir2.1 have been identified in human inherited diseases of
type II Bartter syndrome (V72E and D74Y) (755) and
Andersen syndrome (Y68D, D71V, D71N, T74A, T75A,
T75M, T75R, D78G, D78Y) (44, 123, 139, 622, 767) (see
sects. IIF and VA6). These mutations led to the impairment
of channel activity by either suppressing channel expression in the plasma membrane or inhibiting K⫹ permeability. Therefore, the slide helix has important roles to play
in Kir channel function.
2. Cytoplasmic domain
The crystal structure of the cytoplasmic domains of
Kir channels has been obtained in two ways. Stable crystals of the NH2- and COOH-terminal complexes that make
up the cytoplasmic domains of Kir3.1 (574), Kir2.1 (611),
and Kir3.2 (306) have provided high-resolution information. These results have proven to be comparable to the
lesser resolution data obtained from entire Kir channels
(404, 573). Therefore, conformational information obtained from the cytoplasmic region of Kir channels should
correspond to that of the overall structure of the entire
channel protein. But data concerning the region connecting NH2 and COOH termini and the membrane interface
should be interpreted with caution. It is worth noting that
the amino acid sequence of regions connecting NH2 and
COOH termini to the membrane segment is conserved
among the Kir channel family (Fig. 4D).
The cytoplasmic domain of Kir channels is made up
of the NH2 and COOH termini of four Kir subunits. Each
terminus is rich in ␤-strands (Fig. 4D). These ␤-strands
form three ␤-sheets. The cytoplasmic domain is mainly
formed from the COOH terminus of each subunit, while
the NH2 termini are present between adjacent COOH
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termini and they contribute to formation of the interface
between subunits. Each NH2 terminus contains a single
␤-strand (␤A) that forms a ␤-sheet with two ␤-strands (␤L
and ␤M) in a COOH terminus. This contributes to the
stability of the whole cytoplasmic region. The interaction
between NH2 and COOH termini has been observed in
both Kir3.x and Kir6.x channels. Huang et al. (1995, 1997)
demonstrated that NH2- and COOH-terminal domains of
Kir3.1 and Kir3.4 subunits could bind to each other and
together synergistically enhanced G␤␥ binding (288, 289).
Tucker and Ashcroft (792) demonstrated a similar association between the COOH terminus of Kir6.2 and a conserved region of the NH2 terminus of either Kir6.2 or
Kir2.1, and they suggested that the association of both
termini may be common to all Kir channels.
The four groups of associated NH2/COOH termini
make up a cylinder that surrounds the so-called cytoplasmic pore. This architecture is a characteristic of the Kir
channel family and extends the ion conduction pathway
by ⬃30 Å (574). Therefore, in Kir channels, K⫹ has to pass
over 60 Å through the pore composed of the transmembrane and cytoplasmic domains. The detailed structure of
the cytoplasmic pore and its relevance to channel function and channelopathies (611) will be discussed in reference to Kir2.1 (see sect. II).
Inward rectification of Kir channel currents results
from intracellular Mg2⫹ and polyamine block of the channel pore when Em is more depolarized than EK (see sect.
IC1). Although several residues responsible for this block
have been identified on the inner TM2 helix in the transmembrane domain, others have been identified in the
cytoplasmic domain (195, 230, 231, 417, 757, 865). These
acidic residues have been mapped to the wall of the pore
of the cytoplasmic domain (see sect. II for further details).
PtdIns(4,5)P2 is a molecule that is necessary for the
normal functioning of Kir channels (44, 263, 287, 474, 723,
889), as for other ion-transport proteins such as the Na⫹/
Ca2⫹ exchanger, voltage-gated Ca2⫹ channels, and transient receptor potential channels (263, 264, 748) (see sect.
IC1). In Kir channels, a number of basic and uncharged
residues in the cytoplasmic region of the channel are
proposed to be involved in the action of PtdIns(4,5)P2
(146, 163, 204, 287, 474, 487, 653, 699, 722, 723, 886, 889).
These are mostly to be found upon the surface of the
cytoplasmic region which faces the membrane (Fig. 4D).
Residues in the transmembrane region may also participate in the association between the channel and
PtdIns(4,5)P2; they include basic residues clustered in
lower part of the inner TM2 helix and an uncharged
residue in the slide helix (474, 660). However, the mechanism of action of PtdIns(4,5)P2 has not been fully elucidated. The crystal structure of the KirBac3.1-Kir3.1 chimera was obtained in the presence of PtdIns(4,5)P2 (573),
but the amino acid residues at the membrane-cytoplasm
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interface as well as the crystal of PtdIns(4,5)P2 itself
could not be modeled due to their weak electron density.
The cytoplasmic domain pore abutting to the transmembrane domain is formed by a loop [HI (G) loop]
between ␤H and ␤I strands (Fig. 4D). The cross-sectional
area of the space surrounded by the HI loops is wide in
Kir3.2 and narrow in Kir2.1. Mutations in the HI loop
disrupted gating and affected inward rectification of
Kir2.1 (611) (see sect. IIB for details). In the crystallization
of the KirBac3.1-Kir3.1 chimera, two different conformations were obtained (573). A prominent difference between the two is found in the apex of the cytoplasmic
pore formed by the HI loop (G-loop). In one conformation
the apex is dilated, whereas in the other it is constricted.
These two conformations of the chimera channel may
represent open and closed states of the cytoplasmic pore,
respectively. In support of this idea, several loss-of-function mutations in Kir channelopathies are found in the
residues forming the apex (611) (see sect. II).
The cytoplasmic domains are involved in the control of
Kir channel gating by Na⫹, nucleotides, and G proteins.
Structural analyses of Kir3.x strongly suggest that G␤␥ and
Na⫹, like PtdIns(4,5)P2, interact with the cytoplasmic region
(78, 246, 288, 289, 305, 326, 392, 402, 734, 863) (see sect. IIIB).
In the case of KATP channels, homology modeling predicts
that one NH2 terminus residue and three COOH terminus
residues create the binding pocket for ATP in the Kir6.x
subunit (15) (see sect. IVB). Biochemical assays show that, in
Kir2.3, acidification strengthens the interaction between
NH2 and COOH termini, which is suggested to be involved in
channel closure by an unidentified mechanism (634). The
modulation of the conformation of NH2 and COOH termini
and their interaction clearly play pivotal roles in the regulation of Kir channel gating.
E. Pharmacology
Blockers most commonly used for Kir channels are
Ba2⫹ and Cs⫹ (189, 234 –236, 266, 315, 592). Tetraethylammonium (TEA) and 4-aminopyridine (4-AP) are known as
the inhibitors of Kv channels but have little effects on Kir
channels (236, 598). Ba2⫹ and Cs⫹ effectively block the
majority of Kir channels. Although high concentrations of
Ba2⫹ can also block Kv channels, at micromolar concentrations this cation is relatively specific to Kir channels (188,
636). Ba2⫹ and Cs⫹ are often used to examine the physiological roles of Kir channels in native cells and tissues.
Externally applied Ba2⫹ and Cs⫹ suppress Kir currents in a
voltage-dependent manner. They inhibit Kir channels more
strongly as the membrane is hyperpolarized (189, 235, 236,
592). In addition, the blocking effect of Cs⫹ and Ba2⫹ decreases substantially as [K⫹]o increases (235, 236). However,
there are different aspects in the behavior of the two blockers. First, following a voltage step, the approach to steady-
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state block is much faster for Cs⫹ than Ba2⫹. Second, the
dissociation constant for Cs⫹ is dependent on extracellular
K⫹ concentration ([K⫹]o) (236), but that for Ba2⫹ is independent of this factor (235).
In spite of a limited number of blockers of Kir channels (see above), pharmacological and physiological assays have revealed other compounds that can affect particular types of Kir channels. Tertiapin is a toxin that was
isolated from honey bee venom which blocks KG and
Kir1.1 channels (342). The oxidation-resistant tertiapinQ
(343) blocks KG current, but not KATP current or IK1 in
isolated cardiac myocytes (136, 372) (see sect. IIIE). The
majority of reagents affecting KATP channel activity react
on the auxiliary SURx subunits. KATP channels are the
targets of antidiabetic sulfonylurea compounds (219) as
well as the class of compounds known as “K⫹ channel
openers” (18) (see sect. IVE). These reagents are also
useful tools to examine basic properties of KATP channels.
II. CLASSICAL Kir CHANNELS (Kir2.x)
A. Historical View and Molecular Diversity
The first member of this family to be cloned was from
a mouse macrophage cell line and named IRK1/Kir2.1/
KCNJ2 (394). Afterward, three more subunits were identified as members of this family. They are Kir2.2(IRK2)/
KCNJ12 (389, 758), Kir2.3(IRK3, BIR11)/KCNJ4 (56, 64, 543),
and Kir2.4(IRK4)/KCNJ14 (782). The amino acid sequence of
mouse brain Kir2.1 shares 70, 61, and 63% identity with
Kir2.2, Kir2.3 and Kir2.4, respectively. The sequences are
most highly conserved in the TM1, TM2, and H5 regions.
Initially Kir2.x subunits were thought to be made up of
only homomeric complexes (780). However, recent studies
have revealed that Kir2.x subunits can function as heterotetramers both in vitro and in vivo. Indeed, in vitro electrophysiological experiments have shown that each of Kir2.1, Kir2.2,
and Kir2.3 can assemble with any one of the other subunits,
and the respective heteromer exhibits different properties
from that of their homomers (626) (Table 1). Some of these
heteromers have been identified in native tissues. For example, heteromeric assemblies of Kir2.1/2.2 and Kir2.1/2.3 channels are expressed in cardiac myocytes (see sect. IID), and
Kir2.1/2.4 has been found in the brain (695).
B. Pore Function and Structure Bases
⫹
The inward rectifier K channel in skeletal and cardiac
muscle belongs to the Kir2.x channel family. The channels of
this family are constitutively active and exhibit strong inward rectification. They contribute to the establishment of
highly negative Eres and long-lasting action potential plateau
in various cells including cardiac myocytes.
TABLE
1.
1. Rectification and activation
The basic architecture of Kir channels with transmembrane and cytoplasmic regions and pore structure
(see Fig. 4 and sect. ID) is conserved among all Kir2.x
subunits. Inward rectification is caused by intracellular
Biophysical properties of Kir2.x channels
Channel Name
Unitary Conductance, pS
Kir2.1
Kir2.2
23–30.6
34.2–40
Kir2.3
Kir2.4
Kir2.1-Kir2.2
(Concatamers)
Kir2.1-Kir2.3
(Concatamers)
Kir2.2-Kir2.3
(Concatamers)
Kir2.1⫹Kir2.2
(Coexpression)
Kir2.1⫹Kir2.3
(Coexpression)
Kir2.2⫹Kir2.3
(Coexpression)
13–14.2
15
30
28.1
NE
NE
NE
NE
Ba2⫹ Sensitivity (IC50)
3.2 ␮M
(60 mM 关K⫹兴o
8 ␮M
(96 mM 关K⫹兴o
0.5 ␮M
(60 mM 关K⫹兴o
6 ␮M
(96 mM 关K⫹兴o
10.3 ␮M
(60 mM 关K⫹兴o
390 ␮M
(96 mM 关K⫹兴o
0.68 ␮M
(60 mM 关K⫹兴o
3.39 ␮M
(60 mM 关K⫹兴o
1.73 ␮M
(60 mM 关K⫹兴o
0.64 ␮M
(60 mM 关K⫹兴o
6.32 ␮M
(60 mM 关K⫹兴o
1.94 ␮M
(60 mM 关K⫹兴o
394, 461, 626, 782
in XO)
in XO)
461, 626, 758, 782
in XO)
in XO)
461, 543, 626
in XO)
782
in XO)
626
in XO)
626
in XO)
626
in XO)
626
in XO)
626
in XO)
626
in XO)
NE, not examined; XO, Xenopus oocytes.
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STRUCTURE AND PHYSIOLOGICAL FUNCTION OF Kir CHANNELS
ions such as Mg2⫹ (508, 802) and polyamines (471, 856)
(see sect. IC). Studies of mutated Kir2.x channels have
revealed that the subunits possess more than one binding
site for Mg2⫹ and polyamines. The strong rectifier Kir2.1
has the negatively charged residue Asp at the D/N site.
Another residue in the TM2 helix, S165 in Kir2.1, has been
reported to be crucial for block by Mg2⫹ but not by
polyamines (196). The crystal structure of KirBac1.1 suggests that these residues face the transmembrane pore
cavity formed by the inner TM2 helixes.
Further site-directed mutagenesis has identified negatively charged amino acids (Glu) at two different positions (E224 and E229 for Kir2.1) in the COOH terminus of
the cytoplasmic domain that are critically involved in both
Mg2⫹ and polyamine sensitivity (396, 756, 757, 865). Mutagenesis and substituted cysteine accessibility experiments suggest that these residues directly interact with
Mg2⫹ and polyamines (482, 534). Structural information
from KirBac, Kir3.1, and Kir2.1 supports the pore-lining
location of these amino acids: the side chains of Glu point
to the center of the cytoplasmic domain conduction pathway in each subunit, forming rings of negatively charged
residues that create a complimentary electrostatic match
301
for the binding of a positively charged polyamine (404,
574, 611).
The reason why Kir2.1 has residues that bind Mg2⫹
and polyamines in both the transmembrane and the cytoplasmic domains is still elusive. It is possible that these
two types of binding sites play distinct roles: polyamines
may plug the pore in the transmembrane domain (plugging site), whereas the cytoplasmic region would serve as
an intermediate binding site (nonplugging site) that increases the local concentration of polyamines around the
plugging site (396, 434, 472, 844).
Analysis of the crystal structure of the cytoplasmic
domain of Kir2.1 has recently identified an intrinsically
flexible loop around the membrane face of the cytoplasmic pore (611). The loop constricts the cytoplasmic pore
to ⬃3 Å and forms a girdle around the central pore axis.
The girdle, which consists of a loop between ␤H and ␤I
strands and is called the “G-loop,” forms the narrowest
portion of the ion conduction pathway in the cytoplasmic
region (Fig. 5) (see also sect. ID). The narrowest part of
the G-loop is made up by A306 and to a lesser extent by
E299, G300, M301, and M307. A306 is localized at the apex
of the G-loop. The substitution of Glu, Cys, or Thr for
FIG. 5. The structure of the cytoplasmic pore region of Kir2.1. Side (left and bottom right) and top-down (top right; membrane to cytoplasm) views
of the cytoplasmic region (NH2 and COOH termini) of the Kir2.1 structure, highlighting amino acids lining the permeation pathway. C␤ atoms (A306) of
the G-loop make up the narrowest ⬃3-Å region of the pore and are shown as open circles for clarity. Other residues near the G-loop are labeled. This
illustration was constructed from protein database ID 1U4F (611). [From Pegan et al. (611), with permission from Nature Publishing Group.]
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HIBINO ET AL.
A306 abolished Kir2.1 current. Because the side chain of
these residues is larger than that of Ala, these substitutions would result in the physical occlusion of the G-loop
without changing its backbone conformation. When another constituent of the G-loop M301 was mutated to Ala,
an enhancement of inward rectification was observed.
Thus the G-loop also contributes to inward rectification in
Kir2.1. Charged amino acids, R228, D255, D259, and R260,
face the cytoplasmic pore (Fig. 5). Charge reversal at
D255 (D255R) or at D259 (D259R) and charge neutralization at D259 (D259A) decreased inward rectification,
whereas substitutions of R228 and R260 with Ala had little
effect on the rectification profile. Collectively, the diaspartate cluster (D255/D259) that faces the cytoplasmic
pore is involved in inward rectification of Kir2.1 channels.
Kir2.x are activated by PtdIns(4,5)P2 (264, 287, 760)
(see sect. I, C and D). The sequence that was first found to
be associated with activation of Kir2.1 by PtdIns(4,5)P2
was the group of positively charged residues, KKR (177–
179 in Kir2.1) just beyond the TM2 region (287, 474, 722,
737). Cytoplasmic NH2 and COOH termini also harbor
residues involved in PtdIns(4,5)P2 regulation. In Kir2.1,
these residues are H53, R67, K187, K188, R189, K219,
R228, and R312 (474).
Andersen syndrome is caused by dysfunction of
Kir2.1 (see sect. IIF for the phenotypes) (Table 2). Early
studies identified a variety of mutations, including R67W,
D71V/N, T75R, ⌬95–98, S136F, G144S, G146D, P186L,
R189I, T192A, G215D, N216H, R218W/Q, G300V/D, V302M,
E303K, R312C, and ⌬314 –315 (11, 139, 281, 622, 787).
They generally disrupt Kir2.1 activity via dominant negative interactions in the formation of heteromeric assemblies (44, 281, 622, 626, 787). Of the more than 30 mutations that are currently known to be associated with
Anderson syndrome (120, 682), the majority occur in the
cytoplasmic domain: some are located around the top region
of the cytoplasmic structure (R189, T192, R218, G300, V302,
E303, R312, ⌬314 –315) close to the PtdIns(4,5)P2 binding
TABLE
2.
site (11, 139, 474, 622, 787), and others (G215, N216) are
included in ␤C-␤D loop near G-loop. Four locations, G300,
V302, E303, and the site of the deletion mutant ⌬314 –315,
are in the G-loop region (Fig. 5), implying a crucial role of
this region in the K⫹-conduction pathway. For example,
V302 is located near the apex of the cytoplasmic structure. A loss of function mutation, V302M (787), rendered
the channel unable to conduct K⫹ without affecting subunit assembly or attenuating trafficking, but by impairing
PtdIns(4,5)P2 sensitivity (487).
2. Other regulatory factors
Kir2.x subunits heterologously expressed in Xenopus
oocytes and mammalian cells elicit strongly rectifying K⫹
currents. As summarized in Table 1, the channels made up
of various homo- and heteromultimers of Kir2.x subunits
exhibit unique single-channel conductance, respectively
(394, 461, 543, 626, 758, 782). The steady-state open probability (Po) of Kir2.2 decreases with hyperpolarization,
whereas that of Kir2.1 or Kir2.3 remains unchanged. In
addition, a remarKABle feature of Kir2.3 is its activation by
intracellular as well as extracellular alkalization (pKa ⫽
6.76 and 7.4, respectively) (106, 634, 895). The determinant of this pH sensitivity is a single His residue (H117 in
Kir2.3) in the TM1 to H5 linker region (106). Extracellular
alkalization also enhances human Kir2.4 channel current
with a pKa of 7.14 (290). This subunit has His residue
(H130) in the position corresponding to H117 in Kir2.3.
The activity of some Kir2.x channels can be modified
by kinases. The K⫹ current associated with Kir2.3 but not
that of Kir2.1 is suppressed by PKC activators, phorbol
12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) (251, 357). Apparently, T53 in the NH2terminal region of Kir2.3 is phosphorylated by PKC (896).
Kir2.2 current can also be suppressed by PMA treatment
(901). A study analyzing regulation of Kir2.x channels by
␣1-adrenergic receptors has recently shown that, while
Human Kir channelopathies
Disease
Channel
Bartter’s syndrome (type II)
Andersen syndrome (LQT7)
Short Q-T syndrome
Generalized seizures
SeSAME
Persistent hyperinsulinemic hypoglycemia of infancy
Persistent hyperinsulinemic hypoglycemia of infancy
Developmental delay, epilepsy and neonatal diabetes
syndrome
Permanent neonatal diabetes
Permanent neonatal diabetes
Epilepsy, dysmorphic feature, and neonatal diabetes
Dilated cardiomyopathy
Snowflake vitreoretinal degeneration
Condition
Reference Nos.
Kir1.1
Kir2.1
Kir2.1
Kir4.1
Kir4.1
SUR1
Kir6.2
Kir6.2
Loss of function
Loss of function
Gain of function
ND
Loss of function
Loss of function
Loss of function
Gain of function
130, 131, 167, 185, 186, 332, 474, 617, 696, 700, 729, 743, 755, 809
11, 44, 120, 139, 281, 622, 626, 682, 787
628
72
52, 693
294, 776
252, 293, 560, 647, 777
210, 504, 629, 631, 675
SUR1
Kir6.2
Kir6.2
SUR2A
Kir7.1
Gain of function
Gain of function
Gain of function
Loss of function
Gain of function
33, 155
151, 209-211, 504, 675, 804
629, 631
48
250
ND, not determined.
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Kir2.3 channels are inhibited via activation of PKC, Kir2.2
channels are inhibited via activation of src kinases in a
PKC-independent manner (900). Although it was initially
reported that PKA activators had little effect on Kir2.1 or
Kir2.3 currents (251), Kir2.1 was found to interact with A
kinase-anchoring protein 79 (AKAP79). Of interest, Kir2.1
current was increased by cAMP when the channel was
coexpressed with AKAP79 and treated with the phosphatase inhibitors okadaic acid or cypermethrin (118). Activation of PKA was also shown to increase Kir2.2 current
(901). Physiologically, the phosphorylation of these channels by these kinases may be involved in receptor-dependent modulation of various excitable cells.
A recent study has further suggested that cytoplasmic
regulatory factors such as phosphorylation and pH might
modulate channel function by affecting the channelPtdIns(4,5)P2 interaction. Among the Kir2.x subunits,
Kir2.1 interacts more strongly with PtdIns(4,5)P2 than
Kir2.3 does. Du et al. (146) made two point mutants,
Kir2.1(R312Q) and Kir2.3(I213L), which were found to
weaken and strengthen channel-PtdIns(4,5)P2 binding, respectively (146). When they compared the function of the
mutant channel with their corresponding wild-type channels, they found that inhibition induced by phospholipase
C (PLC)-␤ PLC-␥, protein kinase C (PKC), lipid phosphatases, and protons correlates inversely with the apparent
affinity of the channels for PtdIns(4,5)P2.
C. Intracellular Localization
1. Channel trafficking to the membrane
Classical Kir channels possess an amino acid sequence responsible for recruitment of the channel from
the endoplasmic reticulum (ER) to the cell surface. This
ER export signal is conserved in all subunits of the Kir2.x
subfamily and corresponds to the amino acid sequence
F-C-Y-E-N-E in the cytoplasmic COOH terminus (488,
745). Disruption of this motif by mutagenesis causes retention of Kir2.1 in the cytosol. It is of note that Kir1.1 has
different ER export signals (V-L-S and E-X-D) in its
COOH-terminal region (488). It is therefore likely that the
surface expression of channels of each Kir subfamily is
regulated by different machineries.
In general, after synthesis in ER, membrane proteins
are transported to the Golgi apparatus for posttranslational modification, and then carried onto the cell surface.
In Kir2.1, particular parts of the NH2-terminal region have
been identified to be responsible for Golgi export (744).
The double mutation R44A, R46A in the NH2 terminus
resulted in disappearance of Kir2.1 from the cell surface
and its accumulation in the Golgi complex. In addition,
Y-X-X-⌽ (position 242–245 in Kir2.1) was found to be an
essential element to allow Golgi export (275). Thus two
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independent motifs seem to regulate trafficking of Kir2.1
from Golgi complex to cell surface.
All the Kir2.x subunits have these ER- and Golgi-export
signals (275, 488, 745). However, when Kir2.4 was expressed
in cultured cells, it distributed mainly in the Golgi complex
with little expression on the cell surface. Mutagenesis assays
revealed that the flanking sequence upstream of the Y-X-X-⌽
motif in Kir2.4 would interfere with its trafficking (275).
Some of the loss-of-function mutations in Kir2.1 that
are linked to Andersen syndrome (see sect. IIF and Table
2) impair trafficking to the cell surface. They are V302M,
⌬95–98 in the TM1 region, and ⌬314 –315 in the COOHterminal region (622, 787), and they lead to Kir2.1 being
retained in the cytoplasm (44; but see Ref. 487 and sect.
IIB for the role of V302M in inhibition of currents through
the K⫹-conduction pathway). Therefore, the export signals described above are not alone in determining a channel’s trafficking to the cell surface.
Activity of Kir2.1 on the cell surface may be negatively controlled by its internalization, which depends on
small GTPase Rho family proteins. Kir2.1 exhibits a high
degree of internalization mediated by dynamin, a protein
crucial for endocytosis (59, 447). A dominant negative
mutant of RacI belonging to Rho family, when coexpressed with Kir2.1 in HEK293T cells, doubly increases
K⫹ current by inhibiting its internalization, and this effect
is likely to involve disruption of function of dynamin (59).
The RacI-dependent regulation is detectable in neither
Kir2.2 nor Kir2.3 and thus specific to Kir2.1. Although a
previous study showed that another small GTPase, RhoA,
might be crucial for internalization of Kir2.1 in tsA201
cells (345), a recent report has been unable to reproduce
the same result (59). This discrepancy would be due to
difference of the cell types.
2. Localization in the membrane macrodomains
Kir2.x subunits are identified in specific locations in
cells such as neurons and epithelial cells. They occur in
somata and dendrites of neurons (302, 633). In mouse
olfactory bulb, Kir2.3 is specifically expressed at the
postsynaptic membrane of dendritic spines of granule
cells, which receive mostly excitatory synaptic inputs
(302). The postsynaptic localization of Kir2.x subunits
may be determined by mechanisms such as protein-protein interactions. The PDZ-proteins are known to bind
various molecules such as glutamate receptors and Kv
channels and target them to postsynaptic sites (99, 109,
370, 555, 710). Interaction occurs between the PDZ domains and consensus motifs at the COOH-terminal end of
the receptors and channels. Since Kir2.x subunits possess
a class I PDZ domain recognition sequence (X-S/T-X-V/I)
(736), PDZ-proteins are strong candidates for determining
postsynaptic localization of Kir subunits. Several studies
support this idea by demonstrating the following:
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1) Kir2.1 can interact with PSD-95 and PSD-93/
chapsyn110 (99, 447, 555); 2) Kir2.2 can associate with
SAP97, CASK, Velis, and Mint-1 (441, 442); and 3) Kir2.3
can bind to PSD-95, PSD-93/chapsyn110, and SAP97 (99,
302, 442, 555). Notably, association of PSD-95 with Kir2.3
causes reduction of its single-channel conductance (555),
which could contribute to control of synaptic excitability
in the brain. Since these PDZ-proteins simultaneously
gather different molecules such as nitric oxide(NO) synthase (66, 67), SynGAP (371), and AKAPs (100), they may
not only determine the postsynaptic localization of Kir2.x
channels but also make up functional units that mediate
efficient signaling. However, much of this regulation via
protein-protein interaction remains to be demonstrated in
vivo.
When expressed in cultured epithelial cells such as
Madin-Darby canine kidney (MDCK) and pig kidney epithelial cell line LLCPK, Kir2.3 is sorted to the basolateral
membrane surface (430, 431, 595). In kidney, functional
Kir2.3 was found at the basolateral membrane of epithelial cells in collecting ducts (430, 532, 827). At least two
processes are thought to be involved in the basolateral
targeting of Kir2.3. The first step may be coordinated by
PDZ-proteins. In MDCK cells, truncation of the PDZ-binding motif of Kir2.3 (E-S-A-I) resulted in its missorting to
intracellular vesicle compartments that contained recycling endosomes (595). Biochemical assays revealed that
Kir2.3 interacts with a PDZ-protein, Lin-7, which is expressed at the basolateral membrane and also associates
with another PDZ-protein, CASK (9, 595). Collectively,
this interaction would retain Kir2.3 at the membrane surface. The second step is mediated by a membrane targeting sequence in the COOH-terminal region of Kir2.3. It
was found that deletion of 11 amino acids, which neighbor the PDZ-domain binding motif at the COOH-terminal
end, mislocates Kir2.3 from basolateral to apical membrane (431). This sequence arrangement is unique to
Kir2.3, and therefore, this channel could utilize a particular mechanism to determine its localization in these
epithelial cells. It is still not known why a deletion mutant
that lacks both of the PDZ and membrane targeting motifs
(15 amino acid truncation) is not internalized and remains
at the apical membrane surface (431). Other binding proteins and/or posttranslational modification may be involved in the trafficking process of Kir2.3. Further studies
are needed to clarify the mechanism determining the
localization of Kir2.3 in epithelial cells.
D. Physiological Functions in Cells and Organs
1. Heart
The classical Kir current IK1 is abundantly expressed
in cardiac myocytes, including Purkinje fibers as well as
ventricular and atrial tissues (41, 406, 514, 575, 667) but
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not in nodal cells (578). This conductance dominates the
resting conductance of these tissues and is defined as a
time-independent background K⫹ current. It exhibits a
large inward current at the Em more negative than EK and
a relatively large outward current at the Em slightly more
positive than EK. This feature is essential to stabilize the
Eres of cardiac myocytes near EK. But, due to inward
rectification, as the membrane is further depolarized the
IK1 current progressively decreases. This characteristic
generates a region of so-called negative slope of the conductance of IK1. The current becomes practically zero at
positive Em (Fig. 6A). The lack of outward conductance
through IK1 at positive potentials prevents K⫹ efflux during the action potential plateau, resulting in the maintenance of depolarization (Fig. 6, B and C). When repolarization is initiated by activation of the Kv channels, as Em
repolarizes through the negative slope region relatively
large outward currents pass through IK1, which accelerates the final stage of repolarization. Hence, IK1 is critically involved in determining the shape of the cardiac
action potential, namely, 1) setting the resting potential,
2) permitting the plateau phase, and 3) inducing rapid
final stages of repolarization (Fig. 6).
In the heart, analyses of mRNA transcripts by in situ
hybridization histochemistry, RNase protection assay,
and RT-PCR demonstrated that cardiomyocytes express
Kir2.1, Kir2.2, and Kir2.3 (37, 63, 134, 461, 638, 821, 830).
In contrast, Kir2.4 is restricted to neuronal cells (461).
The precise molecular make up of the cardiac IK1
channel in mouse has recently been identified by the
analysis of Kir2.1 and Kir2.2 knockout animals. Whereas
Kir2.2 knockout mice showed ⬃50% reduction in the IK1
current, no detectable current was observed in Kir2.1
knockout mice (881). It is therefore likely that Kir2.2
subunits assemble with Kir2.1 to form the IK1 current.
Also, it is possible that homomeric Kir2.2 channels which
form functional K⫹ channels in heterologous expression
systems are nonfunctional in native cardiac myocytes. In
rabbit ventricular myocytes, expression of the dominant
negative form of either Kir2.1 or Kir2.2 suppressed ⬃70%
of the IK1 current, supporting the idea that IK1 is generated
by heteromeric Kir2.1/2.2 channels (902). These results
suggest that among the Kir2.x subfamily, it is Kir2.1 that
may be the core subunit generating the IK1 current. The
cardiac phenotypes observed in Kir2.1-knockout mice are
as follows (see also sect. IIF). The majority of ventricular
myocytes isolated from wild-type mice were quiescent
with Eres approximately ⫺72 mV. In contrast, most of the
cells from Kir2.1 knockout mice showed spontaneous
rhythmical action potential firing so that it was difficult to
measure a true Eres. In addition, Kir2.1 knockout myocytes exhibited significantly broader action potentials
than wild-type myocytes. Such phenotypes were not observed in Kir2.2 knockout mice. On the other hand, the
electrocardiograms of Kir2.1 knockout mice showed nei-
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305
ther ectopic beats nor reentry arrhythmias, indicating that
their cardiac abnormalities did not include alteration of
the sinus pacing of the heart. Surprisingly, Kir2.1 knockout mice consistently had a slower heart rate, which was
attributed to an indirect effect as the result of disruption
of the channel activities in organs other than the heart.
Similar phenotypes were detected in animals that overexpressed a dominant negative form of Kir2.1 (518, 526).
Notably, all Kir2.1-knockout animals exhibited the complete cleft of the secondary palate. This abnormality prevented the pups from nursing and causes the aspiration of
oral secretions. The mice died within 12 h after birth
because of the respiratory problems (880).
The molecular constituents of guinea pig IK1 has
remained uncertain. The single-channel conductance of
IK1 in ventricular cells of guinea pig heart is reported to be
20 – 40 pS when pipette solution contains 145–150 mM
[K⫹] (406, 507, 679, 716). A detailed study compared the
single-channel properties of cloned Kir2.x subunits and
IK1 in native cells (461). Guinea pig myocytes display
three populations of Kir channels with mean conductance
of 34.0, 23.8, and 10.7 pS (461). The cloned guinea pig
Kir2.1, Kir2.2, and Kir2.3 channels showed conductances
of 30.6, 40.0, and 14.2 pS (461). The Ba2⫹-block profile of
the native 34.0-pS channel was virtually identical to that
of the cloned Kir2.2. Consistently, hyperpolarization of Em
decreases Po of cardiac myocytes of guinea pig (406),
which is the characteristic observed only in cloned mouse
Kir2.2 but neither mouse Kir2.1 nor Kir2.3 (315, 758).
Another study identified a heteromeric assembly of
Kir2.1/2.3 in guinea pig cardiomyocytes by biochemical
assays (626). Further studies are needed to clarify the
precise subunit components of guinea pig IK1.
j
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FIG. 6. Excitability of cardiac myocytes. A: current-voltage relationship and the action potential in the ventricular myocyte. The currentvoltage relationship of the instantaneous current is indicated by the
solid line and that for the steady-state current at 1 s by the broken line
(top panel). The action potential (bottom panel) is drawn sideways to
relate it to the current-voltage relationship. Gray area indicates IK1
current. B: regional variation in action potentials in the heart. Their
relation to a typical electrocardiogram is indicated at the bottom.
C: comparison of action potentials in different heart regions. Eres in
sinoatrial (SA) and atrioventricular (AV) nodes is not as hyperpolarized
as in other regions. The Em in SA node shows a “phase 4” depolarization
that triggers the next action potential. In SA and AV nodes, the rate of
rise of the action potential is slow. The SA action potential stimulates
atrial myocytes. Atrial myocytes exhibit a hyperpolarized Eres of approximately ⫺90 mV without automatic excitation and a relatively short action
potential. Eres of His bundle and Purkinje fibers is also hyperpolarized to
approximately ⫺90 mV. Their action potential duration is significantly
longer than that of atrial myocytes. Ventricular myocytes show hyperpolarized Eres of about ⫺90 mV and yield action potentials with a sustained
plateau phase. The relative amounts of IK1, which is made up of classical
Kir2.1 channels, are indicated below. SA and AV nodes express little IK1. IK1
is involved in the formation of deep Eres, the sustained plateau, and rapid
repolarization.
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2. Blood vessels
The major constituents of vasculature are endothelial
and smooth muscle cells. Electrophysiological studies
have demonstrated that both cell types express classical
Kir channels (2, 570). In vascular endothelial cells, they
are considered to be the most prominent channels (570,
571, 811). Functional expression of classical Kir channels
sets the Eres of endothelial cells to a negative potential,
which provides the driving force for Ca2⫹ influx through
Ca2⫹-permeable channels. Indeed, block of endothelial
Kir channels by Ba2⫹ inhibits not only flow-induced Ca2⫹
influx but also vasodilatation caused by Ca2⫹-dependent
production of NO (420, 828). Since an increase in intracellular Ca2⫹ concentration ([Ca2⫹]i) activates enzymes
such as NO synthase and phospholipase A2 (PLA2) and
thus induces the secretion of vasoactive factors, control
of Em by classical Kir channels in endothelial cells is one
of the key regulatory systems for vascular tone. In support of this idea, shear stress induced by laminar flow
evokes classical Kir currents in aortic endothelial cells
(591), which would hyperpolarize the cells and relax
smooth muscle by secretion of NO (198, 523, 608, 803).
The molecular make up of endothelial Kir channels has
not been fully elucidated. In aortic endothelial cells,
whereas Kir2.1 and Kir2.2 proteins are expressed, Ba2⫹
and pH sensitivity and single-channel conductance analyzed by patch-clamp techniques suggested Kir2.2 as the
dominant channel (166). It would be of interest to examine the endothelial function of Kir2.1 and Kir2.2 knockout
mice.
In vascular smooth muscle cells, it has been suggested that classical Kir channels might contribute to
vasodilation in response to an increase in [K⫹]o (152, 374,
515, 516, 556). Although elevated [K⫹]o usually depolarizes smooth muscle cells and is expected to constrict
blood vessels, a mild increase in [K⫹]o from 6 to 15 mM
hyperpolarizes Em and dilates cerebral and coronary arteries (374, 516, 556). The Eres of the arteries’ muscle cells
is known to be about ⫺45 mV, and an elevation of [K⫹]o
to 15 mM hyperpolarized the Em to about ⫺60 mV by
increasing the Kir conductance (374, 556). This hyperpolarization closes voltage-gated Ca2⫹ channels and therefore reduces [Ca2⫹]i, which results in vasodilation (373).
Ba2⫹ blocked both K⫹-induced hyperpolarization and vasodilation of coronary and cerebral arteries (374). In the
cerebral arteries, it was reported that [K⫹]o surrounding
the smooth muscle cells was elevated due to K⫹ secretion
from the end feet of astrocytes (174), and disruption of
the system by various pathological factors may cause
neuronal disease. The local augmentation of [K⫹]o occurred when neurons were stimulated. This system may
therefore couple neuronal activity to control of local
blood flow in the brain (174).
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In vascular smooth muscle cells, Kir2.1 but neither
Kir2.2 nor Kir2.3 was identified (61). Blood vessels in
Kir2.2 knockout mice dilated normally in response to high
[K⫹]o stimulation but not vessels from Kir2.1 knockout
mice (880). Therefore, Kir2.1 seems to be a main subunit
to form the classical Kir current in these cells.
Activity of classical Kir channels is also important for
relaxation and contraction of tracheal smooth muscle and
is controlled by autacoids. It has been found that neurokinin A, a type of tachykinin, inhibits classical Kir channels in tracheal smooth muscle cells (550), although molecular composition of the channels has remained unknown. This process depolarizes the cells and induces
tracheal contraction, which may be involved in pathogenetic process of asthma.
3. Neurons in the brain
Electrophysiological techniques detected Kir currents in hippocampal neurons (68) and neonatal rat spinal
motor neurons (759). The currents, which are generated
by various tetramers of Kir2.x subunits, are considered to
be critically involved in the maintenance of Eres and regulation of the excitability of the neurons. Indeed, Ba2⫹
block of Kir channels in an isolated neuron caused depolarization and initiated action potential firing (121).
In situ hybridization histochemistry and immunohistochemistry show classical Kir channels to be abundantly
and differentially expressed in the brain. Kir2.1 is expressed diffusely and weakly in the whole brain, Kir2.2
moderately throughout the forebrain and strongly in the
cerebellum, Kir2.3 mainly in forebrain and olfactory bulb,
and Kir2.4 in the cranial nerve motor nuclei in the midbrain, pons, and medulla (280, 302, 360, 633, 782). Expression of classical Kir channels is restricted to neuronal
somata and dendrites (302, 633). For example, in mouse
olfactory bulb, Kir2.3 is specifically localized at the
postsynaptic membrane of dendritic spines of granule
cells, which receive mostly excitatory synaptic inputs
(302) (and see sect. IIC). The spinal localization of Kir2.3
is also detected in striatopallidal neurons. In this region,
the current emerging from Kir2.3-containing channels is
significantly suppressed by M1 muscarinic receptor stimulation through depletion of PtdIns(4,5)P2, which results
in enhancement of dendritic excitability (708).
Function of brain classical Kir channels may be further modulated by neurotransmitters. The classical Kir
channels reconstituted with injection of brain poly(A)⫹
RNA into Xenopus oocytes are shown to be inhibited by
isoproterenol, a ␤-adrenergic agonist (322). Pharmacological assays imply that this effect is exerted by increase of
intracellular cAMP and also cGMP but not by activation of
PKA (322). Therefore, ␤-adrenergic inhibition of brain
classical Kir channels seems to be mediated by phosphorylation-independent action of cyclic nucleotides. How-
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STRUCTURE AND PHYSIOLOGICAL FUNCTION OF Kir CHANNELS
ever, the physiological role of this signaling machinery
has not yet been clarified in the brain.
Kir2.1 and Kir2.2 knockout mice show no obvious
phenotypes related to neuronal abnormality (880). Since
in many cases neurons express multiple types of Kir
channel subunits (633), loss of one Kir2.x protein could be
compensated by another subunit(s) in the mutant mice.
It is of interest that, in Schwann cells surrounding peripheral nerve fibers, a Kir current has been recorded (842).
Immunohistochemical analysis identified that Kir2.1 and
Kir2.3 were specifically expressed in the microvilli of
Schwann cells at the nodes of Ranvier (525). Since the villi
are facing towards the axon, these Kir channels may play a
role in maintaining [K⫹]o by absorbing excess K⫹ released
from excited axons. This function is similar to the “K⫹buffering” action of astroglial K⫹ channels and may therefore be important for sustaining proper function of nerve
fibers (see sect. VB).
4. Skeletal muscle
A major determinant of the Eres in skeletal muscle is
a Cl⫺ conductance (274). Classical Kir channels also participate in setting the Eres and shift it toward the direction
of hyperpolarization. All types of Kir2.x subunits are expressed in skeletal muscle at the mRNA level (394, 614,
638, 758, 782). The importance of Kir2.1 in muscle function was highlighted by analysis of Andersen’s syndrome
(622) (see sect. II, B and F, and Table 2). This disease is
accompanied by periodic paralysis. In this case, reduction
of the Kir2.1 conductance should depolarize the Eres,
which would inactivate Na⫹ channels and thus make
them unavailable for the initiation and propagation of
action potentials.
The functional expression of Kir2.1 seems to be necessary not only for differentiation of myoblasts (387) but
also for the fusion of mononucleated myoblasts to form a
multinucleated skeletal muscle fiber (182). Both events
are Ca2⫹-dependent processes and essential for skeletal
muscle development, growth, and repair. During development, the Eres of myoblasts gradually hyperpolarizes from
about ⫺10 to ⫺70 mV (462), due to the developmental
increase in Kir2.1 expression (182). This Kir2.1-induced
hyperpolarization sets Em in a range where Ca2⫹ can
enter the myoblasts through Ca2⫹-permeable channels,
which promotes the differentiation and fusion of myoblasts. In addition, differentiation is at least in part mediated by expression and activity of myogenic transcription
factors, such as myogenin and myocyte enhancer factor-2.
Their expression is triggered by Kir2.1-induced hyperpolarization (387). Although abnormality in morphology of
skeletal muscles is not obvious in either Andersen’s syndrome patients or Kir2.1-null mice, the “slender” build
observed in this patient group could be attributed to mild
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307
impairment of muscle development caused by dysfunction of the channel (346).
E) KIDNEY. The cortical collecting duct (CCD) is a
major site of K⫹ secretion in kidney. Patch-clamp recording showed that apical and basolateral membranes of
CCD express distinct types of inward rectifiers. Whereas
apical channels displayed mild inward rectification with
an intermediate conductance (20 – 45 pS) (192, 820), basolateral channels showed strong rectification with a
small conductance (27–30 pS) (481). Such an asymmetric
profile of Kir channel expression in epithelial membranes
is considered to be important for vectorical K⫹ transport
from the basolateral to apical side of the epithelium.
Basolateral Kir channels, which are made up of Kir2.3
(430, 827), maintain the potential difference across the
basolateral membrane and a favorable electrical driving
force for apical K⫹ exit without significantly recycling K⫹
back to the interstitium (247, 827). The apical K⫹ conductance is yielded by Kir1.1 and plays a central role in
secreting K⫹ to urine and sustaining activity of the Na⫹K⫹-2Cl⫺ cotransporter which is colocalized with the
channel (see sect. VA4) (247).
Another subunit, Kir2.1, is found specifically in juxtaglomerular cells but in neither epithelial cells nor glomeruli (438). Kir2.1 thus seems to be a major determinant
for Eres of approximately ⫺60 mV in juxtaglomerular cells
(70, 183).
E. Pharmacology
Specific blockers and activators for classical Kir
channels are not known. In heterologous expression systems, the homomeric Kir2.x channels differ substantially
in their sensitivity to Ba2⫹ and Cs⫹ (Table 1). In particular, Kir2.4 was much less sensitive to these blockers than
other subunits (Ki values for Ba2⫹ and Cs⫹ of 390 ␮M and
⬃8 mM for Kir2.4, respectively; ⬃8 ␮M and 420 ␮M for
Kir2.1) (782) (Table 1). In addition, when K⫹ currents
generated by coexpression of different Kir2.x subunits or
expression of concatamers (tandem subunits) were analyzed, it was found that their Ba2⫹ sensitivity clearly
differed from that of homomeric Kir2.x channels (Table 1)
(626). These findings suggest that heteromeric assembly
of Kir subunits elicits unique electrophysiological properties that may be involved in the functional roles of the
channels.
Recent pharmacological and electrophysiological
studies have demonstrated that several reagents block
Kir2.x channels, although their effects are not specific to
them. The first-generation blockers of histamine H1 receptors (mepyramine and diphenhydramine) but neither second- nor third-generation drugs moderately inhibited
Kir2.3 current by ⬃25 and ⬃17% at 100 ␮M, respectively
(460). Kir2.3 current was also partially (⬃50% maximum)
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reduced by genistein, an inhibitor of protein tyrosine
kinases, with an IC50 of 16.9 and 19.3 ␮M when the
channels were expressed in Xenopus oocytes and
HEK293T cells, respectively (891). These two types of
reagents had little effect on Kir2.1 currents (460, 891).
Analyses of chimera of Kir2.3 and Kir2.1 suggested that
transmembrane regions (TM1 and TM2) and the pore
region but not cytoplasmic domains were involved in the
action of genistein.
Choloroquine is an important drug for the treatment
of malaria. It is a serious problem that this drug induces
lethal ventricular arrhythmias mainly by inhibiting cardiac IK1 currents (43, 681). This side effect is attributable
to block of Kir2.1 channels. Mutation analyses of Kir2.1
mapped the binding site of chloroquine, unlike most ion
channel antagonists, to the cytoplasmic domain rather
than the transmembrane pore (658). Molecular modeling
implies that chloroquine would plug the cytoplasmic conduction pathway by interacting with E224, E299, and
D259 in the cytoplasmic pore region (see Fig. 5) which
contains the polyamine binding sites.
A class Ia antiarrhythmic agent, quinidine, is also
known to block cardiac IK1 current with IC50 of 4 – 40 ␮M
(268, 558). This action could be involved in the clinical
effects of the drug. The binding site for quinidine in Kir2.x
subunits has not been examined.
F. Diseases
1. Andersen’s syndrome (LQT type 7)
The finding of the mutations responsible for Andersen’s
syndrome (Table 2) has had a great impact on electrophysiological and pathological studies of Kir channels and
provided a novel insight into channelopathies. Andersen’s
syndrome is accompanied by cardiac arrhythmias reminiscent of long Q-T syndrome (LQT7), periodic paralysis,
and dysmorphic bone structure in the face and fingers
(771). The syndrome is an autosomal dominant disorder,
and it is caused by mutations in the KCNJ2 gene which
encodes the Kir2.1 subunit (622). Some of the mutations
elicit dominant negative effects on the K⫹ current (44,
429, 477, 622, 787) by impairing the interaction between
the channels and PtdIns(4,5)P2 (474) or by inhibiting trafficking of Kir2.1 to cell membrane surface (44) (see sect.
II, B and C). The symptoms of Andersen’s syndrome in the
heart are caused by reduction of Kir2.1 function that
prolongs the plateau phase of the action potential and
depolarizes Eres. It may unstabilize Em and trigger arrhythmias. The possible mechanism underlying periodic paralysis is discussed in section IID. Abnormal bone structure
in the disease would be caused by provoking dysfunction
of osteoclasts. Low extracellular pH in the extracellular
matrix maintained by H⫹ secretion via an ATP-driven
proton pump is critical for proper degradation of bone by
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osteoclasts. Since this H⫹ secretion is achieved in exchange for K⫹ transport through Kir channels, disruption
of these channels would cause osteoclast dysfunction
that could lead to severe bone deformity.
2. Kir2.1 and Kir2.2 knockout mice
Kir2.1 and Kir2.2 knockout mice have been generated
and their phenotypes have been studied. These studies
have clarified the point that in smooth muscle Kir2.1
rather than Kir2.2 is an important regulator of vascular
tone (880) (see sect. IID2). In addition, the knockout mice
study has revealed that Kir2.1 is a major constituent of the
IK1 current in heart, although Kir2.2 also contributes to
the formation of the current by assembling with Kir2.1 (881)
(see sect. IID). The phenotypes of blood vessels and heart in
Kir2.1 knockout mice were described in section II D.
Kir2.1 knockout mice mimic some of the symptoms
of Andersen’s syndrome. The mice exhibit a narrow maxilla and complete cleft of the secondary palate (880).
These phenotypes may correspond to the facial dysmorphology observed in humans. Although isolated ventricular myocytes of the knockout mice showed longer action
potentials and prolonged Q-T interval, no arrhythmia was
observed in the mutant mice.
3. Other diseases
A recent study has identified another abnormality of
the Kir2.1 gene that causes a cardiac disease. The short
Q-T syndrome constitutes a new clinical criterion that is
associated with a high incidence of sudden cardiac death,
syncope, and/or atrial fibrillation even among young patients and newborns (688). Genetic analysis of a family
with this syndrome found a G514A substitution in the
KCNJ2 gene that resulted in a change from Asp to Asn at
position 172 (D172N) (628) (Table 2). D172, which is close
to the distal end of the second transmembrane region
(TM2), is a residue crucial for establishment of the strong
rectification profile of Kir2.1 (see sect. IIB). Electrophysiological assays in transfected Chinese hamster ovary
(CHO) cells demonstrated that the mutant exhibited a
larger outward current than the wild type. In silico simulation suggests that the abnormality caused by this Kir2.1
mutation produces an abrupt increase in the rate of final
repolarization of the ventricular action potential and
shortens its duration (628).
As described in section IID3, when M1 receptor in
striatopallidal neurons of brain is stimulated, activity of
Kir2.3-containing channels expressed at these neurons
can be significantly suppressed. This event leads to elevation of dendritic excitability and opening of voltagegated Ca2⫹ channels, which further seems to cause loss of
spines. Several observations imply that this process is
involved in abnormality of neuronal network found in
Parkinson’s disease (519) as follows (708). First, deple-
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STRUCTURE AND PHYSIOLOGICAL FUNCTION OF Kir CHANNELS
tion of neuronal dopamine by administration of reserpine,
the procedure making model animals of Parkinson’s disease, not only induces loss of spines in striatopallidal
neurons but also causes increase of mRNA of Kir2.3 and
decrease of mRNA of Kir2.1 that is also expressed in these
neurons. Second, because Kir2.3 activity is more sensitive
to reduction of membrane PtdIns(4,5)P2 content than
Kir2.1, the change of expression profile of Kir subunits by
reserpine significantly augments the Kir current fraction
suppressed by M1 receptor stimulation. Third, in M1 receptor-null mice, the dopamine depletion by reserpine
barely affects number of their spines. Fourth, injection of
6-hydroxydopamine into the brain, the procedure which
destroys dopamine neurons and thus mimics Parkinsonism, elicits loss of spines in wild-type mice but has little
effect on their number in M1 receptor-null mice. Therefore, modulation of function of Kir2.3-containing channels
by M1 signaling may be critically involved in neuronal
phenotypes of Parkinson’s disease (708).
Interestingly, the cholesterol content of a membrane
affects the function of classical Kir channels, and this
could be involved in some diseases (165, 662, 663). The
application of cholesterol reduced the Kir2.1 current density in CHO cells and native classical Kir channels in
bovine and human aortic endothelial cells (662, 663).
More importantly, analysis of hypercholesterolemic pigs
revealed that an increase in plasma cholesterol levels
strongly reduced endothelial Kir currents and depolarized
the Eres (165). Thus suppression of classical Kir currents
may be a factor not only in hypercholesterolemia-induced
endothelial dysfunction but also in various vascular diseases.
III. G PROTEIN-GATED Kir CHANNELS (Kir3.x)
A. Historical View and Molecular Diversity
The human genome encodes thousands of GPCRs.
They are involved in a wide variety of physiological functions. The activation of a GPCR by its ligand (hormone or
neurotransmitter) results in the liberation of two intracellular effector molecules G␣ and G␤␥ that can influence a
large number of cellular processes. KG channels are one
of the targets of GPCRs (65, 411, 412, 620). In the heart,
muscarinic K⫹ (KACh) channels, a type of KG channels, are
responsible for the effects of ACh and adenosine (Fig. 7,
A and B). Stimulation of these GPCRs results in channel
opening which hyperpolarizes the cells. Similar signal transduction mechanisms are used by different receptors, including somatostatin, 5-hydroxytryptamine-1, ␣2-adrenergic, ␮and ␦-opioid, D2-dopamine, glutamate, and GABAB receptors in neurons and hormone-secreting cells as well as
smooth muscle cells (60, 226, 265, 423, 452, 542, 581, 584,
669, 684, 705, 851).
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The KG channels can be activated by intracellular
GTP (GTPi) in the presence of agonist or by intracellular
GTP␥S even in the absence of agonist (Figs. 7B and 8A).
This can occur in cell-free, inside-out membrane patches
in a “membrane-delimited” manner, suggesting that channel opening is triggered by membrane-bound G proteins
(Fig. 7, B and C) (411, 412, 414). After a long controversy,
it was finally established that KG channels are activated
not by G␣ but by G␤␥ subunits of PTX-sensitive G proteins (Figs. 7B and 8A) (96, 323, 409, 416, 465, 833, 850,
854, 869, 870).
Molecular cloning techniques have identified four KG
channel subunits (Kir3.1, Kir3.2, Kir3.3, and Kir3.4). Physiological assays and studies on knockout mice have revealed that various combinations of subunits from the
Kir3 subfamily form different KG channels. The activity
and the function of KG channels are precisely tuned by
their localization, protein-protein interactions, and a variety of small substances.
1. Molecular characterization
Functional KG channels are tetrameric assemblies of
Kir3 family subunits (307, 333, 382, 391, 444, 453) (see Fig.
2B). The assembly can be either homomeric or heteromeric. The subunit composition of KG channels varies
among different cells and tissues which allows them to
play diverse functional roles.
Kir3.1/GIRK1/KCNJ3 cDNA was isolated from a rat
heart cDNA library, and this was the first Kir subunit shown
to contribute to formation of functional KG channels (119,
397). Kir3.1 shares 39 and 42% amino acid identity to Kir1.1
and Kir2.1, respectively. Although Kir3.1 may form a homomeric assembly in an overexpression system (104), expression of Kir3.1 alone does not produce functional KG channel
current in mammalian cell lines (392, 621, 738, 843, 851) (see
also sect. IIIC). Coexpression of Kir3.1 with Kir3.2 (443) or
Kir3.4 (392) not only enhanced each of their channel currents but also induced different channel properties (150, 382,
733, 805). In Xenopus oocytes, the current could be elicited
by expression of Kir3.1 alone, which was shown to be due to
its heteromeric assembly with Kir3.5/XIR, an intrinsic homolog of Kir3.4 (248). Thus Kir3.1 is generally incorporated
into the heteromers with other Kir3.x subunits to form KG
channels in native cells and tissues.
There are at least four different isoforms of Kir3.2/
GIRK2/KCNJ6. They are generated by alternative splicing
from a single gene, which is composed of more than eight
exons (304, 823, 836). Initially identified as GIRK2A,
GIRK2B, and GIRK2C, the first three isoforms are now
designated as Kir3.2a-c, respectively (315, 316, 443).
Kir3.2b is 87 amino acids shorter than Kir3.2a, and 8
amino acids in the COOH terminus of Kir3.2b are different
from those in Kir3.2a. Kir3.2c was initially named GIRK2A
(444). This subunit was also called KATP-2, because it was
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isolated from an insulinoma cell cDNA library and proposed to constitute KATP channels (746). Among the splicing variants, Kir3.2c is the longest isoform: it possesses an
additional 11 amino acids in the COOH-terminal end that
are not observed in Kir3.2a. Another variant, Kir3.2d, was
isolated from a mouse testis cDNA library (304). Kir3.2d is
18 amino acids shorter than Kir3.2c at its NH2-terminal end,
which is the sole difference between the two isoforms.
Kir3.2 isoforms exhibit differential expression patterns in
various tissues such as brain, pituitary, pancreas, and testis,
where they usually coincide with the expression of other
Kir3.x subunits (307, 360, 375, 453). These histochemical
observations and immunoprecipitation assays from native
tissues suggest that Kir3.2 isoforms usually form heteromers
with other subunits such as Kir3.1 or Kir3.3. Nevertheless, it
is also true that Kir3.2 isoforms form homomeric channels in
particular areas (see sect. IIID) (304, 307).
Kir3.3 was isolated from a mouse brain cDNA library
in 1994 (443). Afterward, a variant of Kir3.3 was also
cloned from mouse brain (334). The lately isolated mouse
Kir3.3 possesses additional 17 amino acids at COOHterminal end and Ser-60 and Ala-77 instead of Arg and Val
at the corresponding positions in the first isolated one
(334, 443). The amino acid sequences of rat and human
Kir3.3 subunits were almost identical to the lately isolated
mouse Kir3.3 (334, 694, 843). These Kir3.3 subunits may
form heteromers with other Kir3.x subunits, but their
functional profile seems to be disputed. As for the first
isolated mouse Kir3.3, one study reported that, when this
subunit was coexpressed with Kir3.1, Kir3.2, or Kir3.4,
negligible current was detectable (444). On the other
hand, another study showed that a considerable KG current was observed when the first isolated Kir3.3 and Kir3.1
were expressed together, and coexpression of this Kir3.3
dramatically suppressed Kir3.2 current (382). As for the
lately isolated mouse Kir3.3, some works reported that,
when this subunit was coexpressed with Kir3.1 (334, 484)
or with Kir3.2 (333, 484), a large KG current was detected;
in these cases, single-channel property of Kir3.1/3.3 was
similar to those of Kir3.1/3.2 and Kir3.1/3.4 (334) and
mean open time of single channel of Kir3.2/3.3 was longer
than that of Kir3.2 homomer (333). Coexpression of Kir3.1
with rat Kir3.3 whose sequence is similar to the lately
isolated mouse Kir3.3 produced a considerable current in
one report (843) but displayed little channel activity in
another report (489), although both papers demonstrated
that the expression of the Kir3.3 reduced heteromeric
FIG. 7. Activation of KG channels by G protein-coupled receptors
and G proteins. A: the time-dependent response to 11 ␮M acethylcholine
(ACh) of a whole cell KG channel current in a guinea pig atrial myocyte.
In normal extracellular solution which contained 5.4 mM K⫹, the cell
membrane potential was voltage clamped at ⫺53 mV. The pipette contained (in mM) 150 KCl, 2 MgCl2, 5 EGTA, 5 HEPES, and 0.1 GTP (pH
7.4). [From Kurachi et al. (415), with kind permission of Springer Science ⫹ Business Media.] B: activation of KG channels by ACh or adenosine (Ado) requires GTPi and is blocked by islet-activating protein (IAP,
pertussis toxin). The single-channel recording began in the cell-attached
patch configuration with a pipette solution containing either ACh (1.1
␮M; top panel) or Ado (10 ␮M; middle panel). The inside-out patch was
formed (heavy arrows) and channel activity declined, then GTP (100
␮M) was applied to the intracellular surface. In the bottom panel, KG
channels were recorded in an inside-out membrane patch in the presence of GTP (100 ␮M). The channels were exposed to a mixture of the
A protomer of IAP (1 ␮g/m) and NAD (1 mM). The holding potentials are
indicated to the left of each panel. [From Kurachi et al. (412), with kind
permission of Springer Science ⫹ Business Media.] C: a cartoon representing activation of a KG channel. On the atrial cell membrane, two
different GPCRs (P1-purinergic and muscarinic M2 ACh receptors) activate the channel via GTP-binding proteins GK (412).
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Kir3.1/3.2 channel activity. In addition, human Kir3.3,
even when coexpressed with Kir3.1 or Kir3.2, exhibited
negligible current (694). Together, the functional role of
Kir3.3 and its relationship with the difference of the sequences have not yet been fully determined, although it
seems probable that Kir3.3 makes a complex with other
Kir3.x subunits such as Kir3.2 in in vivo brain (333).
Kir3.4/GIRK4/CIR/KCNJ5 was identified in bovine
atrial membrane by immunoprecipitation with Kir3.1 antibody (391). The subunit assembles with Kir3.1 in vitro
and elicits a current similar to the cardiac K⫹ current
known as IKACh. In support of this idea, Kir3.4-deficient
mice lack IKACh in the atria (835). The heterotetrameric
assembly of Kir3.4 and Kir3.1 and a stoichiometry of 1:1
have been confirmed by various assays including biochemical experiments (104, 305, 391) and electrophysiological analyses (728, 794). A homotetramer of Kir3.4 was
also found in atrial myocytes by biochemical assays, implying that Kir3.4 may form a homomer as well (103).
B. Pore Function and Subunit Structure
1. Activation of KG channels by G␤␥
In the absence of an agonist, G␣ binds GDP, the
G␣␤␥ complex is attached to the receptor, and GTPase
activity is low (207). When an agonist stimulates a GPCR,
the GDP/GTP exchange rate is accelerated, which results
in functional separation of G␤␥ from G␣. Although it has
been reported that both G␣ and G␤␥ subunits are able to
regulate various effectors such as adenylyl cyclases and
phopholipases, KG channels are activated only by G␤␥
(Fig. 8A) (96, 323, 405, 465, 652, 837).
The direct interaction between G␤␥ and KG channel
subunits was shown by biochemical pull down assay,
yeast two-hybrid assay, and electrophysiological experiments: G␤␥ binds to both NH2 and COOH termini of KG
channel subunits (78, 246, 288, 289, 305, 326, 392, 402, 734,
863). Hybridization of the G protein-insensitive Kir2.1 by
inserting the COOH terminus of Kir3.1 led to channel
activation by G␤␥ (734).
Key residues that affect G␤␥ binding and Kir3.x channel activation are H64 and L262 in Kir3.4 (246, 326) and
L344 and G347 in Kir3.2 (181). They correspond to H57,
L262, L333, and E336 in Kir3.1 (see Fig. 4D). These residues are localized in the cytoplasmic region. The crystal
structure of the cytoplasmic region of Kir3.1 has revealed
that two neighboring COOH termini bind to each other
and an NH2 terminus is located between them (574). In
Kir3.1, H57 is located on the ␤A strand of the NH2 terminus, and L333 and E336 are on a loop between the ␤L and
␤M strands of the COOH terminus. These three ␤-strands
form a ␤-sheet located on the external surface of the
cytoplasmic domain facing the cytoplasm. Interaction of
the NH2- and COOH-terminal domains of Kir3.1 and Kir3.4
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synergistically enhances G␤␥ binding (288, 289), and fluorescence resonance energy transfer studies have detected that G␤␥ induces rotation and expansion at the
NH2 and COOH termini of the Kir3.1/3.4 heteromer (655).
Therefore, the cytoplasmic region of KG channels is the
target for G␤␥, and this region seems to be critically
involved in channel gating. The importance of the cytoplasmic region for channel function is likely to be common to all Kir channels, which is supported by the observation that Kir1.1 and KirBac3.1 are insensitive to G␤␥,
but both show considerable conformational changes in
their cytoplasmic regions during gating (403, 645, 697).
Recently, it has been suggested that G␤␥ regulates the
affinity of KG channels for PtdIns(4,5)P2 binding and thus
channel activity (287) (see also sect. IIIB3).
The mechanism underlying control of KG channel
gating by G␤␥ was studied in detail in native KACh channels in inside-out membrane patches from atrial myocytes
(Figs. 7B and 8A) (320, 323, 411, 465, 850, 854). The
single-channel conductance of KACh channels measured at
Em more negative than EK is ⬃40 pS with 145 mM [K⫹]o
(411, 412, 414). The mean channel open time in the presence of ACh is ⬃1 ms, which is several orders of magnitude shorter than the open time of IK1 channels. In addition to the short open time component, the histogram also
reveals a component with a longer mean open time, although its frequency is relatively low. The closed time
distribution has at least two distinct components with
mean closed times of ⬃1 and 100 ms (678). In addition,
there are also rare very long closed events (282, 678).
The addition of G␤␥ but not G␣ to the intracellular
surface of cell membrane activates the KACh channels
(Fig. 8A and see below) (465). We found that intracellular application of GTP, in the presence of extracellular ACh, or that of G␤␥, activates KACh channels in a
positively cooperative manner with a Hill coefficient of
⬃3 (320, 408, 850), although some studies reported that
the coefficient of the relationship between KG channels’
activation and G␤␥ concentration was 1.5–1.7 (334,
392). Our results suggest that G␤␥ binds to multiple
sites on a functional channel unit. To elucidate the
machinery underlying this positive cooperative activation of KACh channels by G␤␥, Hosoya et al. (282)
analyzed channel kinetics at different concentrations of
GTP using spectral analysis techniques (Fig. 8, B and
C). The results could be accounted for by the Monod,
Wyman, and Changeux allosteric model if channels
were presumed to be an oligometric protein composed
of four or more functionally identical subunits, each of
which bound one G␤␥. This observation is consistent
with the results of recent molecular biological studies
and structural analysis.
Several studies report possible roles of G␣ in KG
channel function. Inside-out patch-clamp analysis shows
that intracellular application of the GDP-bound form of
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the transducin ␣-subunit irreversibly inhibits cardiac KG
current activated by either GTP with ACh (in pipette
solution) or GTP␥S (Fig. 8A) (850). This observation indicates that G␣ would chelate G␤␥.
In addition, G␣ is proposed to be involved in the
control of KG channel activity. It has been demon-
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strated by in vitro biochemical assays that GDP-bound
form of G␣ can interact not only with the NH2 and
COOH terminus of Kir3.1 and Kir3.2 but also with a
complex of G␤␥ and KG channels (327, 612, 734). These
results imply that, even in the resting state (without
agonist), KG channels may make a complex with GDP-
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STRUCTURE AND PHYSIOLOGICAL FUNCTION OF Kir CHANNELS
bound G␣, G␤␥ in the cell. This protein complex of
three units (GDP-bound G␣, G␤␥, and KG channels) is
called “preformed” complex. It is also proposed that,
when the ligands bind to the receptor, G␣ would be
released from the preformed complex and simultaneously G␤␥ could become active to stimulate the
channels. In this condition, G␤␥ may be always in very
close vicinity of KG channels even without agonists, and
thus could effectively and quickly activate the channels
as soon as G␣ is dissociated from the preformed complex. Accordingly, formation of the preformed complex
is suggested to be involved in rapid activation of the KG
channels in response to agonists, which is observed in
native tissues (612, 654, 734). In support of this idea, in
heterologous expression system, coexpression of G␣
fastens activation time course of ACh-induced current
of Kir3.1/3.2 channels (327). Moreover, when G␣ is
coexpressed with KG channels in Xenopus oocytes, the
basal current decreases but the total current amplitude
including agonist-induced component is unchanged or
even enhanced (327, 612). It is therefore considered
that, whereas coexpression of G␣ may chelate G␤␥ as
opposed to KG channels and reduce their basal activity
in the absence of agonists, it would simultaneously
augment the number of the preformed complex and
increase the pool of G␤␥ available for the channel’s
activation in response to agonists. More studies will be
necessary to determine the precise roles of G␣ on KG
channels.
2. Regulation of activity and relaxation of KG
channels by regulators of G protein signaling
proteins
Regulator of G protein signaling (RGS) proteins negatively regulates various G protein-mediated signal path-
313
ways by accelerating intrinsic GTPase hydrolysis in the
G␣ subunit (Fig. 9A) (253, 666). Recently, a role for RGS
proteins in controlling KG channel activity has been reported. GPCRs, G proteins, RGS proteins, and KG channels seem to be gathered in close vicinity (95, 576, 654,
890). RGS proteins expressed in Xenopus oocytes and
mammalian cell lines accelerate the time course of agonist-induced activation and deactivation of KG currents
(142, 194, 676). The acceleration of the “turn-off” response
by RGS proteins can be simply explained by the facilitation of the sequestration of G␤␥ when RGS proteins enhance the GTPase activity of G␣ and thus reduce GTPbound G␣. On the other hand, the mechanism underlying
acceleration of activation time course has not been yet
clarified.
We have found that RGS proteins can also be responsible for the relaxation behavior of cardiac KG channels.
The cardiac KG current recorded during a hyperpolarizing
voltage step is composed of two kinetically distinct processes, instantaneous and time-dependent components
(Fig. 9B). The time-dependent alteration of KG currents at
a given potential is called “relaxation.” It reflects the
gradual increase in Po during the hyperpolarizing voltage
step. The instantaneous increase of current on hyperpolarization is seen in almost all Kir channels. It is due to a
relief from the blockade of the channel pore by intracellular cations such as Mg2⫹ and polyamines. The subsequent time-dependent “relaxation” is a characteristic of
KG channels. Interestingly, this characteristic depends on
the concentration of agonist as well as on Em (851).
Relaxation behavior of KG current was first described
in sinoatrial node cells (579), but the mechanism has
remained unknown for a long time. We have found that
the relaxation of KG currents reconstituted in Xenopus
oocytes with Kir3.1/3.4 and the M2 muscarinic acetylcho-
FIG. 8. Regulation of KG channels by different G protein subunits and nucleotides. A: activation of KG channels by GTP and G␤␥. KG channels
in atrial cardiac myocytes were recorded in inside-out patches (excision at vertical arrows) in the presence of ACh in the pipette solution (see inset).
Nucleotides and G protein subunits were applied to the intracellular surface of the excised patches as indicated above each recording. a: Effects
of different concentrations of G␤␥, GTP␥S-bound G␣ subunits, and GTP␥S. b: GDP-bound form of the transducin ␣ subunit (T␣-GDP) was applied
to channels activated by GTP and ACh. Note: T␤␥ opened the channels. c: T␣-GDP was applied to channels preactivated by GTP␥S. [From Yamada
et al. (850), with permission from Elsevier.] B: concentration-dependent effects of GTPi on KG channel in the absence and the presence of ACh. a:
Examples of inside-out patch recordings obtained from guinea pig atrial myocytes. The channel currents were recorded at ⫺80 mV in symmetrical
145 mM K⫹ solutions. Above, no ACh in the pipette; below, 1 ␮M ACh. The bars above each trace indicate the periods of application of different
concentrations of GTP and 10 ␮M GTP␥S to the internal surface of the patch membrane. b: Relationship between the concentration of GTP and the
NPo of KG channels. The data were normalized to the maximum NPo which was induced by 10 ␮M GTP␥S in each patch. Symbols are means.The
continuous lines indicate the fit of the relationship between GTP and channel activity in the presence of each concentration of ACh with the following Hill
equation: f ⫽ Vmax/{1 ⫹ (Kd/[GTP])n}, where f is the relative NPo, Vmax is the maximum NPo available in the presence of 10 ␮M GTP␥S, Kd is the apparent
dissociation constant of GTP, and n is the Hill coefficient. [From Ito et al. (320), copyright 1991. Originally published in The Journal of General Physiology.]
C: relationship between GTP concentration and the fraction of the “available” state in KG channels according to the concerted allosteric model of Monod,
Wyman, and Changeux. a: The fraction of the “available” state [A/(A ⫹ U)] was estimated from inside-out membrane patch experiments. Symbols represent
data concerning the relationship between GTP concentration and the calculated fraction of the “available” state. Lines indicate fits to the data of the Monod,
Wyman, and Changeux allosteric model with different assumed values of n. b: A schematic representation of the Monod, Wyman, and Changeux allosteric
model. In this scheme, each channel is assumed to be an oligomer composed of four identical subunits (n ⫽ 4). Each subunit is in either the tense (T)
or the relaxed (R) state represented by squares and circles, respectively. Each subunit in the T or R state binds one dissociated G␤␥ subunit (solid circles)
independently of each other with a microscopic dissociation constant of either KT or KR. In this model, all subunits in the same oligomer must change their
conformations simultaneously. Therefore, the whole channel complex can be either T4 or R4. T4 and R4 are in equilibrium according to the allosteric
constant L. [From Hosoya et al. (282), copyright 1996. Originally published in The Journal of General Physiology.]
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line receptor emerges when RGS proteins are also expressed (194) and that the interaction of the RGS domain
with the G␣ subunit mediates the phenomenon (303) (Fig.
9, A and C). Then it was found that the apparent voltage
dependence of relaxation is caused by voltage-dependent
Ca2⫹ influx across the cell membrane (Fig. 9B) (313). In
the resting conditions, RGS protein activity is inhibited by
binding of PtdIns(3,4,5)P3. On depolarization, Ca2⫹ enters
into atrial myocytes and forms a complex with calmodulin
(CaM). Ca2⫹/CaM complex then interacts with RGS to
remove PtdIns(3,4,5)P3 in a competitive manner. The activated RGS then accelerates the hydrolysis of GTP on
G␣, reduces the amount of free G␤␥, and decreases the
activity of KG channels (Fig. 9C) (303, 314). The voltage
step to a hyperpolarized voltage stops Ca2⫹ entry, decreases Ca2⫹/CaM complex, and allows reassociation of
RGS protein, and PtdIns(3,4,5)P3, reduces GTPase activity
of G␣, increases the amount of free G␤␥, and enhances KG
channel activity. “Relaxation” thus may represent the time
course of a series of biochemical reactions and not a
gating process intrinsic to the KG channel.
315
shown to be involved in controlling the affinity of
PtdIns(4,5)P2 (889) as well as Na⫹
i (see below) to KG
channels.
Na⫹
i can activate KG channels which contain Kir3.2 or
Kir3.4 subunits (270, 271, 444, 619, 749, 889). Asp in the
CD loop between ␤C-and ␤D-strands (D228 in Kir3.2 and
D223 in Kir3.4) is considered to be the Na⫹
i sensor (270,
889). The corresponding residue in Kir3.1 is N217 (see Fig.
4D). This supports the notion that Asp in the CD loop
plays an important role in Nai-induced activation of KG
channels containing Kir3.2 or Kir3.4.
A recent structure-based study indicates an intimate
relationship between the effects of PtdIns(4,5)P2 and Na⫹
i
on certain Kir3.x channels (665). Molecular dynamic analysis suggests that, without Na⫹
i , the side chains of D223
and R225 in Kir3.4 are connected via an ionic bond, and
this connection may prevent the Arg residue from controlling PtdIns(4,5)P2 sensitivity of the channel. When
Na⫹
i is present, it is expected to be coordinated by side
chains of D223 and H228 and free R225 to increase the
channel’s sensitivity to PtdIns(4,5)P2. Similar results were
obtained for Kir3.2 (665).
3. Other modulators of KG channel gating
G␤␥.
A) PTDINS(4,5)P2 AND MODULATION OF ITS EFFECT BY Na
⫹
AND
Like other Kir channels, KG channels require
PtdIns(4,5)P2 to maintain their activity (287) (see also
sects. ID and IIB). When PtdIns(4,5)P2 is removed in inside-out patches, KG current runs down completely (750).
The channels in this condition cannot be opened by addition of G␤␥ alone. Application of PtdIns(4,5)P2 slowly
causes openings of the channels over a time scale of
minutes. Unlike most Kir channels, KG channels’ activation by PtdIns(4,5)P2 is significantly augmented and accelerated by additional gating molecules such as G␤␥ and
Na⫹
i , which seem to strengthen the interaction between
the channels and PtdIns(4,5)P2 (146, 287, 750). Consistently, the coexpression of G␤␥ with Kir3.1/3.4 channels
dramatically slows the rate of the loss of channel activity
induced by a PtdIns(4,5)P2 antibody (287). The loop between ␤C- and ␤D-strands in the cytoplasmic domain is
4. Oxidation-reduction and acidification
Application of the reducing agent dithiothreitol
(DTT) causes openings of KG channels (Kir3.1/3.4) without affecting their permeation or rectification properties
(883). DTT seems to directly act on the channels because
its effect was abolished when Cys in the NH2-terminal
cytoplasmic region was mutated. Control of KG channel
activity by redox signaling may protect cells and tissues
under hypoxic or ischemic insult.
KG channels are also inhibited by intracellular acidification. The pHi sensitivity relies on a few His residues in
their NH2 and COOH-terminal cytoplasmic domains (H57,
H222, and H346 in Kir3.1; H64, H228, and H352 in Kir3.4)
and does not seem to depend on G proteins (496). This
modulation may be involved in the control of respiratory
activity and CO2 chemoreception in the brain stem (36,
216, 553).
FIG. 9. Mechanism of relaxation behavior in KG channels. A: a schematic representation of the mode of action of RGS proteins. RGS proteins
stabilize the transition state (G␣-GTP) of GTP hydrolysis on the G␣ subunit, which results in the acceleration of intrinsic GTPase-activity. B: effects
of extracellular Ca2⫹ on KG currents. a: Voltage-clamp protocol (top) and a typical trace of whole cell ACh-induced KG current in an isolated atrial
myocyte (bottom). Inward current on stepping membrane voltage to ⫺100 mV first changed instantaneously (Iins) and then slowly increased to a
steady state (Imax). b: KG currents evoked by 10⫺7 M (left) or 10⫺6 M (right) ACh in control conditions. Currents at ⫺100 mV were recorded after
prepulses to between ⫺100 and 40 mV in steps of 20 mV (inset). c: Relationship between prepulse voltage and Iins/Imax ratio for currents elicited
by either 10⫺7 M (open circle) or 10⫺6 M (solid circle) ACh (n ⫽ 10). d and e: KG current evoked by 10⫺7 M ACh when either extracellular free Ca2⫹
was chelated by EGTA (d) or intracellular Ca2⫹ was chelated by BAPTA (e). f: Relationship between prepulse voltage and Iins/Imax ratio with
intracellular BAPTA. KG currents were elicited by 10⫺7 M (open circle) and 10⫺6 M (solid circle) ACh (n ⫽ 8). In each current trace, arrowheads
indicate the zero-current level; vertical scale bars represent 500 pA. C: a schematic representation of voltage-dependent relaxation of KG resulting
from Ca2⫹/CaM-dependent facilitation of the action of RGS proteins. In a hyperpolarized state, the action of RGS is inhibited by PtdIns(3,4,5)P3.
Once the intracellular Ca2⫹ concentration is elevated, e.g., upon depolarization, Ca2⫹/CaM binds to RGS proteins and reverses the inhibitory effect
of PtdIns(3,4,5)P3, which results in the negative regulation of the G protein cycle. When the [Ca2⫹] decreases to the steady-state level again, CaM
dissociates from RGS proteins and their action is once again inhibited by PtdIns(3,4,5)P3.x is an unknown Ca2⫹ transport apparatus. [A and B from
Ishii et al. (313).]
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5. Ion permeation, gating, and inward rectification
KG channel currents show strong inward rectification. The mechanisms underlying rectification and its
structural basis are similar to those of other Kir channels
(see sects. I and II) (143, 172, 336, 338, 404, 471, 507, 539,
574, 611, 802, 831, 856, 894). One difference is that Kir3.2,
Kir3.3, and Kir3.4 have Asn instead of a negatively
charged Asp as a polyamine binding site in the TM2
transmembrane helix, although Kir3.1 possesses Asp in
this position (611, 733).
In terms of ion selectivity, KG channel subunits share
the signature sequence (T-X-G-Y/F-G). The loss of K⫹
selectivity in the weaver mouse (see sect. IIIF) is caused
by the mutation of G156 to Ser in the selectivity filter of
Kir3.2 (606), which permits nonselective cation permeation (383, 554, 733). Residues outside the selectivity filter
of Kir3.x channels have also been found to contribute to
K⫹ selectivity. First, mutations at E139 and R149 of Kir3.1,
E152 of Kir3.2, and E145 and R155 of Kir3.4 cause dramatic loss of K⫹ selectivity in KG channels (492). Molecular modeling suggests that these residues form a salt
bridge behind the selectivity filter and probably act to
maintain the structure rigid. Second, substitution of S177
in TM2 of Kir3.2 located at the base of the selectivity filter
abolished K⫹ selectivity (872). Equivalent results were
obtained at S166 in Kir3.1 and A172 in Kir3.4 (492).
The roles of the TM2 helices in the gating of Kir3.x
channels were described in detail in section ID1. In summary, G␤␥-induced channel openings may depend on a
number of processes. G␤␥ binding to the cytoplasmic
domain induces a configuration change that is transduced
to the lower TM2 region and rotates it (340, 404). This
might also result in bending of the TM2 helix at the Gly
hinge (97, 872). This hypothesis is very convincing, but it
needs to be verified in future studies.
C. Channel Localization
1. Membrane trafficking machinery
When Kir3.1 is expressed alone in heterologous expression systems, it is not sorted to the cell membrane
surface; instead, it remains in ER compartments (367, 368,
489). On the other hand, when coexpressed with Kir3.2 or
Kir3.4, Kir3.1 is efficiently transported to the membrane
surface. Homomeric channels composed of Kir3.2 or
Kir3.4 do appear on the cell surface (489). Immunolabeling analysis of various mutated Kir subunits suggests that
wild-type Kir3.2 and Kir3.4 are expressed on the cell
surface due to two forward trafficking signals in their
cytoplasmic regions. One is an ER export signal in their
NH2 terminus (N-Q-D-M-E-I-G-V in Kir3.2; D-Q-D-V-E-SP-V in Kir3.4), and the other is a post-ER trafficking signal
that is composed of a cluster of acidic residues in the
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COOH terminus (489). Both of these signals seem to be
necessary to traffic the subunits to the plasma membrane.
Of note, some population of Kir3.2, when expressed
alone in COS7 cells, is prominently localized in intracellular vesicles (489). In cultured hippocampal neurons, a
majority of endogenous Kir3.2 exists in cytoplasmic region, although it is modestly expressed on the cell surface
(90). This subunit has a sequence of T-E-S-M-T-N-V-L
(amino acid 7–14) in NH2 terminus, which is similar diLeu endocytosis signal [(D/E)-X-X-X-L-L]. Mutation of V-L
to A-A results in disappearance of vesicular localization of
Kir3.2 and increase of its surface expression, implying
that these amino acids act as an internalization motif
(489). Recent studies have shown physiological regulation
of intracellular localization of Kir3.2-containing channels
in neurons (90) (see sect. IIID3).
Coexpression with Kir3.3 results in a large decrease
in the KG currents normally yielded by homomeric Kir3.2,
heteromeric Kir3.1/3.2, and Kir3.1/3.4, which is attributed
to retention of the channels in the ER (382, 444, 489, 694).
This phenotype depends on the following two mechanisms: 1) Kir3.3 lacks an ER export signal, and 2) Kir3.3
has a lysosomal targeting signal, Y-W-S-I (Y-X-X-⌽), in its
COOH terminus. These signals target channels that contain Kir3.3 from the ER to lysosomes without delivering
them to the plasma membrane. Even when the membrane-targeting region of Kir2.1 is spliced onto Kir3.3, the
hybrid Kir3.3 remains mainly in the cytoplasm (489). In
this case, the hybrid channel protein is detected mainly in
the ER, probably because the proteins that moved to
lysosome may be rapidly degraded. Together, the lysosomal targeting signal seems to have a strong effect on
localization of the channels containing Kir3.3.
2. Localization in native tissues
Kir3.1, Kir3.2, and Kir3.3 proteins are expressed
throughout the brain (225, 307, 390, 453). But the expression of Kir3.4 is found in only limited areas (297, 834).
Studies using light and electron microscopy have revealed
the precise localization of Kir3.x subunits in neurons.
Kir3.1 and Kir3.2 are localized at both postsynaptic (see
Fig. 10C) and presynaptic regions (see Fig. 10D), whereas
Kir3.3 has been detected in certain axons (225, 307, 390,
401, 453, 544, 623). Kir3.4 immunoreactivity was observed
at the axonal terminus (297, 834). Thus KG channels may
be involved not only in slow inhibitory postsynaptic potentials but also in the presynaptic modulation of neuronal activity.
Neuronal KG channels are either homomeric or heteromeric complexes of Kir3.x subunits, but the relationship between the specific subunit combination and the
subcellular localization is not entirely clear (307, 390, 444,
453). A Kir3.2 homomer composed of Kir3.2a and Kir3.2c
was found to occur at the inhibitory postsynaptic region
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317
FIG. 10. Intracellular localization of KG channels and their functional roles. A: Kir3.1 in the rat anterior pituitary lobe. Immunogold electron
microscopy shows that Kir3.1 occurs on the intracellular secretory vesicles of the thyrotroph. Kir3.1 signals (15-nm particles) and TSH signals
(10-nm particles) were detected on the same vesicles (inset) (a). Scale bar, 0.5 ␮M (inset, 0.15 ␮m). After TRH administration (b), a population of
Kir3.1-positive vesicles aligns near the plasma membrane, and immunogold particles were detected on the membrane surface (arrows).
B, a: simultaneous recordings of membrane capacitance (Cm), conductance (Gm), and membrane current (Im) during stimulation of a thyrotroph
cell. The application of 10 nM bromocriptine had no effect on Cm. The addition of 3 ␮M TRH (arrow) transiently increased Cm. Im was little
influenced by bromocriptine. The addition of TRH induced a marked increase of inward Im at the holding potential ⫺100 mV. Arrowhead indicates
the zero-current level. Solid circles show calibration signals for Cm [250 femtofarads (fF)]. The scale bar for Cm is 250 fF, which corresponds to 2.35
nS, and the scale bar for Im is 250 pA. Time scale bar ⫽ 1 min. b: A schematic illustration of TRH-induced recruitment of KG channels which contain
Kir3.1 to the plasma membrane via exocytotic fusion. DA, dopamine; SS, somatostatin. [A and B from Morishige et al. (542), with permission from
The American Society for Biochemistry and Molecular Biology.] C: postsynaptic localization of Kir3.2 in the brain. Kir3.2 immunoreactivity was
localized on the postsynaptic membranes in the dendrites contacted by several axonal termini in substantia nigra pars reticulata. [From Inanobe et
al. (307), with permission from The Journal of Neuroscience.] D: presynaptic localization of Kir3.1 in brain. Kir3.1 immunoreactivity in axon
terminals within the paraventricular nucleus of the hypothalamus (a and b). The labeling appears to be condensed in the intracellular region. D,
dendrite. [From Morishige et al. (544), with permission from Elsevier.]
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HIBINO ET AL.
in dopaminergic neurons of the substantia nigra, and this
localization may be determined by protein-protein interactions (307). PDZ-proteins are considered to be one of
the major determinants of postsynaptic localization of
various proteins (711). There is a PDZ-domain binding
motif at the distal COOH-terminal region of Kir3.2c but
not Kir3.2a. The Kir3.2 complex in substantia nigra would
be sorted to the postsynaptic site via interaction between
Kir3.2c and PDZ. This type of interaction may further
modulate the function of KG channels containing Kir3.2c.
Under certain conditions, a KG channel current could be
recorded from Xenopus oocytes expressing Kir3.2c and a
GPCR only when a PDZ-protein SAP97 was coexpressed
(258).
A proteomics approach has recently identified a new
regulator for trafficking and function of KG channels. An
intracellular PDZ-protein called sorting nexin 27 (SNX27)
is reported to directly interact with Kir3.3 (484) (this
Kir3.3 corresponds to the lately isolated Kir3.3; see sect.
IIIA1). Biochemical assays indicate that the interaction
occurs between the PDZ domain of SNX27 and a PDZbinding motif, ESKV, at the COOH-terminal end of Kir3.3.
In HEK293 cells, coexpression of SNX27 largely suppresses the currents due to Kir3.1/3.3 and Kir3.2c/3.3 heteromers. The effect is due to the recruitment of the Kir3.3
subunit from the cell surface to the early endosome.
Although SNX27 binds to Kir3.2c via the same PDZ-binding motif, its coexpression affects neither surface expression of Kir3.2c nor its current amplitude. This implies that
SNX27 specifically regulates the activity of KG channels
containing Kir3.3.
Another mechanism regulating the trafficking of neuronal KG channels involves the neural cell adhesion molecule (NCAM). Cultured hippocampal neurons from mice
lacking NCAM exhibit a larger KG current, which is probably composed of Kir3.1/3.2, than that in wild-type neurons. When NCAM140 or NCAM180 was coexpressed with
Kir3.1/3.2 in Xenopus oocytes or CHO cells, the surface
expression of the channels was decreased. Consistently,
whereas in cultured hippocampal neurons of NCAM-null
mice the cell surface expression of Kir3.1/3.2 is significantly augmented, a considerable amount of Kir3.1/3.2 in
the neurons from wild-type mice is expressed in Golgi
apparatus. The effect of NCAM was not observed for
Kir3.1/3.4 channels. The regulatory process of Kir3.1/3.2
expression by NCAM is likely to involve the role of DRM
(lipid-raft) microdomains. Biochemical assays show that
both Kir3.1 subunits and NCAM occur in DRM of transfected CHO cells and the brain. Mutational and pharmacological disruption of the association of NCAM140 in
DRMs abolished its effect on the surface delivery of
Kir3.1/3.2 channels. Thus, in intracellular compartments
such as Golgi apparatus, NCAM incorporated into DRMs
would somehow affect Kir3.1/3.2 and prevent its trafficking to the plasma membrane (127).
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A KG channel with an identical subunit composition
sometimes shows a different localization in different tissues. For example, the KG channels of atrial myocytes and
thyrotrophs in the anterior pituitary are both heteromers
of Kir3.1/3.4. In atrial myocytes, they are found on the cell
membrane (391). On the other hand, in resting thyrotrophs, the channel is found exclusively on intracellular
secretory vesicles (Fig. 10A) (542). The mechanism underlying such distinct localization of Kir3.1/3.4 channels
in different cell types has not been clarified yet.
D. Physiological Functions in Cells and Organs
1. Heart
In the heart, ACh released from the vagal nerve terminals decelerates the heart beat (see Fig. 1B) (464). This
ACh-induced bradycardia was found to be due to an increase in K⫹ efflux through the membrane (295) and
membrane potential hyperpolarization (73, 125). Then,
Trautwein colleagues (579, 601, 678, 786) identified KACh
channel current in the rabbit sinoatrial node. They proposed that ACh induces activation of a specific population
of K⫹ channels, cardiac KG channels which we now know
are composed of Kir3.1 and Kir3.4 subunits (391).
Kir3.4 knockout mice were generated and analyzed
to elucidate the role of the channels in the heart (835). As
expected, in knockout atrial myocytes, no KG current
(IKACh) was detected. Although there was no difference of
the resting heart rate between wild-type and knockout
mice, the decrease of heart rate in response to parasympathetic stimulation was considerably diminished in
knockout mice. Strikingly, the knockout mice lost heart
rate variability, i.e., the beat-to-beat fluctuations determined by the balance between sympathetic and parasympathetic influences.
2. Brain
In the brain there are heteromeric complexes of at
least Kir3.1/3.2 (307, 453), Kir3.2/3.3 (110, 307, 333), and
Kir3.2/3.4 (444) (as for the possibility of Kir3.1/3.3 complex, see below). Also, homomeric expression of Kir3.2
isoforms, i.e., complexes of Kir3.2a and Kir3.2c, were
identified in dopaminergic neurons of the substantia nigra
(307). A number of GPCRs have been shown to activate
KG channels at postsynaptic as well as at presynaptic sites
(407, 535, 778, 779, 873). Postsynaptic KG channels produce a slow inhibitory postsynaptic potential (sIPSP) and
suppress their excitability. Several studies using the
knockdown strategy suggest that Kir3.2 was critically involved in the formation of IPSP in hippocampal and cerebellar neurons (485, 726). The Kir3.2 knockout mice
develop spontaneous seizures and are more susceptible
to pharmacological maneuvers to induce seizures (726),
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which is quite different from the case of Kir3.3-gene ablation causing no obvious phenotype (783). Recent analyses of knockout mice have further defined physiological
roles of neuronal KG channels in different regions of
brain. In locus ceruleus, administration of opioid agonists
such as [Met5]-enkephalin (ME) is known to hyperpolarize the neurons (613, 839, 840). KG conductance and/or
cAMP-dependent cation conductance has been suggested
to cause this effect. Three Kir3.x subunits, i.e., Kir3.1,
Kir3.2, and Kir3.3, are expressed in these neurons (82,
360). Compared with the case of wild-type mice, although
ME-induced hyperpolarization and whole cell current are
not significantly altered in Kir3.3-null mice, they are reduced by 40% in Kir3.2-null mice and by 80% in Kir3.2/3.3
double-null mice (783). These results indicate that the
ME-induced hyperpolarization is mainly mediated by KG
channels containing at least Kir3.2 but not by cAMPdependent cation conductance. Kir3.3 also seems to be
involved in formation of KG current, although the detailed
mechanism is unknown. The phenotype mentioned above
would indicate a possibility that both Kir3.1/3.2 and
Kir3.1/3.3 exist and function in neurons (the former could
be more dominantly expressed than the latter) but, when
either Kir3.2 or Kir3.3 is lacked, the remainder would
compensate the function. The similar results are also
observed in hippocampal CA1 neurons where GABABreceptor agonists induce KG current to elicit postsynaptic
inhibitory effects (390). Although Kir3.4 expression in the
hippocampus is low (297, 834), Kir3.4-deficient mice show
impaired performance in spatial learning and memory
tasks (834).
A few studies have demonstrated an involvement of
neuronal KG channels in the mechanism underling drug
abuse and addiction. The release of dopamine in response
to activation of mesocorticolimbic system, which originates in the ventral tegmental area (VTA), is considered to
be critical for induction of compulsive addictive behavior
(656). All addictive drugs react on mesocorticolimbic dopamine system (486, 559). Although a low-affinity agonist
of GABAB receptor, ␥-hydroxybutyric acid (GHB), can
cause strong abuse (567), baclofen, which also activates
the same receptor, shows anticraving effect (107). This
difference seems to depend on a distinct combination of
Kir3.x subunits among Kir3.1, Kir3.2c, and Kir3.3 in VTA
where dopaminergic neurons are negatively regulated by
GABAergic interneurons (110). In brain slices, both dopaminergic and GABAergic neurons postsynaptically express GABAB receptor and KG channels. However, EC50
for baclofen-induced activation of the channels is much
higher in the dopaminergic neurons (⬃15 ␮M) than in the
GABAergic ones (⬃1 ␮M). Indeed, a low dose of baclofen
(0.1 ␮M) suppresses excitability of GABAergic interneurons by considerably activating KG channels but barely
affects GABAB receptor in dopaminergic neurons. This
results in a significant increase of firing frequency of
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319
action potential in dopaminergic neurons. On the other
hand, the high dose (100 ␮M) of baclofen abolishes the
action potential probably by direct activation of KG channels in dopamine neurons. As for expression profile of
Kir3.x subunits, dopaminergic neurons have Kir3.2c and
Kir3.3, whereas GABAergic neurons harbor Kir3.1, Kir3.2c,
and Kir3.3. In heterologous expression system, EC50 for
baclofen-induced activation of Kir3.2c/3.3 is significantly
higher than that of each Kir3.1/2c, Kir3.1/3.3, and Kir3.2c
channels (110; see also Ref. 333). A low-affinity GABAB
agonist GHB at concentrations obtained with recreational
use (10 mM) activates strongly KG channels in GABAergic
neurons but only slightly those in dopaminergic neurons.
Therefore, differential sensitivity of KG channels to GABAB
agonists, which seems to be elicited by distinct combination
of Kir3.x subunits, is at least partially responsible for the
opposite pharmacological effect on drug abuse between
high-dose baclofen and GHB.
Interestingly, a subsequent study has suggested that
modulation of KG channels in dopaminergic neurons by
RGS2 is involved in tolerance of GHB-induced addiction
observed after a certain period of its chronic administration (421). Inhibition of RGS family proteins by injecting
PtdIns(3,4,5)P3 and gene ablation of RGS2 prominently
reduces the EC50 value of KG channels’ activation by
baclofen to ⬃5 ␮M in dopaminergic neurons. This would
be a condition that a low-affinity GABAB agonist GHB at
concentration of recreational use can decrease excitability of dopaminergic neurons by directly activating Kir3.2c/
3.3 and prevent addictive behavior. After animals are
chronically exposed to GHB, their dopaminergic neurons
decrease not only expression of RGS2 but also the EC50 of
KG channels’ activation, which coincides with induction
of tolerance for the drug. Therefore, regulation of KG
channels by RGS may provide a key process in occurrence of drug abuse and its tolerance.
3. Vesicular and intracellular KG channels
A heteromeric complex of Kir3.1 and Kir3.4 has been
identified in the secretory vesicles of thyrotrophs of the
rat pituitary gland (Fig. 10A) (542). When these thyrotrophs are stimulated with thyrotropin-releasing hormone, the secretory vesicles fuse with the plasma membrane and this enhances dopamine- and somatostatininduced KG currents (Fig. 10B) (542). This enhancement
of KG currents results in hyperpolarization of the thyrotroph Em and may reduce or suppress thyrotrophin secretion by stopping Ca2⫹ influx via Ca2⫹ channels. The amplitude of KG currents induced by the inhibitory neurotransmitters is therefore related to the magnitude of
thyrotroph stimulation. This would provide an effective
negative-feedback loop to control blood plasma hormone
concentration (Fig. 10B). We suggest that a similar mechanism could function in the nervous system where KG
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HIBINO ET AL.
channels are localized in the cytosolic regions of axonal
termini (Fig. 10D) (390, 544, 623).
A recent study actually shows that trafficking of
Kir3.2-containing channels may be dynamically controlled
by neuronal activity. In cultured hippocampal neurons,
the heteromer of Kir3.1 and Kir3.2 forms a functional KG
channel, but the majority of these subunits exist in the
cytoplasmic region. Activation of NMDA receptor by application of glutamate augments surface expression of
Kir3.1 and Kir3.2 in soma, dendrites, and dendritic spines
doubly within 15 min (90). This regulation is abolished by
mutation of the V-L internalization motif in NH2 terminus
of Kir3.2 (position 13 and 14) and accelerated by dephosphorylation of Ser near the motif (position 9) (90), implying that phosphorylation status of Ser modulates functionality of the V-L motif similar to the case of diLeu-mediated internalization of other proteins (57). Ser is
dephosphorylated by protein phosphatase-1, and this enzyme is activated when NMDA receptor is stimulated.
Thus excitation of neurons by glutamate would simultaneously prepare an inhibitory machinery by increasing
surface KG channels that will cause membrane hyperpolarization in response to neurotransmitters. Interestingly,
activation of NMDA receptor increases the population of
KG channels that can be activated by stimulation of adenosine A1 but not GABAB receptor (89).
The similar system may also be involved in excitatory
synaptic plasticity. In hippocampal neurons, depontentiation of long-term potentiation of field excitatory postsynaptic potential requires activity of NMDA receptor, A1
receptor, and protein phosphatase-1 (285, 286), which are
the elements necessary for surface trafficking of Kir3.2containing channels and their opening (see above). Disruption of Kir3.2 by gene-targeted ablation or application
of a KG channel blocker tertiapin inhibits the depontentiation in hippocampal slice (89). These two lines of experimental results suggest that NMDA receptor-dependent increase of Kir3.2 expression on cell surface could
be involved in the depontentiation process, one form of
synaptic plasticity.
4. Pancreas KG channels
Several neurotransmitters and hormones are known
to inhibit secretion of insulin and glucagon from pancreatic islets. Catecholamines and somatostatin suppress insulin secretion from pancreatic ␤-cells, and somatostatin
can also inhibit glucagon secretion from ␣-cells. These
regulators act on the islet cells through multiple signaling
mechanisms (706), one of which may involve the hyperpolarization of Em by an increase in K⫹ conductance (1,
328, 664, 706, 724, 878). In both ␣- and ␤-cells, this hyperpolarization seems attributable to activation of KG currents. Kir3.2c and Kir3.4 subunits were found to occur in
islet cells (55, 171, 746, 878), and the expression of Kir3.1
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and Kir3.3 has also been suggested (328). Homo- or heteromultimers of these subunits may form functional KG
channels to regulate hormone secretion from islet cells.
E. Pharmacology
Tertiapin is a potent inhibitor of Kir3.1/3.4 and
Kir1.1 channels with nanomolar affinity (342, 372) (see
sect. IE). In Kir1.1, tertiapin binds in the outer vestibule
of the channel pore. Though since this position is only
partially conserved within tertiapin-sensitive Kir channels (343) (see sect. VA5), the toxin may associate with
the channels via multiple contacts. Jin and Lu (343)
created an oxidation-resistant toxin, tertiapinQ, which
is more stable than tertiapin in solution. The modified
toxin blocks IKACh, but not other currents in isolated
cardiac myocytes (136, 372).
n-Alcohols such as methanol, ethanol, and 1-propanol can activate KG channels made up of different combinations of Kir3.1, Kir3.2, and Kir3.4 subunits, although
Kir1.1 and Kir2.1 are insensitive (376, 446). KG channels
containing Kir3.2 show the greatest enhancement by ethanol.
KG channels in the heart (KACh channels) are blocked
by quinidine and quinine with an IC50 of ⬃10 ␮M (362,
413, 551). Verapamil also inhibits ACh-activated cardiac
KG channels with an IC50 of 1 ␮M, although its effect is
partially mediated by block of the M2-muscarinic receptor/G protein system (321). KG channels are rather insensitive to other well-known K⫹ channel blockers such as
TEA, 4-AP, apamine, charybdotoxin, disopyramide, and
procainamide (309, 362, 422, 423, 551). The antimuscarinic effect of disopyramide and procainamide is mediated mainly by blocking at the M2-receptor but not at the
KG channel itself (551).
Heterologous expression of Kir3.x subunits in Xenopus oocytes has shown that KG channels made up of
various combinations of Kir3.x subunits can be modulated by many compounds, including antipsychotic
drugs such as haloperidol, thioridazine, pimodine, and
clozapine acting on Kir3.1/3.2 and Kir3.1/3.4 channels
(377); antidepressants such as imipramine, desipramine, amitriptyline, nortriptyline, clomipramine, maprotiline, citalopram, and fluoxetine acting on Kir3.1/3.2, Kir3.2, and Kir3.1/
3.4 channels (378, 380); volatile anesthetics such as halothane, F3 (1-chloro-1,2,2-trifluorocyclobutane), isoflurane,
and enflurane (533, 824, 860) acting on Kir3.1/3.2, Kir3.2, and
Kir3.1/3.4 channels; and the local anesthetic bupivacaine
acting on Kir3.1/3.2, Kir3.2, Kir3.1/3.4, and Kir3.4 channels
(893). These compounds block KG channel activity, but they
clearly do not show any subunit combination specificity. It is
also the case that the selective dopamine D1 receptor antagonist R-(⫹)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (419), the NMDA
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receptor antagonists MK-801 (383) and ifenprodil (379), the
inhaled drug of abuse toluene (126), the Ca2⫹ channel
blocker verapamil, and the classical cation channel blocker
QX-314 (383) have been shown to inhibit various combinations of Kir3.x subunits. Although the binding sites for most
of these compounds have not been examined, those for
halothane and bupivacaine are estimated to be in the cytoplasmic regions of the channels (533, 860, 893).
321
3. Neuronal disorders
Knockout of Kir3.2 in mice is accompanied by the
reduction of Kir3.1 protein expression in the brain, the
development of spontaneous seizures, and a range of
phenotypes related to sporadic seizures (726). These mice
also show an increased susceptibility to convulsant
agents (726). These phenotypes would result from the
loss of IPSP and the resultant neuronal hyperexcitability
(see also sect. IIID).
F. Diseases
IV. KATP CHANNELS (Kir6.x/SURx)
1. Atrial fibrillation
Atrial fibrillation (AF) is the most common cardiac
arrhythmia in clinical practice. AF can become persistent
due to remodeling of atrial electrophysiology. The
changes in the electrical properties of atrial cardiomyocytes, such as a decrease of L-type Ca2⫹ current density
(800) and an increase of IK1 current density (137), are
thought to contribute to the pathology of persistent AF.
Recently, IKACh has been found to play an important role
in chronic AF (cAF) and thus could form a possible target
for AF therapy. Dobrev et al. (136) have reported that
electrical remodeling in AF patients causes an increase of
a constitutively active component of IKACh but a decrease
of its ACh-induced component. This switch from a ligandgated current to constitutively active behavior would lead
KG/Kir3.x channels to shorten atrial action potential duration and refractory period in cAF patients. Although the
molecular basis of this abnormality in cAF patients remains elusive, IKACh composed of Kir3.1/3.4 could be a
therapeutic target for AF prevention and treatment. Tertiapin has been shown to suppress AF in canine models
(77, 244). In this context, it should be remembered that
IKACh can be inhibited by classical antiarrhythmic agents
such as quinidine (413) and AN-132 [3-(diisopropylaminoethylamino)-2⬘,6⬘-dimethylpropionanilide] (410).
2. Weaver mice
A mutation in weaver (wv) mice occurs in the signature amino acid sequence of the selectivity filter of Kir3.2
(606). This results in nonselective cation permeation
rather than K⫹ selectivity (383, 554, 733). The distribution
of Kir3.2 clearly overlaps tissues showing defects in wv
mice. The mutation results in phenotype abnormalities
such as severe locomotor defects, a deficiency in the
migration of granule cells of the cerebellum (640), and
cellular deficits in the midbrain dopaminergic system
(691). Because of abnormal selectivity of KG channels that
contain mutant Kir3.2, inhibitory transmission in wildtype turns into excitatory signaling, which results in neuronal death. The failure of sperm production in homozygous male wv mice (243) probably results from the mutation of Kir3.2d which is expressed in the acrosome of
spermatids (304).
Physiol Rev • VOL
A. Historical View and Molecular Diversity
1. Identification of KATP channels in native tissues
KATP channels were first identified in cardiac myocytes (577, 788). KATP channels are also found in pancreatic ␤-cells (22, 102), skeletal muscle (739, 740), vascular
smooth muscle (40, 349, 353, 741), and neurons (24, 25). In
the inside-out membrane patch, KATP channels open spontaneously. Thus the KATP channels should be classified
together with the classical Kir2.x channels that have constitutive activities. The openings are inhibited by ATPi
(except for in smooth muscle) and activated by NDPsi
such as ADP (21, 773). These are the hallmarks of KATP
channels and therefore from the onset the channels were
proposed to be associated with cellular metabolism and
membrane electrophysiology.
The heteromeric complex nature of KATP channels was
first suggested by Tung and Kurachi (795) showing that the
inhibitory effect by ATP and the stimulatory effect by NDP
are mediated by reaction of these nucleotides on distinct
sites of the cardiac KATP channel. They proposed that a KATP
channel comprises a gate, an ATP-binding unit, and a transducer unit which transduces signals from the ATP-binding
unit to the gate (773, 795). KATP channels are now known to
be functional octomers composed of four Kir channel subunits forming the ion channel pore and four auxiliary proteins: the sulfonylurea receptors, SURx (Fig. 11A). The Kir
subunits are responsible for ATP inhibition and the SUR
proteins for NDP activation. A variety of pharmacological
agents can either stimulate or inhibit KATP channels by binding to SUR (Fig. 12). The inhibitory agents include sulfonylureas, such as chlorpropamide, tolbutamide, and glibenclamide (144, 154, 832), which are used in the therapeutic
treatment of type II diabetes (122, 144, 689, 690, 832) (see
sect. IVE). The stimulatory agents are known as K⫹ channel
openers (KCOs), and they include pinacidil, nicorandil, and
diazoxide (21, 773) (see sect. IVE).
Physiological and pharmacological analysis has characterized distinct types of KATP channels in different tissues (Fig. 11A). Native KATP channels display quite different unitary conductance, for instance, ⬃70 –90 pS in car-
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diac muscle, ⬃55–75 pS in skeletal muscle, and ⬃50 –90
pS in pancreatic ␤-cells (21, 577, 635, 858). In vascular
smooth muscle, the initial report of a large-conductance
KATP channel (741) has not been reproduced (see also
Ref. 40). Instead, small-conductance (⬃15 and 25 pS) K⫹
channels are activated by KCOs (349, 353), and the presence of an NDP is indispensable for KCO-mediated activation of the channel. These are called KNDP channels,
and their opening is insensitive to ATPi. The KNDP channels are the major representative of the KATP channel
family in vascular smooth muscle cells. KATP channels in
different tissues also exhibit considerable variation in
their pharmacological properties. The affinity for a radiolabeled analog of glibenclamide classifies SURs into at
least two types (202, 690). One type shows a high affinity
for sufonylureas and resides in pancreatic ␤-cells. Another type with a lower affinity is present in the heart,
skeletal muscle, and brain. These early findings implied a
molecular heterogeneity of KATP channels. Subsequent
cDNA cloning identified the molecular constituents of the
KATP channels and their diversity (3, 4, 299 –301, 317, 853).
2. Cloning of the constituents of KATP channels
In 1995, cDNA of uKATP-1 was isolated from rat pancreatic islets (301). This channel possessed the primary
structure common to Kir subunits, but its heterogeneous
expression did not yield any functional K⫹ currents.
An SUR was first cloned from cDNA libraries of rat
and hamster insulinoma cell lines (4). The deduced amino
acid sequence defined the receptor as a member of the
ATP-binding cassette (ABC) transporter superfamily with
two nucleotide-binding domains (NBD) (Fig. 11B). The
FIG. 11. Molecular constituents of KATP channels. A: KATP channels
are composed of pore-forming Kir6.x subunits and SUR subunits. SUR
subunits contain 17 transmembrane regions grouped into 3 transmembrane domains TMD0, -1, and -2 (top panel). Each SUR unit has two
nucleotide-binding domains (NBD, depicted as hemispheres) between
TMD1 and TMD2 (NBD1) and in the COOH terminus following TMD2
(NBD2). SUR2A and SUR2B differ only in the COOH-terminal 42 amino
acids (C42). A functional KATP channel is a heterooctamer made up of
four Kir6.x subunits and four SUR subunits (bottom left). Combinations
of different SURx and Kir6.x subunits form distinct types of KATP channels. Bottom right images show top and side views of the entire KATP
channel complex analyzed at 18 Å resolution. Blue represents Kir6.x.
Yellow represents TMD0 of SUR. Red represents the rest of SUR. [From
Mikailov et al. (528), with permission from Nature Publishing Group.]
B: homology between SUR subunits. TM, transmembrane region; TMD,
transmembrane domain; NBD, nucleotide binding domain. [From Isomoto et al. (315).]
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FIG. 12. Determinants of the characteristics of KATP channels. The
single-channel characteristics (unitary conductance and rectification) of
KATP channels are determined by the pore-forming Kir6.x subunits.
Drugs that modify channel activity, i.e., sulfonylureas and K⫹ channel
openers, act on SURs. Nucleotides can influence both Kir6.x and SUR
subunits to regulate channel behavior.
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STRUCTURE AND PHYSIOLOGICAL FUNCTION OF Kir CHANNELS
receptor possesses 17 TM regions that can be grouped
into transmembrane domain 0 (TMD0: TM1-TM5), TMD1
(TM6-TM11), and TMD2 (TM12-TM17) (Fig. 11A). This
SUR (SUR1), like other ABC proteins, could bind and
hydrolyze ATP (47, 511). Unlike a Cl⫺ channel ABCC7
that is also called cystic fibrosis transmembrane conductance regulator (CFTR), however, SUR1 did not show
catalysis-coupled ion transport activity (6), and the injection of SUR cRNA alone into Xenopus oocytes generated
neither ATP-sensitive nor glibenclamide-sensitive K⫹ currents (4).
Inagaki et al. (301) cloned another Kir channel subunit (BIR) from a human genome library using uKATP-1
cDNA as the probe. The amino acid sequences of uKATP-1
and BIR were highly homologous with ⬃70% shared
amino acid identity. These two subunits also shared ⬃40 –
50% identity with other Kir channels. It was therefore
suggested that the two clones belonged to the same Kir
subfamily, Kir6.x (see Fig. 2B). Today uKATP-1 and BIR
are known as Kir6.1/KCNJ8 and Kir6.2/KCNJ11, respectively.
Kir6.2 mRNA is expressed abundantly in pancreatic
islets and various pancreatic cell lines and moderately in
the heart, skeletal muscle, and brain (301). Kir6.2 and
SUR1 mRNAs were found to occur together in a variety of
tissues and cell lines, and coexpression of Kir6.2 and
SUR1 in COS-1 cells reproduced the main physiological
and pharmacological properties of the KATP channels observed in pancreatic ␤-cells (300).
Several tissues such as heart, skeletal muscle, brain,
and smooth muscle that possess functional KATP channels
do not express the SUR1 subunit. A homolog of SUR1,
SUR2A, was isolated (299) (Fig. 11B). When SUR2A and
Kir6.2 were coexpressed, the functional KATP currents
were less sensitive to either ATPi or glibenclamide than
the currents yielded by the channels composed of SUR1
and Kir6.2 (299). Kir6.2/SUR2A channels were activated
by the “cardiac” KCOs, cromakalim and pinacidil, but not
by diazoxide, which suggested that Kir6.2/SUR2A may
form cardiac and skeletal muscle type channels (299). A
variant of SUR2A, SUR2B, was cloned from a mouse heart
cDNA library (Fig. 11B) (317). Only the last 42 amino acid
residues in the COOH terminus (C42) differ between
SUR2B and SUR2A, indicating that they are formed by
alternative splicing of a single gene (Fig. 11B) though C42
of SUR2B shares considerable homology with C42 of
SUR1. Coexpression of SUR2B and Kir6.2 in HEK 293T
cells elicits KATP currents that are activated by pinacidil
and diazoxide (317). On the other hand, diazoxide cannot
stimulate channels composed of Kir6.2/SUR2A (114).
Therefore, under physiological conditions, C42 of SUR2B
may be involved in diazoxide activation of KATP channels
in smooth muscle cells (317, 853).
The conclusion of the cloning and reconstitution
studies is that the KATP channel is a complex of four
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pore-forming Kir6.x subunits and four auxiliary SURx
proteins (98, 721) (Fig. 11A). SUR1, SUR2A, and SUR2B
represent pancreatic, cardiac, and vascular smooth muscle types of SUR, respectively, and different combinations
of Kir6.1 and Kir6.2 and the SURx determine the properties of each native channel (3). Although it has proven to
be possible to construct heteromers of Kir6.x and different SUR subunits which show variant characters (79), the
presence of Kir6.1/6.2 heteromeric channels in native tissues is disputed (625, 703).
B. Pore Structure and Function
1. Intrinsic pore function
A) THE STRUCTURAL BASIS OF THE UNITARY CONDUCTANCE OF
KIR6.1 AND KIR6.2.
The single-channel conductance of Kir6.2
is ⬃80 pS, whereas that of Kir6.1 is ⬃30 pS. Studies using
chimera and point mutations of the channel revealed that
extracellular links between the two transmembrane segments and the H5 pore region were the critical elements
(386, 651). Homology modeling based on the KcsA structure (143) suggests that M148 in Kir6.1 and V138 in Kir6.2
occupy the same position in the extracellular linking region between the H5 pore helix and the second transmembrane segment (TM2) and face the external entrance of
the pore (651). The side chain of M148 is larger than that
of V138, which may lead to differences in K⫹ flux through
the pore mouth. Also, in the linking region between the H5
pore helix and the first transmembrane helix (TM1), at
least three sequential residues (S113-I114-H115 of Kir6.2;
N123-V124-R125 of Kir6.1) were found to be involved in
the difference of conductance between the two channel
types. While homology modeling of Kir6.2 suggests that
such triplet residues may be localized far from the channel outer entrance, it also suggests a hydrogen bond
interaction between S113 with R136, a residue that is
highly conserved among Kir channels and which is considered to play a key role in maintenance of the stability
of pore selectivity filter (867). Therefore, via this interaction, the three residues could control the conformation of
the permeation pathway and determine the different conductance of Kir6.1 and Kir6.2 pores (651).
B) INWARD RECTIFICATION. KATP channels exhibit weak
inward rectification. The Kir6.2 subunit contains an Asn in
the TM2 inner helix (N160) at the D/N site (see sects. IC1,
IIB, and VA2). Mutation of N160 in Kir6.2 to Asp or Glu
resulted in generation of KATP channels showing strong
rectification (721).
2. Regulation of KATP channel function
KATP channel activity is regulated by intracellular
nucleotides and by various pharmacological agents. The
physiological and pharmacological control of KATP chan-
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nel activity has to be interpreted in terms of conformational changes in the Kir6.x pore through protein-protein
interaction with SUR as well as direct action of agents
such as ATP on the pore subunit itself (Figs. 12 and 13).
A) REGULATION OF GATING BY INTRACELLULAR NUCLEOTIDES.
I) Differential action of intracellular nucleotides on
KATP channel activity. Physiologically, regulation of KATP
channel activity by intracellular nucleotides is the most
significant profile. ATPi is the main regulator and can
exert two distinct functions: 1) it closes the channel, and
2) it maintains channel activity in the presence of Mg2⫹.
The KATP channel stays closed as long as ATP is
bound to the channel. In native pancreatic ␤-cells, the
presence of Mg2⫹ shifted the IC50 value of KATP channels
for ATPi from 4 to 26 ␮M, suggesting that ATP4⫺ in its free
acid form is a more potent inhibitor of the channel and
that Mg-ATP has little inhibitory effect (19). In smooth
muscle cells of portal vein, Mg-ATP is also less effective
than ATP4⫺ (349, 557). Cardiac-type KATP channels are
inhibited by both ATP4⫺ and Mg-ATP in a similar manner
(175, 351, 432).
Cardiac and ␤-cell KATP channels spontaneously
open when their cytoplasmic side is exposed to ATP-free
solution. Then, the channel activity declines with a variable time course. This “rundown” can be accelerated by
divalent cations such as Mg2⫹. Treatment of rundown
membrane patches with Mg-ATP but not with nonhydrolyzable ATP restores spontaneous channel activity (54,
178, 179, 536, 586, 761, 788). This is a phenomenon that is
common to all Kir channels (287); Mg-ATP maintains KATP
channel activity through PtdIns(4,5)P2 generation from
phosphatidylinositol by ATP-dependent lipid kinase (see
sect. I, C1 and D2).
NDPs such as ADP are also essential for the physiological opening of KATP channels, which would otherwise
be permanently closed by their overt sensitivity to ATPi.
There was evidence that NDPs such as ADP increased
KATP channel activity against the ATPi-induced inhibition
of channel opening (149, 176, 178, 350, 432, 536). Therefore, it was initially proposed that NDPs would activate
KATP channels by competing with ATP-binding to the
channels. However, NDPs can also open KATP channels in
the absence of ATPi (149, 176, 178, 350, 432, 536). The
positive effects of NDPs require the presence of Mg2⫹, in
the absence of Mg2⫹ they enhance the inhibitory effect of
ATPi (149, 176, 178, 350, 432, 536). Of importance, when
rundown channels were activated by NDPsi, they could be
subsequently inhibited by ATPi with a Ki value similar to
that in the absence of the NDPsi (795). Based on these
FIG. 13. Modeling opening and closing of a SUR2x/Kir6.2 channel.
A: an allosteric model of SUR2x/Kir6.2 channels. Here, we assume the
following: 1) a SUR2/Kir6.2 channel has a hetero-octameric structure
composed of four SUR2x and four Kir6.2 subunits; 2) each SUR2x has a
receptor (indent) for a ligand (solid circle); 3) SUR2x has two distinct
conformations that are able (R conformation) and unable (T conformation) to open the channel pore formed from Kir6.2 subunits; 4) the T and
R conformations are in equilibrium determined by the allosteric constant L, where L is the ratio of R to T in the absence of any ligands; 5) the
structure of the receptor with regard to the ligand is distinct in the R and
T conformations and has higher affinity for the ligand in the R rather
than the T conformation; and 6) as a result, the ligand shifts the T-R
equilibrium toward the R conformation. The ligand can be regarded as
either nicorandil, ATP, or ADP. In the case of nicorandil, the indent
represents the drug receptor site. For ATP and ADP, the indent corresponds to either NBD1 and/or NBD2. B: hypothetical conformational
change of SUR2x induced by dimerization of NBDs. The illustration
demonstrates two opposing Kir6.2 subunits and one SUR2x interacting
with one of the Kir6.2 subunits. SUR interacts with Kir6.2 through TMD0
and L0, a cytoplasmic linker between TMD0 and TMD1 (29, 80). In this
model, NBD1 and NBD2 face each other. ATPi induces dimerization of
these NBDs and thereby a conformational change of TMD1 and TMD2.
This conformational change is transferred to Kir6.2 through TMD0-L0
(green arrows), resulting in opening of the channel pore formed by
Kir6.2. The conformation of SUR2x with and without NBD dimer could
be considered equivalent to the R and T conditions. [A and B from
Yamada et al. (852).]
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STRUCTURE AND PHYSIOLOGICAL FUNCTION OF Kir CHANNELS
observations, it was proposed that NDPs might not compete with ATP at its binding site but may affect other
site(s) to control the function of the channels (795). KATP
channels are therefore likely to bear two distinct sites for
channel gating, an inhibitory ATP-binding site and a stimulatory NDP-binding site, which could be regulated by
NDPs in two different ways, i.e., antagonism and agonism,
respectively (775, 795).
II) Modulation of channel kinetics by nucleotides. In
the absence of ATPi, cardiac and pancreatic KATP channels exhibit spontaneous bursts of rapid openings and
closings (fast kinetics), which are separated by long
closed intervals (slow kinetics) (8) (see also sect. IVA).
The major effect of ATP upon gating is to increase
“long” closed lifetimes (7, 8, 145, 156 –158). ATP may also
decrease the duration of the bursts of rapid openings
which suggests that ATP destabilizes the channel’s open
state as well as stabilizes its closed state (156, 162, 448,
566, 785). On the other hand, NDPs eliminate the long
closed state between bursts and promote channel opening in sustained bursts without changing channel short
open time (7). Therefore, ATPi and NDPsi principally
modulate slow-gating but not fast-gating processes. NDPs
also counteract the effects of ATPi. A kinetic model has
suggested that NDP allows KATP channels to operate in an
ATP insensitive state (7), which could explain how NDP
allows KATP channel opening in the presence of otherwise
inhibitory concentrations of ATPi. KNDP channels are also
regulated by ATPi and ADPi, but the mechanism of this
regulation remains elusive.
III) Structural basis of channel closure by ATP. The
site for ATPi inhibition of KATP opening locates on the
Kir6.2 subunit (791). A number of basic residues and
uncharged residues, R50 in the NH2 terminus and I182,
K185, R201, and G334 in the COOH terminus, are involved
in ATP-mediated inhibition of channel opening (15, 111,
344, 450, 632, 649, 790 –792). Homology modeling suggests
that these five residues form an ATP binding pocket located at the interface of the NH2 and COOH termini (15,
158, 238, 564, 784). The stoichiometry of ATP binding in
one Kir6.2 subunit provides one high-affinity ATP-binding
site, and therefore, one functional KATP channel bears
four sites (501).
This structural information indicates that four ATP
molecules could bind to one KATP channel. ATP inhibited
the native KATP channel of cardiac myocytes or skeletal
muscle, and SUR2A/Kir6.2 as well as SUR2B/Kir6.2 channels, with a Hill coefficient significantly larger than unity
(⬃1.8 –3) (21, 177, 299, 317, 589, 825, 826, 858). On the
other hand, in pancreatic ␤-cell KATP channels, SUR1/
Kir6.2, and Kir6.2⌬C, the truncated form of Kir6.2 that can
function in absence of SUR, the Hill coefficient was close
to 1 (15, 25, 218, 300, 501, 565, 680, 785, 791). These
findings suggest that the type of SUR associated with
different Kir6.x may influence the effectiveness of ATP
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325
binding. In vitro reconstruction of KATP channels with
different SUR subunits coexpressed with Kir6.2 showed
channels with different opening kinetics in the absence of
ATPi (32, 91) which may relate to their different sensitivity to ATPi. But a recent study by Craig et al. has found
that, in the case of Kir6.2/SUR1 channel, the binding of
one ATP to a single Kir6.2 subunit, by interaction between
several of the associated cytoplasmic domains, should
suffice to induce channel closure (108). The reason that a
Hill coefficient of ATP-induced inhibition of native KATP
channels in cardiac myocytes and skeletal muscle is
larger than unity remains unknown.
Mutations in Kir6.2 that change intrinsic channel gating may have a secondary effect to reduce ATP sensitivity
(145, 157, 158, 719, 785, 792), probably because ATP preferentially binds to the closed channel (8, 157, 720).
PtdIns(4,5)P2 is responsible for sustaining spontaneous KATP channel activity in excised membrane patches
(see sect. IC1). PtdIns(4,5)P2 binds to positive charges in
the cytoplasmic region of the Kir6.2 subunit (R54, R176,
R177, and R206) close to the ATP binding site (39, 722,
723). If PtdIns(4,5)P2 sustains KATP channel activity by
stabilizing their open state, it may not be surprising that it
can also decrease channel ATP sensitivity. Assays suggest
competitive binding between PtdIns(4,5)P2 and ATP to
the channel (39, 158, 239, 564, 723).
IV) Mechanisms of activation of KATP channels by
NDPs. The site for NDP activation of KATP channel opening is not located on the Kir6.2 subunit (791). An SUR
subunit has two NBDs with Walker-A and Walker-B consensus motifs (G-X-X-X-X-G-K-T/S) (815). NBD1 is located
on the cytoplasmic loop between the TM11 and TM12, and
NBD2 is situated on the cytoplasmic COOH terminus
following the TM17 (Fig. 11A). Mutations on the Walker-A
motif of NBD1 prevent nucleotides from binding to both
NBDs (796) and interfere with the stimulatory effect of
NDPs (28, 114, 221, 512, 720). Thus NDPs activate KATP
channels via their binding to the SUR, while ATP inhibits
channel activity by binding directly to Kir6.2 (Fig. 12), and
there is cooperation between the two NBDs to function as
nucleotide receptors (511, 797, 898).
NBDs of other ABC proteins form a dimer when
exposed to nucleotides (81, 278, 735). Homology models
of the three-dimensional structure of NBDs of SUR2A and
SUR2B (855, 857) were made with using the monomeric
structure of HisP and the ATP-mediated dimer structure
of MJ0796 (292, 735). When mutations equivalent to those
that prevent dimerization of MJ0796 were introduced into
NBDs of SURs, the NDP-mediated activation of KATP
channels was impaired (852). When mutations equivalent
to those that permit MJ0796 to be dimerized by Na⫹-bound
ATP were introduced into NBDs of SUR2, Na⫹
i increased the
activity of KATP channels in the presence of ATPi (852).
Homology models of the dimerized structure of the NBDs in
SUR1 were generated and mutations introduced into the
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putative interface of the dimerized NBDs altered their
affinity for Mg-NDP (75, 503). These results indicate that
the two NBDs form a dimer in SUR proteins to contribute
to gating of the ion channels. A similar mechanism was
reported to control CFTR Cl⫺ channel activity where
NBDs are dimerized when bound to ATP (806).
In all ABC proteins studied so far, NBD1 and NBD2
sandwich Mg-ATP in the dimer (276). The dimer is needed
to generate two catalytic sites for ATP hydrolysis. These
sites are formed by the Walker-A motif (or P-loop) of one
NBD and the ABC signature sequence motif (L-S-G-G-Q
motif or C-loop) of the other NBD. Of the two catalytic
ATP-binding sites, that formed by the Walker-A motif of
NBD2 and the C-loop of NBD1 shows the most prominent
ATPase activity (47, 510). The ATP hydrolysis activity of
NBD2 of SUR1 is also higher than that of SUR2A (502)
which may contribute to the differences between Kir6.2/
SUR1 and Kir6.2/SUR2 gating properties. An analysis of the
effects of NDPs on KATP channels which contained either
SUR2A or SUR2B with the Monod-Wyman-Changeux
(MWC) model (538, 852) (Fig. 13A) suggested KATP channel activation via conformational changes in the SUR
subunits transiting between relaxed (R, able to open) and
tense (T, unable to open) conformations (Fig. 13) (852,
855, 857). The T-R transition in nucleotide-bound forms of
the NBDs was higher in SUR2B than in SUR2A (512, 513,
855). The 42 residues in the COOH terminus of SUR2B
(C42) are located at the interface between the NBD dimer
(Fig. 13B) where the terminus could interact with the
NBD dimer (855, 857). The mechanism of action of NDP
added to the internal surface of a membrane-bearing KATP
channels may be to lock the NBDs in a “posthydrolytic”
configuration that is associated with the open channel
(897).
V) Mechanisms underlying SUR modulation of Kir
pore function. It still remains unclear how SUR can modulate the pore function of KATP channels. The unique
TMD0 domain of SUR was reported to anchor SUR to the
outer TM1 helix and NH2 terminus of Kir6.x (29, 164, 283,
386) (Figs. 11A and 13B). The adjacent L0 linker functionally links Kir6.2/TMD0 with the SUR protein’s core (Fig.
13B) (30, 649). This arrangement seems to be involved in
both opening and closure of KATP channels. Thus ligandinduced conformation changes in SUR may be transduced
via L0 and TMD0 to the Kir6.x channel pore. Recently, the
Asp/Glu-rich domain (ED domain), a stretch of 15 negatively charges residues adjacent to NBD1 in SUR2A, was
also suggested to have a role transducing conformational
rearrangements of the sulfonylurea receptor to the Kir6.2
channel pore (356).
3. Other means of regulation of KATP channel gating
A) PHOSPHORYLATION. KATP channels can be phosphorylated by Gs-mediated PKA signaling pathways, and impor-
Physiol Rev • VOL
tance of this modulation has been highlighted particularly
in smooth muscles (42, 458, 557, 635, 637, 713). PKA
phosphorylation sites include S385 in Kir6.1 and T633 and
S1465 in SUR2B (637), and S1387 in NBD2 of SUR2B
(713). Kir6.2 can be phosphorylated at T224 by PKA (458)
and at T180 by PKC (455). It is still not clear how phosphorylation of these residues activates the channels.
B) CYTOSKELETON-DEPENDENT GATING OF KATP FUNCTION. Gating of the atrial KATP channel is mechanosensitive, and
mechanical pressure applied to a cardiac cell leads to an
increase in their activity (798, 799). In general, the actin
cytoskeleton plays an important role in the detection
and/or transduction of mechanical stress to ion channels
(203, 810). The disruption of actin filaments with DNase I
or cytochalasin B antagonized ATPi-mediated inhibition
of KATP channels (774). The same treatment impaired
sulfonylurea-induced closure of cardiac KATP channels
(62, 874). Although the molecular mechanism remains
elusive, this modulation may help to open the cardiac
KATP channels during ischemia or hypoxia when mechanical disturbances of the cytoskeleton can occur. The
mechanosensitivity of KATP channels is enhanced under
ischemic conditions (799).
C. Intracellular Localization
1. Trafficking to the plasma membrane
Only the octameric KATP channel complex can reach
the cell plasma membrane (4, 300, 680, 887). However,
Kir6.2⌬C26 is transported and forms a functional K⫹
channel on the membrane without any SUR subunits
(791). The truncated region contains an ER-retention sequence, R-K-R (887). SUR1 also contains the R-K-R sequence which prevents its surface expression, and mutation allows the receptor to traffic to the cell surface in the
absence of Kir6.x channels (887). Thus the ER retention
motifs in Kir6.x and SUR must be mutually masked to
permit membrane surface expression (887). These sequences are different from known ER retention signals,
i.e., the luminal K-D-E-L and cytoplasmic K-K-X-X motifs
(772).
2. Mitochondrial KATP channels
In 1991, Inoue et al. (308) identified KATP channels in
rat liver mitochondrial inner membrane by single-channel
recording. These mitoKATP channels were inhibited by
ATP applied to the luminal matrix face with a Ki of ⬃0.8
mM. The channels were also inhibited by the K⫹ channel
blocker 4-AP and by 5 ␮M glibenclamide. The mitoKATP
may be made up of Kir and SUR subunits. Kir6.1 antibody
labeled the mitochondria and bound to a 51-kDa protein
in the mitochondrial membrane fraction, suggesting that
the Kir6.1 subunit is a possible component of mitoKATP
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STRUCTURE AND PHYSIOLOGICAL FUNCTION OF Kir CHANNELS
(752). Other studies have suggested that Kir6.1, Kir6.2,
and SUR2A subunits are present in the mitochondria of
the heart and brain (426, 427). However, because neither
overexpression of a dominant negative form of either
Kir6.1 or Kir6.2 in which the pore signature sequence
G-F-G was mutated to A-F-A, nor targeted ablation of the
Kir6.1 or the Kir6.2 gene could disrupt mitoKATP function
(531, 703, 754), the molecular identity of the mitoKATP
channel is still controversial.
D. Physiological Functions in Cells and Organs
1. Pancreas
In pancreatic insulin-secreting ␤-cells, KATP channels
that are made up of Kir6.2 and SUR1 (299, 300) not only
set the Eres (about ⫺70 mV) but also serve to couple blood
glucose concentration and insulin secretion (19, 20). Insulin secretion is stimulated by high blood glucose levels.
At substimulatory blood glucose levels, pancreatic ␤-cell
KATP channels are open and maintain negative Em. As
blood glucose levels increase, glucose uptake and metabolism in ␤-cells are initiated. Then, ATPi concentration is
augmented, whereas ADPi concentration is reduced.
These changes attenuate KATP channel activity which results in cell depolarization and activation of L-type voltage-gated Ca2⫹ channels (VGCC). Ca2⫹ influx via VGCC
leads to the fusion of vesicles containing insulin to the
membrane and release of the hormone.
Consistent with this predicted function, genetic modification of Kir6.2 or SUR1 results in various phenotypes of
glucose homeostasis disorder. Mice lacking either Kir6.2 or
SUR1 showed transient neonatal hypoglycemia (530, 702). In
humans, a number of polymorphisms in Kir6.2 and SUR1
have been identified, and many of them are associated with
hypoglycemia and diabetes. These channelopathies will be
discussed in section IVF (see also Table 2).
KATP channels are also expressed in other endocrine
cells such as glucagon-secreting ␣-cells and somatostatinsecreting ␦-cells (212, 213). They may be involved in the
control of secretion of these hormones.
2. Heart
Early studies assumed that cardiac KATP channels
played a cardioprotective role in ischemic conditions
(319, 354, 566, 577, 773). KATP channels would shorten the
cardiac action potential duration during ischemia (see
Fig. 1B) and reduce harmful Ca2⫹ influx through VGCC.
Physiological and pharmacological assays revealed the
similarity between native cardiac KATP channels and the
current elicited by coexpression of Kir6.2 and SUR2A in
heterologous expression systems (27, 299). The myocardium of Kir6.2-knockout mice lacks functional KATP channels (451, 530, 753, 781). Thus, although this strongly
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suggests that the cardiac KATP channel contains Kir6.2,
the SUR partner is less clear. It has been recently reported
that Kir6.2 may be expressed with SUR1 in atrial myocytes and with SUR2A in ventricular myocytes of the
mouse (184). This observation needs to be extended to
other species.
In contrast to pancreatic ␤-cell KATP channels, cardiac KATP channels are closed under physiological conditions, presumably because of the high ATPi concentration
in this tissue (354, 566, 773). Though how this equates
with in vitro observations of quite extreme rightward
displacement of the ATP channel-closure dose-response
curve by NDPs and in particular PtdIns(4,5)P2 (39, 158,
239, 564, 723) remains to be examined. The channels
would be opened by metabolic insult such as increased
cardiac work load, hypoxia, or ischemia. Elevation of the
S-T segment in the electrocardiogram, a hallmark of acute
myocardial ischemia, was markedly reduced during ischemia in Kir6.2-knockout mice, implying that opening of
KATP channels underlies the S-T elevation (451, 754).
Brief episodes of ischemia result in subsequent protection of the myocardium against later, more severe
ischemic insult. This phenomenon is called ischemic preconditioning (547, 871). There is a general consensus that
KATP channels play a key role in triggering this event,
although whether this involves sarcolemmal KATP channels or mitoKATP is disputed. Gross et al. (224) first proposed that opening of KATP channels was involved in
protective effects of preconditioning and KCOs mimic
preconditioning, whereas KATP channel blockers eliminate the protective role during ischemia (113, 224, 698,
801). In Kir6.2-knockout mice, preconditioning disappeared (229, 754). A large number of mediators and signal
transduction pathways seem to be engaged downstream
of the opening of KATP channels (871).
To understand physiological and pathological roles
of cardiac KATP channels, analysis of results obtained
from Kir6.2-knockout mice are informative. So far, cardiac KATP channels have been implicated in the maintenance of cellular functions and stress adaptation in hyperadrenergic conditions such as physical exertion and
decompensated heart failure, which are well-established
precipitators of arrhythmia. Kir6.2-knockout mice showed
an increased vulnerability to sympathetic stress and vigorous sympathetic challenge caused arrhythmia and sudden
death (899). Similar phenotypes were observed in the
mice whose cardiac KATP channels were conditionally
ablated; they were less tolerant to exercise stress, and
their mortality increased compared with control littermates after the age of 4 –5 mo (781). In wild-type mice,
␤-adrenergic-mediated phosphorylation of KATP channels
(42, 458) or depletion of ATP under the cell membrane
(686) might enhance channel opening and contribute to
shortening of cardiac action potentials. Catecholamine
challenge shortens action potential duration in wild-type
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but not Kir6.2-knockout hearts where an inadequate repolarization causes early afterdepolarization and ventricular arrhythmia (463). Under equivalent hemodynamic
stress, Kir6.2-knockout mice showed an aberrant prolongation of the action potential with pathological Ca2⫹
overi
load compared with wild type, leading to cardiac remodeling, heart failure, and death (355, 859). These findings
indicate broad roles for KATP channels in heart disease
and suggest possible clinical benefits from KATP channel
modulators (see sect. IVF).
3. Smooth muscle
Kir6.1/SUR2B channels resemble native KATP channels
observed in vascular smooth muscle in having a unitary
conductance of ⬃35 pS and no spontaneous opening in the
absence of ATPi (315, 317, 853, 858). KATP channels are
expressed in vascular smooth muscles throughout the body.
Opening of vascular KATP channels hyperpolarizes Em towards EK. It results in closure of VGCC and hence relaxation
of the smooth muscle of the blood vessels, especially that of
veins (vasodilatation). In smooth muscle cells, the physiological function of KATP channels depends on their sensitivity to cellular metabolic state. Under normal conditions,
regulation of smooth muscle contraction may be attributed
not only to the direct effects of cellular metabolism itself
(e.g., ATP/ADP) on KATP channel activity, but also to the
effects on the channels of vasodilators such as prostaglandin, CGRP, and adenosine or vasoconstrictors including endothelin, vasopressin, 5-HT, and histamine secreted from
surrounding cells (635). These vasodilators and vasoconstrictors may exert their action at least in part by phosphorylating KATP channels via PKA (42, 458, 635, 637, 713).
KCOs are clinically used as hypotensive agents. Nicorandil, besides its role as a KATP channel opener, can directly supply NO, a potent vasodilator substance. Some
KCOs are used to treat angina since they dilate coronary
vessels. Uterine myometrial smooth muscles express Kir6.1/
SUR2B KATP channels, and their number gradually increases
during pregnancy (112, 685). KCOs can inhibit uterine contraction during late pregnancy and might prove to be useful
agents for prevention of premature labor (685).
4. Brain
In the hypothalamus there are a variety of “glucosesensitive” neurons (24, 668). For example, in the mouse
lateral hypothalamus, orexin/hypocretin neurons that regulate wakefulness, locomotor activity, and appetite are
inhibited by an increase in glucose concentration. Melanin-concentrating hormone neurons that are involved in
sleep and energy conservation are excited by an increase
in glucose concentration. In the hypothalamus arcuate
nucleus, an excitatory action of glucose on the anorexigenic pro-opiomelanocortin (POMC) neuron has been reported, while the appetite-promoting neuropeptide Y (NPY)
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neuron may be directly inhibited by glucose. These findings
emphasize the fundamental importance of the glucose-sensing systems of hypothalamic neurons for orchestrating
sleep-wake cycles, energy expenditure, and feeding behavior. Some neurons that are excited by glucose express KATP
channels and are thought to employ a glucose-sensing strategy similar to pancreatic ␤-cells. In a majority of these
neurons, elevated ATPi closes KATP channels, which causes
membrane depolarization and thus increases excitation (24),
and these neurons contain Kir6.2 (359, 529, 882). Some
glucose-excited neurons in the ventromedial hypothalamus
and glucose-sensitive striatal cholinergic neurons express
KATP channels composed of Kir6.1 and SUR1 (435, 436). The
mechanism of glucose-induced inhibition of neural activity
is less understood. It has been proposed to involve modulation of the Na⫹-K⫹-ATPase (597) and activation of a hyperpolarizing Cl⫺ current that is presumed to involve CFTR-like
Cl⫺ channels (668).
KATP channels may play a protective role in neurons
under pathological conditions (35, 848). During severe
cellular metabolic impairment such as ischemia and hypoxia, most mammalian neurons are known to depolarize
and die. In the substantia nigra pars reticulate (SNr) (261,
545), KATP channels suppress neuronal activity during
hypoxia by opening postsynaptic KATP channels (335, 546,
849). In contrast, in Kir6.2 knockout mice, these neurons
exhibit no such hyperpolarization and are depolarized
(849). The SNr acts as a central gating system in the
propagation of seizure (128, 296), and the Kir6.2 knockout
mice were extremely susceptible to generalized seizure
after brief hypoxia (849).
E. Pharmacology
KATP channels are the targets of two major classes of
therapeutic compounds, sulfonylureas and KCOs (18, 219).
The most clinically important are the sulfonylurea KATP
channel blockers that are used to stimulate insulin secretion
in patients with type 2 diabetes mellitus (219). KCOs activate
KATP channels with a broad range of potential therapeutic
applications (18). All of these drugs affect KATP channels via
their action on the SUR.
1. Sulfonylureas
A) PHARMACOLOGICAL ACTION OF SULFONYLUREAS. Sulfonylureas such as acetohexamide, tolbutamide, glipzide, glibenclamide, and glimepiride contain a S-phenylsulfonylurea
structure (144, 154, 832). The antidiabetic action of sulfonylureas was first reported in studies for sulfonamide antibiotics that were originally explored as an effective treatment
for typhoid. The compound was unexpectedly found to induce hypoglycemia (607). Sulfonylureas are used exclusively for treatment of type 2 diabetes mellitus.
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Sulfonylureas bind to SURx of KATP channels (see
Fig. 12). In pancreatic ␤-cells, this binding inhibits K⫹
efflux through the KATP channels, thus causing cell membrane depolarization, opening of VDCC, the induction of
Ca2⫹ influx, and insulin secretion.
B) INTERACTIONS BETWEEN SUR AND SULFONYLUREAS. KATP
channels reconstituted by the coexpression of Kir6.2
with SUR1 or SUR2A exhibit different responses to
sulfonylureas like native KATP channels in ␤-cells and
cardiac myocytes. Kir6.2/SUR1 is more sensitive to sulfonylureas than Kir6.2/SUR2A (4, 299). The concentration-response curve for tolbutamide strongly suggests
that Kir6.2/SUR1 channels harbor two independent
binding sites with very different affinities (Ki ⫽ 2.0 ␮M
for a high-affinity site and Ki ⫽ 1.8 mM for a low-affinity
site). The high-affinity tolbutamide binding site S1237 is
located in cytoplasmic loop 8 (CL8) between TM15 and
TM16 (23, 527). Glibenclamide binding involves two
sites on SUR1: the CL8 loop described above and the
CL3 loop between TM5 and TM6 (23, 527). Mutation of
S1237 in SUR1 to its SUR2 counterpart (Tyr) abolished
both high-affinity tolbutamide block and 3H-labeled
glibenclamide binding (23). On the other hand,
Kir6.2⌬C36, the functional unit of Kir6.2 without SUR,
was still blocked by high doses of tolbutamide (Ki ⫽ 1.7
mM). Thus the low-affinity block of KATP channels by
tolbutamide seems to be due to its direct interaction
with the Kir6.2 subunit (222, 223).
C) EFFECT OF INTRACELLULAR NUCLEOTIDES ON THE ACTION
OF SULFONYLUREAS. Under physiological conditions, a
number of additional factors are involved in the action
of sulfonylureas. Native KATP channels, analyzed in
cell-attached configuration, are almost completely inhibited by sulfonylureas, whereas in excised membrane
patches, the drugs can block the same channels by only
50 –70% (219). Such a difference may be attributable to
the presence of cytoplasmic nucleotides such as MgADP, which itself influences Kir6.2/SUR1 via two mechanisms, strong activation through SUR1 and weak inhibition through the ATP-binding site on Kir6.2, with the
former but not the latter counteracted by sulfonylureas.
Therefore, the inhibitory effect of sulfonylureas on native ␤-cell KATP and Kir6.2/SUR1 channels is apparently
enhanced by abolishing the stimulatory effect of MgNDP (31, 222). The interference of the binding of nucleotides to SUR1 and SUR2 by sulfonylureas has been
confirmed in biochemical studies using 3H-labeled glibenclamide and 32P-labeled azido-ATP (241, 569, 701,
797). There may also be reciprocal interactions where
Mg-ATP and Mg-ADP reduce binding of glibenclamide
to SURs (242). On the other hand, Matsuo et al. (509)
showed that glibenclamide abolished the effect but not
the binding of ATP and ADP to SURs.
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2. KCOs
A) PHARMACOLOGICAL ACTIONS OF KCOS. KCOs include cromakalim, pinacidil, nicorandil, diazoxide, and minoxidil
sulfate. In vascular smooth muscle, KCOs such as pinacidil, nicorandil, and diazoxide cause relaxation by opening
KATP channels and reduce blood pressure (153, 317).
Some KCOs have found a therapeutic role against hypertension. Nicorandil is clinically used to treat angina pectoris in Japan as well as in Europe. KCOs are also suitable
for management of a variety of other diseases including
acute and chronic myocardial ischemia, congestive heart
failure, bronchial asthma, urinary incontinence, and certain skeletal muscle myopathies. Furthermore, openers of
␤-cell KATP channels such as diazoxide are clinically useful for the treatment of hypersecretion of insulin associated with insulinoma and persistent hyperinsulinemic hypoglycemia of infancy (see sect. IVF).
Native KATP channels in different tissues display distinct sensitivity to KCOs. The pancreatic ␤-cell KATP channels are readily activated by diazoxide, weakly activated
by pinacidil, and unaffected by cromakalim or nicorandil
(18, 20). In contrast, cardiac KATP channels are activated
by pinacidil, cromakalim, and nicorandil but not by diazoxide (318, 773). The smooth muscle KATP channel is
activated by all of these drugs (318, 635). These differences are attributed to the SUR subtypes expressed in
each tissue, i.e., pancreatic SUR1, cardiac SUR2A, and
smooth muscle SUR2B. Coexpression of Kir6.2 with an
appropriate SUR can generate a KATP conductance with
the pharmacological properties observed in each native
tissue (218, 240, 299, 300, 317, 589, 680, 715).
B) KCO BINDING TO SUR. In SUR2A and SUR2B, the binding sites for KCOs such as cromakalim, nicorandil, and
pinacidil are within TMD2, which contains the cytoplasmic loop between TM13 and TM14 and the region encompassing TM16, TM17, and a short segment of NBD2 (28,
114, 797). Two residues in TM17 (L1249 and T1253 in
SUR2A, T1286 and M1290 in SUR2B) have been shown to
be necessary and sufficient for KCO action (540, 541). In
SUR1, the segment including TM6-TM11 and NBD1 may
contribute to the sensitivity to diazoxide (28). The binding
sites for substrates in other polyspecific ABC proteins,
such as multidrug resistance protein MRP1 and MRP3
(117, 324, 361, 497, 590, 650, 888) and multidrug resistance
P-glycoprotein (468 – 470), are located in regions homologous to TM17 of SUR. This suggests that KCOs may act as
pseudo-substrates in SURs and occupy a substrate-binding pocket which is structurally conserved among SURs
and multidrug resistance proteins (540) (see Fig. 12).
C) INTERACTIONS BETWEEN KCO DRUGS AND INTRACELLULAR
NUCLEOTIDES.
The presence of intracellular nucleotides affects the action of KCOs on SURs (Fig. 14) (220, 241, 683,
857). Electrophysiological studies suggest that interaction
of Mg-nucleotides with NBDs not only increases the basal
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FIG. 14. Effects of KCOs on KATP and KNDP channels. SUR2B forms KATP channels with Kir6.2 and makes up KNDP channels with Kir6.1. The
graphs schematically indicate the action of a KCO on the concentration-dependent effect of ATP in each channel type.
activity of KATP channels but also enhances their sensitivity to KCOs. For example, diazoxide cannot induce opening of cardiac-type KATP channels composed of Kir6.2/
SUR2A in the absence of ADP but can do so in the
presence of ADP (114). In addition, nicorandil requires
the presence of ADP to activate cardiac-type channels
(330, 709, 857). KCOs may also activate SUR2B/Kir6.1
(KNDP) channels by synergistically interacting with intracellular nucleotides (Fig. 14) (853). KCOs may increase
ATP hydrolysis at NBD2 (47), and mutations which prevent ATP or ADP binding in NBDs in SUR2A and SUR2B
abolished channel activation by nicorandil (646). Together, these results suggest that the mechanism of action
of KCOs involves allosteric interactions between nucleotides and the NBDs and KCO binding sites in SUR (857).
In summary, KCOs decrease the threshold concentrations of intracellular nucleotides for opening of Kir6.1
KNDP channels and also increase the maximum amplitude.
On the other hand, KCOs decrease the sensitivity of Kir6.2
KATP channels to ATPi inhibition in a concentration-dependent manner that is mediated at least in part by NDP
acting on the NBD dimer of SURs.
D) OTHER EFFECTS OF KCOS. It is well known that some,
but not all, KATP channel openers have hypertrichosis as a
side effect. Minoxidil was first used to treat high blood
pressure (147), but currently it is also used to treat baldness (627). Although the mechanism remains unclear, the
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effect is detected at low concentrations where minoxidil
cannot augment the blood flow (71, 829). Minoxidil may
have a role in maintaining vascularization of the hair
follicle. The hair dermal papilla, which controls hair
growth, is characterized in the anagen phase by a highly
developed vascular network. In these cells minoxidil upregulates VEGF mRNA (424) whose product may promote
vascularization (677).
3. Other drugs
A) NONSULFONYLUREA KATP CHANNEL BLOCKERS. Meglitinide,
repaglinide, nateglinide, and mitiglinide are nonsulfonylurea acylaminoalcyl benzoic acid derivatives which represent a new chemical class of drugs for treating type 2
diabetes (51, 428, 495). They inhibit pancreatic ␤-cell KATP
channels (5, 115, 154, 218, 524, 648, 751) and preferentially
block Kir6.2/SUR1 channels (115, 587, 648, 751). Mitiglinide inhibits the binding of [3H]glibenclamide to microsomes from insulin-secreting HIT-15 cells and SUR1 (587,
751), suggesting that it interacts with the sulfonylurea
binding site in SUR1 but not with the benzamide binding
site in SUR2A (Fig. 12). PNU-37883A, PNU-89692, PNU97025E, and PNU-99963 are blockers of a new generation
that have been derived from a cyanoguanidine “KATP
opener,” P1075. These compounds antagonize the effects
of the vascular KCOs (228, 369, 522).
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F. Diseases
To date, the majority of channelopathies involving
KATP channels have involved pancreatic ␤-cells (16, 17,
629). Some mutations that can cause severe dysfunction
of reconstituted channels (such as Q52R and V59G) may
also disrupt KATP channel function in muscle and brain
(629).
1. Loss-of-function mutations
Persistent hyperinsulinemic hypoglycemia of infancy
(PHHI), which is also known as congenital hyperinsulinism of infancy (CHI), is characterized by excessive secretion of insulin despite hypoglycemia (148). The hypoglycemia causes irreversible brain damage. PHHI mutations
include loss-of-function mutations in SUR1 (ABCC8) (294,
776), which account for ⬃50% of the cases (148) and
loss-of-function mutations in the gene encoding Kir6.2
(KCNJ11) (16, 252, 293, 560, 647, 777). The pathophysiological process is the loss of control of insulin section by
any physiological process following the impairment of
KATP channel activity. Patients with mild forms of the
pathology may be managed with diazoxide. Some populations of familial PHHI induced by mutations in SUR1
may develop type 2 diabetes in later life (294, 491).
More than 100 mutations in the SUR1 gene and several tens of mutations in the Kir6.2 gene have been identified from the genome of PHHI patients. These mutations
distribute throughout the genes. The KATP channel abnormalities associated with these mutations can be roughly
classified into two categories: 1) defects of KATP channel
trafficking to the surface membrane and 2) disruption of
channel gating by nucleotides (16). Because SUR1 is crucial for the trafficking of Kir6.2 to the cell membrane,
mutations that impair SUR1 synthesis, maturation, or subunit assembly result in the loss of surface expression of
functional KATP channels (16, 148, 605, 769, 862). Mutations that reside in NBDs of SUR1 result in abnormal KATP
channels with impaired opening in response to Mg-ADP
(16, 148, 294). This dysfunction results in continuously
closed KATP channels that no longer respond to alterations in extracellular glucose concentration. Several
other mutations in Kir6.2 have been identified to cause
PHHI not by impairing the trafficking of the channels to
the membrane but by reducing KATP channel activity (16).
Dysfunction of other proteins can also influence KATP
channel activity and cause PHHI. Gain-of-function mutations in the glycolytic enzyme glucokinase (GCK) encoded in GCK and the mitochondrial enzyme glutamate
dehydrogenase (GDH) encoded in GLOUD1 cause PHHI
(16, 148). Their enhanced synthesis of ATPi reduces KATP
channel activity and elicits the phenotype.
PHHI patients with the mutations in SUR1 or Kir6.2
are generally insensitive to diazoxide, and their treatment
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331
often requires subtotal pancreatectomy. On the other
hand, PHHI caused by mutations in the genes such as
GCK or GLUD1 respond well to diazoxide (16, 148). The
genotyping of PHHI patients is therefore important to
determine the effective therapy.
Recently, frame-shift (Fs1524) and missense (A1513T)
mutations of SUR2A, which occur at the catalytic ATPase
pocket of NBD2 within SUR2A, have been found in patients
with dilated cardiomyopathy (48). The abnormal KATP channels (Kir6.2 plus mutated SUR2A) show not only decrease of
cell surface expression to some extent but also disruption
of their functionality. The mutants have normal capability of
ATP binding but show slower kinetics of ATP hydrolysis. As
a result, the ATPi sensitivity of the KATP channels is impaired.
2. Gain-of-function mutations
Gain-of-function mutations in either Kir6.2- or SUR1genes cause neonatal diabetes mellitus with reduced insulin secretion and resultant hyperglycemia (16). Approximately 50% of cases of permanent neonatal diabetes
(PNDM) result from mutations in the Kir6.2 gene (151,
209 –211, 504, 675, 804). Mutations in the SUR1 gene (33,
155) are found in ⬃27% of PNDM patients (16). All PNDM
mutations reduce the sensitivity of KATP channels to ATPi.
Several mutations in Kir6.2 cause extremely severe phenotypes including delayed development of speech and
walking and muscle weakness in addition to neonatal
diabetes. The most extreme cases show DEND syndrome
characterized by marked developmental delay, muscle
weakness, epilepsy, dysmorphic feature, and neonatal diabetes (210, 504, 629, 631, 675). The amount of the reduction in ATP sensitivity of the channels correlates with the
severity of the phenotype (16).
There are at least two different molecular mechanisms underlying abnormality of KATP channel function in
PNDM. First, mutations such as I182V or R201C/H in
Kir6.2, which cause neonatal diabetes alone, directly impair ATP binding (16, 210, 211, 344, 629). These mutations
are located in close vicinity to the putative ATP-binding
site. Second, mutations including Q52R, V59M/G, and
I296L in Kir6.2, which cause the severe neonatal diabetes
observed in DEND syndrome, affect ATP-binding indirectly (629, 631). These mutations are located within or
apposed to the slide helix that may be involved in channel
gating and increase channel opening. In consequence,
they indirectly reduce the sensitivity of the channels to
ATPi (785). Most patients with this type of diabetes mellitus can be treated by oral application of sulfonylureas.
3. KATP channel knockout mice
Studies on genetically modified mice also suggest the
involvement of abnormalities in KATP channels in a number of different diseases. Most Kir6.1-knockout mice die
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between 5 and 6 wk after birth because of a high rate of
sudden death syndrome (531). Their electrocardiograms
show spontaneous elevation of S-T segments followed by
atrioventricular block, indicating that their death is attributable to myocardial ischemia resulting from hypercontractility in coronary arteries (531). This phenotype resembles the symptoms of Prinzmetal’s angina, which is
characterized by recurrent episodes of chest pain under
resting conditions (531). The disruption of SUR2 can also
result in transient, repeated episodes of coronary artery
spasm similar to the phenotype of Prinzmetal’s angina
(92). See section IVD for phenotypes of mice lacking Kir6.2
or SUR1 gene.
V. Kⴙ TRANSPORT CHANNELS (Kir1.1, Kir4.x,
Kir5.x, Kir7.x)
A. Kir1.1
1. Historical view and molecular diversity
Kir1.1/KCNJ1 is the first of 15 members of the Kir
family that have been cloned (272). It was initially described as the “rat outer medullary K⫹ channel” ROMK1.
Six alternative splicing isoforms have been identified so
far (ROMK1-6) (53, 141, 272, 385, 717, 892). The products
translated from mRNA of isoforms ROMK4-6 are the same
as the ROMK2 protein. The NH2 termini of ROMK1
(Kir1.1a) and ROMK3 (Kir1.1c) are, respectively, 19 and
26 amino acids longer than the shortest isoform, ROMK2
(Kir1.1b). Like other Kir channels, the Kir1.1 subunit functions as a tetramer (440). Heteromeric assemblies of
Kir1.1 with members of other Kir families have not been
reported.
2. Pore structure and function
Kir1.1 exhibits weak inward rectification, a singlechannel conductance of ⬃35-pS, and high Po over a wide
range of Em when expressed in Xenopus oocytes (53, 83,
272). The deduced amino acid sequence of Kir1.1 shows
that it harbors a Walker type A ATP-binding motif at its
COOH terminus. The functional role of this region has not
been established (56, 86, 272).
A) RECTIFICATION. The weak inward rectification of
Kir1.1 is attributed to the uncharged N171 at the “D/N
site” of the TM2 helix (see sect. IC1).
B) PH SENSITIVITY. Intracellular, but not extracellular,
pH modulates the activity of Kir1.1. Acidification closes
the channels with a pKa of ⬃6.5 (88, 138, 161, 520, 789,
816). Mutation analyses and homology modeling suggest
that pHi sensitivity of Kir1.1 involves residues in three
categories: 1) positively charged residues R41, K80, and
R311; 2) a residue (L160) in the “bundle crossing” region;
and 3) residues in the TM2 transmembrane region.
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Although R41, K80, and R311, the so-called RKR
triad, were all originally implicated in the sensitivity of
Kir1.1 to pHi (88, 161, 520, 696, 816), homology modeling
rather suggests that they are structurally important elements in the general functionality of Kir1.1, and therefore,
their mutation only indirectly influences the reactions of
the channel to pHi (645). To begin with, R41 and R311 are
too far from K80 (⬎24 Å) to influence its ionization state,
and then, K80 seems to be buried within the lipid membrane and inaccessible to solvent. Instead, the NH2 terminus residue R41 forms a stable intrasubunit salt bridge
with the COOH-terminal residue E318; the COOH-terminal residue R311 forms a stable intersubunit ion pair with
E302 on the COOH terminus of the adjacent subunit in the
tetramer; K80 in TM1 and A177 in TM2 occur in the
“helix-bundle crossing” region, are ⬍3 Å apart and form a
potential intrasubunit interaction. Mutation of any of
these six residues alters the pHi sensitivity of Kir1.1 (645).
R311W and A177T, which are found to cause Bartter’s
syndrome, change pKa of Kir1.1 (⬃6.5) to 9.2 and 7.8 in
Kir1.1, respectively, which results in dysfunction of the
channel in physiological pHi range (617, 645, 696) (see
also sect. VA6 and Table 2).
The hydrophobic residue L160 may be involved in
pHi-dependent gating, replacing L160 by a smaller residue,
Gly, abrogated pHi sensitivity of Kir1.1 (672). Since L160
is homologous to Phe in the bundle-crossing region in
KirBac1.1 (F146) (404) (see sect. ID), closure of Kir1.1 by
acidic pHi could result from steric occlusion of the permeation pathway by the convergence of four Leu residues
at the cytoplasmic apex of the inner (TM2) transmembrane helices.
In Kir1.1b, G148 and G157 (G167, G176 in Kir1.1a) are
located close to the cytoplasmic end of the inner (TM2)
transmembrane helix, and mutation of either G148 or
G157 to Ala shifted pKa from 6.6 (wild type) to 7.1 and 7.3,
respectively (673). These two Gly residues are conserved
in all Kir subunits and may contribute to pHi sensitivity in
Kir1.1.
Intracellular acidification causes irreversible collapse of the Kir1.1 channel pore in the absence of extracellular K⫹. The Kir1.1-A177T mutant channel shows a
strong alkaline shift of pHi sensitivity, and the addition of
a K80I mutation led to recovery from acid inhibition in the
absence of extracellular K⫹ (645). The rate of increase of
the Kir1.1 current on switching of extracellular K⫹ from 1
mM (almost no current) to 100 mM (large increase of the
current) showed that the substitution of L160 for Gly
prominently reduced the rate of increase (671). Although
how L160 in Kir1.1 is involved in gating of Kir1.1 has
remained elusive, a recent study has suggested a mechanism underlying pHi-dependent regulation of the channel’s activity via K80 (on TM1) and A177 (on TM2) in
bundle-crossing region (644). Homology modeling of
Kir1.1 shows that these two residues are bridged by H
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bonding in the bundle-crossing region. Mutations of K80
to other residues that are predicted to disrupt the H
bonding not only lower pHi sensitivity of Kir1.1 but also
speeds up recovery of the channel activity by alkalization
from acidification-induced inhibition. Furthermore, the H
bonding seems to mediate slow kinetics of reactivation of
the rundown channel by intracellular PtdIns(4,5)P2 application. Together, the H⫹ bonding between K80 and A177
at the bundle-crossing region may critically control not
only pH-dependent but also PtdIns(4,5)P2-mediated gating of Kir1.1. An additional finding is that this bonding is
also required for the process of aforementioned K⫹-dependent collapse of Kir1.1 pore, a possible phenotype of
dysfunction of the selective filter. Therefore, the H bonding at the bundle-crossing region may be critically involved in the conformational change of the pore region.
3. Intracellular localization
Kir1.1 has an ER retention signal, R-X-R (R368-A369R370), in the COOH terminus (488). Trafficking to the cell
surface is regulated by posttranslational modification via
several intracellular protein kinases including PKA and
SGK. Therefore, a number of physiological signaling pathways modulate the contribution of the Kir1.1 current by
promoting or suppressing its surface expression.
A) SURFACE EXPRESSION ENHANCERS. I) PKA. Kir1.1 channel activity (Po) requires a PKA-dependent phosphorylation process (521), and Kir1.1 has three PKA phosphorylation sites (S44 in the NH2 terminus, S219 and S313 in the
COOH terminus of Kir1.1a). Mutation of any one of these
sites reduces the whole cell current expressed in Xenopus
oocytes by 35– 40% without affecting single-channel conductance. Mutation of any two of the three sites abolishes
visible channel activity (847). Phosphorylation of S44 is
considered to augment cell surface expression of the
Kir1.1 protein (247). PKA-mediated phosphorylation of
S219 and S313 increases channel Po (490) possibly by
enhancing the interaction between the channel and
PtdIns(4,5)P2 (459).
II) SGK. S44 on the NH2 terminus of Kir1.1 can also be
phosphorylated by SGK-1. This increases surface expression
of Kir1.1 and thus enhances the current (877). Since aldosterone stimulates SGK-1 transcription, the Kir1.1 current,
like epithelial Na⫹ channels, could be regulated by this
hormone. However, another study reported that cell surface
expression and current density of Kir1.1 are not altered by
coexpression of SGK-1 alone but are augmented by the
expression of SGK-1 and a scaffolding protein, the Na⫹-H⫹exchanger regulatory factor-2 (NHERF-2) (879). The mechanism of this augmentation is still elusive, because NHERF-2
alone had little effect on Kir1.1 channel current. NHERF-2,
which binds multiple ion transport systems, and Kir1.1 are
shown to be directly associated with each other by a biochemical assay and colocalized at the apical membrane of
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renal epithelial cells by an immunocytochemical study (712,
876). Thus cell surface expression of Kir1.1 may be regulated
by a combination of SGK-1 and NHERF-2 in vivo.
Two recent studies clarified the mechanism underlying the increase of cell surface expression of Kir1.1 by S44
phosphorylation. S44 phosphorylation suppresses the ER
retention signal, R-X-R, which allows delivery of Kir1.1 to
the cell surface (582). S44 phosphorylation may also override another ER location signal (K370 and R371) and
promote surface expression of Kir1.1 (875).
III) PKC. PKC phosphorylates two Ser residues in
Kir1.1a, S4 and S201 (456). Although the phosphorylation
promoted surface expression of Kir1.1a in Xenopus oocytes and HEK cells (456), in renal CCD cells it inhibited
Kir1.1 channels (819) (see sect. VA4). It has been suggested that the suppression of Kir1.1 by PKC is due to the
reduction of membrane PtdIns(4,5)P2, albeit by an unknown mechanism (885).
B) SURFACE EXPRESSION SUPPRESSORS. I) With-no-K (K⫽Lys)
kinases (WNKs). The localization and function of Kir1.1 are
also controlled by other serine-threonine kinases such as
WNK1, WNK3, and WNK4. All of these kinases reduce Kir1.1
current by reducing the amount of Kir1.1 in the plasma
membrane, although through distinct mechanisms (347, 439,
814). Kir1.1 is retrieved from the cell surface by a clathrindependent mechanism via the internalization motif, N-P-X-Y,
in the COOH-terminal region (884). The effect of WNK4 is
attributed to enhanced clathrin-dependent endocytosis and
not its kinase activity (347). Inhibition of Kir1.1 current by
WNK3 is also independent of its catalytic activity (439). On
the other hand, WNK1 seems to inhibit cell surface expression of Kir1.1 by phosphorylating certain residues within the
channel protein (814).
Mutations in either of the WNK1 or WNK4 genes are
associated with pseudo-hypoaldosteronism type II (PHAII:
also referred to as Gordon’s syndrome) which exhibits hypertension with hyperkalemia in an autosomal dominant
form (841). Abnormal modulation of Kir1.1 function by the
mutated WNKs may be linked with these phenotypes. Mutations of WNK4 that cause PHAII also increase its inhibitory
effect on Kir1.1 (884).
II) Ubiquitination. K22 in the NH2 terminus of Kir1.1
is monoubiquitinated (457). Substitution of K22 by Arg
greatly enhanced the Kir1.1 current by increasing its cell
surface expression. Therefore, ubiquitination acts as a
negative regulator of Kir1.1.
4. Physiological functions in cells and organs
A) KIDNEY. Ion concentrations and the volume of extracellular fluids are finely controlled by the kidney. To perform
this task, various types of ion and water transport mechanisms are expressed and functionally coupled together in
the apical and basolateral membranes of renal epithelial
cells (247, 818). For homeostasis of the urine and blood, K⫹
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channels are crucial in regulating not only [K⫹] but also the
concentrations of other ions such as Na⫹ and Cl⫺ (247).
Kir1.1 is considered to play a key role in this regulation.
Kir1.1 is expressed in renal epithelial cells of the thick
ascending limb of the loop of Henle (TAL), the distal convoluted tubule (DCT), and the cortical collecting duct (CCD)
(Fig. 15A) (161, 247, 437). Its isoforms (Kir1.1a-c) are differentially distributed along the nephron (Fig. 15A) (53). Immunohistochemical examinations show that Kir1.1 is specifically localized at the apical membrane of the epithelial cells
(Fig. 15A) (384, 600, 846). Patch-clamp single-channel recording and knockout mouse studies showed that Kir1.1
FIG. 15. Distribution and localization of Kir channels in the kidney and their physiological impact. A: distribution and localization of Kir1.1
channel isoforms and Kir4.1/5.1 heteromeric channels in the kidney. Each Kir1.1 isoform (Kir1.1a, -b, and -c) is expressed in particular parts of the
renal epithelia (top panels). The distal straight tubule is also known as the thick ascending limb of the loop of Henle (TAL). Bottom panels indicate
the cellular location of each type of channel subunit. Kir1.1 channel isoforms are expressed in the apical membranes of renal epithelia. Kir4.1/5.1
heteromer channels occur in the basolateral membrane. B: ion transport mechanisms associated with Kir channels. In TAL cells (left), Kir1.1
channels in the apical membrane are colocalized with the Na⫹-K⫹-2Cl⫺ cotransporter. In distal convolute tubule (DCT) cells (right), the heteromeric
Kir4.1/5.1 channel is colocalized with the Na⫹-K⫹-ATPase in the basolateral membrane. The process known as “K⫹ recycling” involves these K⫹
channels supplying K⫹ to the extracellular K⫹ site of the transporters.
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formed 35-pS and 70-pS K⫹ channels at the apical membrane
of TAL cells as well as 35-pS K⫹ channels in CCD cells (247,
475, 480).
TAL cells in the kidney reabsorb ⬃25% of filtered Na⫹
from the urine mainly via the Na⫹-K⫹-2Cl⫺ cotransporter
(NKCC) located on the apical membrane. K⫹ channels made
up of Kir1.1 are thought to supply K⫹ to the extracellular K⫹
site of NKCC and keep this transporter active (“K⫹-recycling” action; see sect. VB4) (Fig. 15B) (247, 818). The channels also facilitate Cl⫺ absorption via the transporter and
thus promote uptake of NaCl. Na⫹ accumulated in cytoplasmic region would be subsequently carried to the extracellular fluid via Na⫹-K⫹-ATPase at the basolateral membrane.
Thus functional coupling of Kir1.1 and NKCC at the apical
membrane results in driving the unidirectional transport of
Na⫹ from apical to basolateral sides and sustains the activity
of the ATPase. Furthermore, Kir1.1 hyperpolarizes the Em of
TAL cells, which results in accelerating Cl⫺ exit from the
basolateral membrane via Cl⫺ channels (247). It establishes
“the lumen-positive transepithelial potential,” which is the
main driving force for paracellular Na⫹, Ca2⫹, and Mg2⫹
transport from lumen to the side of blood vessels (50, 217).
These functions were confirmed in Kir1.1 knockout mice,
which exhibited impairment of renal NaCl absorption and
“Bartter’s syndrome”-like phenotypes (475, 480) (see sect.
VA6).
The Cl⫺ channel CFTR occurs together with Kir1.1 in
the apical membrane of TAL cells, and it may regulate the
function of Kir1.1 (478). The apical ⬃30-pS K⫹ channels in
TAL cells of wild-type mice are largely suppressed by
physiological levels of ATPi and blocked by glibenclamide
(817). These two properties seem to result from the presence of functional CFTR because they are not observed in
mice lacking CFTR or in mice homozygous for the CFTR
loss-of-function ⌬F508 mutation (478). In addition, when
CFTR associates with Kir1.1, the activity of this K⫹ channel decreases. The effects of CFTR on Kir1.1 are probably
achieved by their interactions with PDZ-domain binding
protein NHERFs (876), which can be abrogated by activation of PKA (478). The physiological roles of the functional association between CFTR and Kir1.1 may be involved in the effects of the antidiuretic hormone arginine
vasopressin (AVP), which increases the apical K⫹ conductance of TAL (173). When urinary flow is augmented,
secretion of AVP from pituitary is suppressed and the
apical K⫹ conductance is reduced by NHERF-mediated
interaction between CFTR and Kir1.1. This process would
limit K⫹ secretion from renal tubules and thus urinary K⫹
loss.
Kir1.1 also seems to be critically involved in ion
transport in CCD cells which play a central role in secretion of K⫹ into the urine (205, 206). K⫹ secretion occurs in
principal cells of the CCD via 35 pS K⫹ channels (191,
192) which are probably made up of Kir1.1, since principle
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cells of Kir1.1 knockout mice did not exhibit this K⫹
current (480).
B) OTHER ORGANS. In situ hybridization showed the
Kir1.1 transcript to be expressed in neurons of the cortex
and hippocampus but not in other areas of the brain (366).
The physiological function of Kir1.1 in the brain remains
unknown.
5. Pharmacology
In addition to the nonspecific Kir channel blockers
Ba2⫹ and Cs⫹, ␦-dendrotoxin (Kd 150 nM) and tertiapin
(Kd 1 nM) inhibit Kir1.1 channels (298, 342, 494) (see
sects. IE and IIIE). Both toxins bind to the external vestibule of the channel pore. The blocking effect of tertiapin
is sensitive to pH: alkaline pH diminishes the dissociation
constant of the interaction between the toxin and Kir1.1
(642). It seems probable that one tertiapin molecule binds
to one functional channel. The binding site is the K⫹conduction pore formed by the linker between the first
and second transmembrane (TM1-TM2) segments (168,
341, 641). Mutations showed that Met at position 13 in
tertiapin interacts with F148 of Kir1.1 (343), a residue
located near the external opening of the pore (404, 573).
The action of tertiapin is specific to Kir1.1 and Kir3.1/3.4
channels with Kd values of 1–2 nM and 8 –10 nM, respectively. Little effect is observed on other types of Kir
channel (298, 342, 494). Of interest is a mutated tertiapin
where H12 and M13 were, respectively, substituted for
Leu and Gln and which showed a greatly reduced affinity
for Kir3.1/3.4 but not for Kir1.1 (see sect. IIIE) making the
mutated tertiapin a relatively specific blocker for Kir1.1
(Kd for Kir1.1: 1 nM versus Kd for Kir3.1/3.2 and Kir3.1/3.4:
274 and 361 nM, respectively) (643).
6. Diseases
The pathophysiological importance of Kir1.1 is shown
by the phenotypes of the Kir1.1 knockout mouse (475, 480)
and its “loss of function disease,” Bartter’s syndrome. Bartter’s syndrome (38) is an autosomal recessive renal tubulopathy that is characterized by hypokalemic metabolic
alkalosis, renal salt wasting, hyperreninemia, and hyperaldosteronism (26, 227, 358, 618, 659). This disease can be
classified into four types (I–IV) (583). Genetic analysis
revealed that type II Bartter’s syndrome results from various mutations in the Kir1.1 gene (167, 247, 729, 755, 809)
(Table 2). In TAL cells, abrogation of Kir1.1 function
greatly diminishes K⫹ supply to NKCC. In addition, the
lumen-positive transepithelial voltage, which drives ⬃50%
of the paracellular reabsorption of Na⫹, is lost (50, 217).
The mutations in Kir1.1 associated with Bartter’s syndrome cause alterations in PKA phosphorylation, pH
sensing, channel gating, proteolytic processing, and sorting to the apical membrane (130, 131, 185, 186, 332, 474,
617, 696, 700, 743). One study demonstrated that R311 is
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crucial for the interaction between Kir1.1 and PtdIns(4,5)P2 and
that the Bartter’s mutations (R311Q/W) could disrupt this
interaction (474), although this idea is disputed (645).
Ablation of the Kir1.1 gene in mice causes a renal
Na⫹, Cl⫺, and K⫹ wasting phenotype (475, 480), which is
consistent with the channel’s crucial role in salt absorption in TAL. These mutant mice also mimicked the phenotypes of Bartter’s syndrome. In the Kir1.1 knock-out
mice, patch-clamp recording in either TAL or CCD cells
failed to show either 35- or 70-pS K⫹ channels in the
apical membrane (479, 480). Like Bartter’s syndrome patients, knockout mice showed hypokalemia and an excess
of K⫹ in their urine. The mechanism of this phenotype
was initially elusive, since Kir1.1 had been proposed to
provide the major K⫹-secretory pathway in renal tubules
(190, 191, 215, 247). Dysfunction of the channel was expected to cause hyperkalemia and, indeed, infants with
the type II Bartter’s genotype often exhibited transient
hyperkalemia (84, 180). A recent study clarified the mechanism (34): in mice Kir1.1 knockout reduces K⫹ absorption from urine in the loop of Henle by impairing function
of NKCC2 in TAL cells but sustains K⫹ secretion by
continuous K⫹ efflux via maxi-K⫹ (namely, large-conductance Ca2⫹-activated K⫹ or BK) channels in the late distal
tubule.
B. Kir4.x and Kir5.1
1. Historical view and molecular diversity
Kir4.1/KCNJ10 was initially and independently identified from a brain cDNA library by several groups and
assigned different names such as BIR10 (56), KAB-2 (763),
BIRK-1 (64), and Kir1.2 (718). In situ hybridization histochemistry showed that Kir4.1 was predominantly expressed in glial cells of the brain (763) (see sect. VB4).
Therefore, Kir4.1 has been considered to form a glial K⫹
conductance that is involved in so-called “K⫹-buffering”
which plays a pivotal role in controlling neuronal function
(400, 563) (see sect. VB4). The salmon homolog of mammalian Kir4.1 is known as Kir4.3 (395). Kir5.1/KCNJ16 was
first described as BIR9 (56).
The deduced amino acid sequence of Kir4.1 has 53,
43, and 43% identity with Kir1.1, Kir2.1, and Kir3.1, respectively, whereas Kir5.1 shares 39, 50, and 40% identity.
When Kir4.1 is expressed alone in a heterologous expression system, it forms a tetramer and elicits a K⫹ current
(616). In native tissues, the channel also exists as a tetramer (254, 348). When Kir5.1 is expressed alone in a
heterologous expression system, it is nonfunctional, but
coexpression with Kir4.1 forms a functional heteromer
whose biophysical properties and pHi sensitivity differ
from those of the Kir4.1 homomer (see sect. VB2) (56, 616,
765, 793). The Kir4.1/5.1 heteromer also exists as a tetramer (616), and it has been detected in various tissues
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including the kidney and the brain. The Kir5.1 homomer is
expressed in the cytoplasm of cells such as fibrocytes in
the cochlea where it does not seem to form functional
channels on the cell membrane surface (see sect. VB3).
Kir4.2/Kir1.3/KCNJ15 was first isolated from a human
kidney cDNA library (718). Another group identified the
Kir4.2 gene by sequencing the Down syndrome chromosome region 1 on subband q22.2 of chromosome 21 (214).
Kir4.2 has 62% identity to human Kir4.1. Kir4.1 has a
Walker type A ATP-binding cassette in its COOH terminus
(763). Kir4.2 does not possess this motif (609). Kir4.2
forms a functional channel when expressed alone in heterologous expression systems (609, 615). It also forms a
functional heteromer when coexpressed with Kir5.1 with
a different single-channel conductance and greater cell
surface expression resulting in increased whole cell current (609, 615) (see sect. VB2). Kidney, liver, embryonic
fibrocytes, and microvascular endothelial cells are reported to express Kir4.2 (124, 425, 609, 718).
2. Pore function and structure
A) KIR4.1 AND KIR5.1. There have been differing reports of
the single-channel conductance of the Kir4.1 homomer
and Kir4.1/5.1 heteromer channels, possibly because they
can show multiple subconductance states. The conductance of the Kir4.1 homomer ranges between 20 and 40
pS, while that of the Kir4.1/5.1 heteromer ranges between
40 and 60 pS (476, 616, 763, 765, 868). The Kir4.1 homomer
exhibits intermediate inward rectification, whereas the
rectification profile of the Kir4.1/5.1 heteromer is much
stronger. The residues of the D/N site are Glu in Kir4.1
(E158) and Asn in Kir5.1 (N161) (see sect. IC1). In excised
membrane patches, even high concentrations of spermine
left 10 –15% residual conductance of the Kir4.1 homomer.
This apparent insensitivity may be due to partial permeation of spermine via the Kir4.1 channel pore because
philanthotoxin, a polyamine with a bulky tail, more completely blocked outward current through Kir4.1 (399). The
E177 residue in the proximal region of the COOH terminus just under the TM2 region in Kir4.1 is required for
heteromer formation with Kir5.1 (388).
An even more remarKABle difference between homomeric Kir4.1 and heteromeric Kir4.1/5.1 channels is their
different sensitivity to changes in pHi. In the physiological
range of pHi between 6.5 and 8.0, the channel activity of
the Kir4.1 homomer is inhibited by acidification with a
pKa of ⬃6 (56, 616, 765, 868). In contrast, over the same
pHi range, the activity of the heteromeric channels is
dramatically suppressed by only a slight intracellular
acidification and enhanced by alkalization with a pKa of
⬃7.5 (765, 868). Several residues in Kir4.1, E158 in the
TM2 region (845), K67 in the cytoplasmic NH2-terminal
region (868), and H190 in the COOH-terminal region (76)
are reported to be involved in the pHi sensitivity of not
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only homomeric Kir4.1 but also heteromeric Kir4.1/5.1
channels. The mechanism underlying the modification of
pHi sensitivity of Kir4.1 by Kir5.1 remains elusive.
Of all Kir channel subunits, Kir4.1 has been found to
have the strongest affinity for PtdIns(4,5)P2 (146).
The activity of the heteromeric Kir4.1/5.1 channel is
enhanced by Na⫹
i (665). This property seems to be exerted by D205 in the cytoplasmic COOH terminus of
Kir5.1, since the corresponding residue confers Na⫹ sensitivity to both Kir3.2 and Kir3.4 ion channels (D226 and
D223, respectively) (see sect. IIIB). This residue is not
conserved in Kir4.1 (N202), and the Kir4.1 homomer channel is not sensitive to Na⫹
i .
B) KIR4.2. Kir4.2 displays a different biophysical profile. The single-channel conductance of the homomer of
Kir4.2 expressed in Xenopus oocytes is ⬃25 pS. The
amino acid sequence and rectification profile of Kir4.2 are
similar to Kir4.1, but Kir4.2 is more sensitive to pHi
(Kir4.2: pKa ⫽ 7.1, Kir4.1: pKa ⫽ 6). Coexpression with
Kir5.1 has only minor effects on the pHi sensitivity of
Kir4.2 (Kir4.2/5.1: pKa ⫽ 7.6). The heteromeric channel
exhibits a larger unitary conductance, ⬃54 pS (615). The
Kir4.2/5.1 heteromer is more sensitive to Ba2⫹ block than
the Kir4.2 homomer (609). Interestingly, mutation K66M
in the NH2-terminal region of Kir4.2 almost abrogated its
pHi sensitivity (615). This is consistent with the mutation
of the corresponding residue (K80), which impairs pHi
sensitivity in Kir1.1 (88, 161, 520, 696) (see sect. VA2). It is
however impossible to explain the difference between the
pHi sensitivity of Kir4.1 and Kir4.2 solely on the role of
this Lys residue, because the intracellular COOH-terminal
parts of these subunits also appear to be responsible for
the difference (615). The crucial residues in this region
have not yet been identified.
3. Intracellular localization
A) KIR4.X AND KIR5.1. I) Localization in cell membranes.
A) Epithelial tissues. When Kir4.1 is expressed in epithelial MDCK cells, it is sorted to the basolateral membrane
(764) by association with a PDZ-protein membrane-associated guanylate kinase with inverted domain structure 1
(MAGI-1a long isoform) (766). The interaction occurs between the fifth PDZ domain of MAGI-1a and the PDZbinding motif in the COOH terminus of Kir4.1. The phosphorylation of S377 in the PDZ-binding motif of Kir4.1
disrupts this interaction and moves Kir4.1 from the basolateral membrane to perinuclear compartments in the
transfected MDCK cells (766). When Kir5.1 is expressed
alone in MDCK cells, it remains in the intracellular compartment, but coexpression with Kir4.1 targets it to the
basolateral membrane (764). Consistent with this pattern,
immunohistochemical, biochemical, and patch-clamp
studies have demonstrated that Kir4.1 and Kir5.1 form
heteromers in the basolateral membrane of some renal
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epithelial cells (325, 476, 765, 793) (Fig. 15A) (see sect.
VB4).
On the other hand, in the stomach, homomeric Kir4.1
is specifically distributed at the apical membrane of parietal cells (193, 364). Careful analysis revealed that the
channel was located at the microvilli of the apical membrane surface but not in the tubulovesicles. The mechanism for determining such specific localization of Kir4.1 in
parietal cells remains unknown.
The distribution pattern of Kir4.1 and Kir5.1 in the
cochlea of the inner ear, which contains a variety of
epithelial cells, is unique (260). Each subunit is expressed
in the cochlear lateral wall but in different components.
Kir4.1 is found in epithelial tissue in the stria vascularis
(257). High-resolution immunolabeling shows that this
channel is specifically expressed on the apical side of
intermediate cells (14, 255). Kir5.1 is expressed in fibrocytes of the spiral ligament and localized in intracellular
compartments rather than on the membrane surface
(255).
B) Glial cells. In the central nervous system (CNS)
and retina, Kir4.1 and Kir5.1 subunits are predominantly
expressed in astroglial cells. They form both Kir4.1 homomers and Kir4.1/5.1 heteromers, each of which exhibits
a unique distribution pattern in distinct cell types.
Kir4.1 is widely distributed in astrocytes of the CNS
in both brain and spinal cord (262, 348, 449, 561, 594, 624,
763). Kir5.1 is also expressed in brain astrocytes (45, 254,
454). Both subunits are present in Müller cells of the
retina (311, 312, 549). In the cortex of the brain, both
Kir4.1/5.1-heteromeric and Kir4.1-homomeric channels
are detected in astrocytic processes surrounding synapses (perisynaptic processes), whereas only the heteromer is present in processes attached to pia and
blood vessels (end feet) (Fig. 16A). Retinal Müller cells
express the Kir4.1 homomer at the end feet which face
vitreous humor and blood vessels, and the Kir4.1/5.1
heteromer in the perisynaptic processes (311) (Fig.
16B).
The machinery responsible for sorting each channel
to different membrane macrodomains in astroglial cells of
either brain or retina have not yet been completely clarified. Several studies suggest that the dystrophin-associated protein complex plays a role in determining localization of Kir4.1 in the end feet. Mutant mice lacking dystrophin (called “mdx3Cv mice” or knockout mice) show
reduced Kir4.1 expression in the end feet of Müller cells
(101, 116) and display a diffuse localization pattern in
isolated cells (187). On the other hand, in mdx3Cv mice,
the expression of Kir4.1 at the perisynaptic membrane
surface is unaffected, suggesting that other mechanisms traffic Kir4.1 channels to this area.
Satellite cells are another subset of glial cells which
also harbor Kir4.1 channels (256, 807, 808, 903). These
cells surround the cell bodies of various ganglion neurons
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FIG. 16. Localization of astroglial Kir channels. A: brain astrocytes. Astrocytes harbor and localize homomeric Kir4.1 channels and heteromeric
Kir4.1/5.1 channels in particular parts of their membrane: both the homomer and the heteromer are present in perisynaptic processes, and the
heteromer is situated alone in the end feet. B: retinal Müller cells. These cells express the heteromeric Kir4.1/5.1 channel at perisynaptic sites and
the homomeric Kir4.1 channel at perivascular processes and in end feet. In both the astrocytes and Müller cells, the Kir channels coexist with the
astroglial water channel aquaporin 4 (AQP4) in the same membrane domains (A and B).
with multiple layers of myelin sheath. Kir4.1 is expressed
specifically on the sheath membrane surrounding cochlear spiral ganglions, trigeminal ganglions, and superior
cervical ganglions (256, 807, 808, 903) (see sect. VB4 for
functional roles).
II) Localization in membrane microdomains. Kir4.1 is
concentrated in particular microdomains of the astroglial
membrane known as DRMs (see sect. IC2) (259). Biochemical and immunohistochemical assays found that Kir4.1
proteins were abundant in noncaveolar DRMs in the brain
as well as in cultured astrocytes and when exogenously
expressed in HEK293T cells. In HEK293T cells, depletion
of membrane cholesterol by methyl-␤-cyclodextrin
(M␤CD) resulted in loss of the association between Kir4.1
and DRMs and channel activity (Fig. 17A). Therefore,
localization of Kir4.1 in M␤CD-sensitive noncaveolar
DRMs seems to be mandatory for channel function. The
astroglial water channel AQP4 also occurs in DRMs, but
these microdomains are M␤CD insensitive and thus suggested to contain little cholesterol (Fig. 17B). Immunolabeling showed that these different DRMs occurred in
close proximity in the astrocytic membrane (Fig. 17,
Physiol Rev • VOL
C–E), and such spatial organization may be involved in
the coupling of K⫹ and water transport (see sect. VB4).
B) KIR4.2. When Kir4.2 is expressed alone in heterologous
systems, it elicits a relatively small current (131, 609, 718).
This seems to be due to the intracellular localization of a
large population of the channel. The intracellular localization of Kir4.2 may result from two independent mechanisms,
which is suggested by the following observations. First,
mutation of a single residue (K110N) in the extracellular
domain between the first transmembrane helix and the pore
helix/selectivity filter dramatically increases the membrane
current (131). Since there is no difference in Po between
K110N-Kir4.2 and wild-type Kir4.2 channels (131), this mutation should facilitate the trafficking of the channel protein
to the plasma membrane. Second, deletion or mutation of a
potential Tyr phosphorylation motif, K-(X)2–3-E-(X)2-Y, in
the COOH terminus also augmented the membrane current,
which may also be due to facilitation of trafficking of the
channel to the membrane surface (610). However, it is not
known how K110N and the phosphorylation motif regulate
the trafficking of Kir4.2 protein. Of note is the fact that none
of the PDZ-domain binding motif (S-X-V), the dileucine tar-
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339
FIG. 17. Kir4.1 and AQP4 exist in different membrane microdomains. A: the subcellular localization of Kir4.1 (top panel) and AQP4 (bottom
panel) in the brain. Mouse brains treated with 1% Triton X-100 were fractionized on a discontinuous sucrose gradient. Each fraction was collected
from the top of the gradient and immunoblotted with specific antibodies against the proteins indicated on the left. The DRM fractions concentrate
Kir4.1 and AQP4. Note that Kir4.1 is also found in non-DRM compartments. Western blotting reveals monomer (x1), dimer (x2), and tetramer (x4)
forms of Kir4.1. B: DRM localization of Kir4.1 and AQP4 expressed in HEK293T cells and their cholesterol dependence. The fractions obtained from
cells transfected with Kir4.1 (top panels) or AQP4 (bottom panel) cDNA were probed with anti-Kir4.1 or anti-AQP4 antibody. Control, nontreated
cells; M␤CD, cells treated with M␤CD, a reagent that depletes membrane cholesterol (4 mM, 1 h). Two bands show glycosylated (top) and
unglycosylated (bottom) Kir4.1. M␤CD treatment disrupts DRM association of Kir4.1 but not that of AQP4. C: localization of AQP4 and Kir4.1 in in
vivo astrocytes. Cryosections from mouse brain were treated with anti-Kir4.1 antibody, followed by incubation with Alexa488-labeled secondary
antibody and Alexa568-labeled anti-AQP4 antibody. a and b show images obtained by focusing at different depths. Solid arrowheads indicate
“line-shaped” (a) and arrows (b) indicate “clustered” labeling of AQP4 (red) and Kir4.1 (green). In b, the edge of the wall of a blood vessel is visible
(open arrowheads), confirming that the microscope was focused on the surface of the wall. D: a high-magnification image of the boxed region in
Cb. AQP4 and Kir4.1 signals are not colocalized but can exist in close vicinity (arrowheads). [A–D from Hibino and Kurachi (259), with permission
from Wiley-Blackwell Publishing.] E: a schematic representation of the localization of microdomains which contain Kir4.1 and AQP4 on the astroglial
membrane. Kir4.1 and AQP4 are expressed respectively in M␤CD-sensitive and M␤CD-resistant DRMs, which are occasionally in close proximity.
geting motif [D/E-(X)3–5-L-L], and the ER retention signal
(R-X-R), all of which are found in the COOH terminus of
Kir4.2, seem to be involved in its intracellular localization
(610).
Kir4.2 is expressed at the basolateral membrane of
renal epithelial cells when it forms a heteromer with
Kir5.1 (476).
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4. Physiological functions in cells and organs
A) KIR4.X AND KIR5.1. I) Kidney and stomach. In the
kidney, the epithelium of DCT plays an important role in
reabsorption of Na⫹ (247). This is mediated by epithelial
Na⫹ channels at the apical membrane (105) coupled with
the activity of the basolateral Na⫹-K⫹-ATPase (Fig. 15B)
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HIBINO ET AL.
(see sect. VA4). K⫹ channels in the basolateral membrane
are key players in this process because they supply K⫹ to
the extracellular K⫹ site of the Na⫹-K⫹-ATPase to maintain its activity. This action is called “K⫹ recycling” (Fig.
15B). Consistently, blocking the basolateral K⫹ conductance of renal epithelial cells with Ba2⫹ inhibits vasopressin-stimulated Na⫹ transport (687).
Kir channels with a unitary conductance of 37– 40 pS
have been identified in the basolateral membrane of DCT
in rabbit (768) and mouse (476). In the latter case, the
channels are inhibited by intracellular acidification with a
pKa of ⬃7.6 (476). It was shown that these channels
consisted of heteromers of Kir4.1/5.1 and/or Kir4.2/5.1
(Fig. 15B) (476, 765, 793). Their pHi sensitivity strongly
suggests that they act as pHi sensors and are involved in
pHi-dependent regulation of ion transport in the kidney.
In the DCT, either Kir4.1 or Kir4.2 may directly interact with a Ca2⫹-sensing receptor in the basolateral membrane (284). In heterologous expression systems, this interaction significantly reduces the current elicited by either subunit. Although the physiological role of this
interaction is unknown, it may be involved in control of
salt homeostasis in the kidney.
Recently a type of K⫹ channel with a unitary conductance of ⬃40 pS on the basolateral membrane of principal
cells in CCD has been shown to be made up of a Kir4.1/5.1
heteromer (425), which may there play a similar role to
the channels in the DCT.
In gastric parietal cells, the Kir4.1 homomer is colocalized with a H⫹-K⫹-ATPase at the apical membrane
(193, 364). This pump secretes protons in exchange for
K⫹. Thus Kir4.1 may be involved in maintaining the activity of the H⫹-K⫹-ATPase by providing K⫹ for the pump,
which is similar to the possible role of this channel in
functioning Na⫹-K⫹-ATPase in renal epithelial cells (see
above). In support of this idea, the application of Ba2⫹ to
the parietal cells diminished their proton secretion (193).
II) Cochlea. The cochlea of the inner ear contains
two extracelluar fluids, perilymph and endolymph. The
ionic composition of perilymph is almost identical to
usual extracellular fluid. On the other hand, the endolymph contains ⬃150 mM K⫹ and possesses a highly
positive potential of ⬃⫹80 mV with reference to either
blood or perilymph. This potential is referred to as the
“endocochlear potential” (260, 812, 813). The unique ionic
and voltage environment of the endolymph is essential for
proper audition.
The endocochlear potential is thought to be maintained by K⫹ circulation from perilymph to endolymph
through the cochlear lateral wall. The lateral wall is made
up of two components, the stria vascularis and the spiral
ligament. The application of Ba2⫹ to the stria vascularis
suppressed the endocochlear potential (499). Kir4.1 is the
only Kir channel subunit expressed in the stria vascularis
(257). It is likely that Kir4.1, which is specifically exPhysiol Rev • VOL
pressed at the apical membrane of intermediate cells in
the stria (14), elicits a large K⫹ diffusion potential across
this membrane and forms a significant fraction of the
endocochlear potential (260, 572, 762). In support of this
concept, Kir4.1 knockout mice are deaf and exhibit an
endocochlear potential of almost 0 mV with an ⬃50%
reduction of [K⫹] in the endolymph (500).
In the spiral ligament, fibrocytes that are bathed in
perilymph express Kir5.1 homomeric channels (255). The
majority of these channels seem to be localized in intracellular compartments. But since perilymphatic perfusion
of Ba2⫹ slightly increased the endocochlear potential, it is
possible that a small population of Kir5.1 homomers on
the plasma membrane of the ligament could negatively
regulate K⫹ circulation (255, 498).
III) Glial cells. Brain astrocytes and retinal Müller
cells project their processes not only to synapses and
soma of neurons but also to blood vessels, pia matter, and
vitreous humor (Fig. 16). These astroglial cells play various roles in the control of synaptic functions. One of the
most important tasks of astrocytes is to maintain the ionic
and osmotic environment in the extracellular space. They
conduct a large Kir current involved in transporting K⫹
from regions of high [K⫹]o, which results from synaptic
excitation, to those of low [K⫹]o (400, 563). As an excess
[K⫹]o would interfere with normal signaling of neurons by
depolarizing them continuously (563, 599), its rapid clearance by astrocytes is essential for the proper function of
synapses. This polarized transport is referred to as “spatial buffering of K⫹” in the brain, and “K⫹-siphoning” in
retinal Müller cells. To achieve these “K⫹ buffering” functions, astroglial cells use Kir channels locating at specific
membrane domains.
Kir4.1 is abundantly expressed in astroglial cells and
forms a major part of their basal K⫹ conductance in brain,
spinal cord, and retina (262, 311, 312, 348, 449, 549, 561,
594, 624, 763). Consistently, the knockout or the knockdown of the Kir4.1 gene causes a large increase of input
resistance, decrease of K⫹ conductance, and/or depolarization of Eres in astroglial cells (135, 381, 561, 594). The
phenotypes of Kir4.1 knockout mice highlight the significance of Kir4.1 in astroglial K⫹ buffering. In the retina of
mutant mice, the slow PIII response of the light-evoked
electroretinogram (ERG) is absent (381). Since this response is thought to be generated by a light-evoked decrease in [K⫹]o in the distal portion of the retina, it can be
attributed to K⫹ flux from the distal to the proximal end
of the retina in response to hyperpolarization of the photoreceptors in which Kir4.1 plays a role. The hippocampus in Kir4.1 knockout mice also shows an impaired K⫹
uptake in response to neuronal excitation (135), which
suggests that Kir4.1 is also involved in K⫹ buffering in the
brain.
Homomeric Kir4.1 and heteromeric Kir4.1/5.1 channels display distinct distribution patterns in astroglial
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STRUCTURE AND PHYSIOLOGICAL FUNCTION OF Kir CHANNELS
cells of the brain and retina (254, 311). In cortical astrocytes of the brain, both heteromer and homomer channels
are located in the perisynaptic processes, whereas only
heteromeric channels are expressed at the end feet (Fig.
16A). This suggests that, in cortical astrocytes, K⫹ is
taken up through heteromeric and homomeric channels
and excreted via heteromeric channels. On the other
hand, the astrocytes in the thalamus and hippocampus,
with abundant synapses, express predominantly the
Kir4.1 homomer. In the case of the retina, Müller cells
harbor Kir4.1/5.1 at their perisynaptic processes and
Kir4.1 at their end feet facing vitreous humor and small
blood vessels (Fig. 16B). This suggests that the former
takes up K⫹ and the latter secretes K⫹.
The physiological significance of these differences in
different organs, tissues, and regions is not known. However, it may offer unique characteristics to each cell. The
major difference between the Kir4.1 homomer and the
Kir4.1/5.1 heteromer is their sensitivity to pHi (see sect.
VB2). Astrocytes express various ion transport systems
including a Na⫹-HCO3⫺ cotransporter. The activation of
this transporter induces intracellular alkalinization and
membrane hyperpolarization because it takes up one Na⫹
and two or three HCO3⫺ (12, 49). Excess extracellular K⫹
due to synaptic activation accelerates the transporter by
depolarizing the astrocytic membrane. The resulting intracellular alkalinization would enhance the activity of heteromeric Kir4.1/5.1 channels and facilitate the uptake of K⫹. In
contrast, astrocytes in the thalamus and hippocampus,
which mainly bear the Kir4.1 homomer, express little Na⫹HCO3⫺ cotransporter (692). The physiological role of heterogeneity of Kir channels expressed in the perisynaptic processes in brain astrocytes is unclear.
In the perivascular processes of brain astrocytes and
retinal Müller cells, Kir channels are colocalized with the
water channel AQP4 (254, 311, 548, 549) (Fig. 16). Deletion of the AQP4 protein slows the rate of astrocytic K⫹
uptake in mice (602). ␣-Syntrophin knockout mice, which
retain Kir4.1 but lack AQP4 in astroglial perivascular processes, show delayed clearance of excess K⫹ induced by
neuronal stimulation (10). These observations reflect a
link between K⫹ and water transport. Kir4.1 and AQP4 are
localized in distinct noncaveolar microdomains as well as
M␤CD-sensitive and -resistant DRMs, which occur in
close vicinity (Fig. 17) (259). This apposition of Kir4.1DRM and AQP4-DRM may underlie the effective cotransport of K⫹ and water in the astrocytes.
Kir4.1 may also be functionally coupled to glutamate
transporters. Kir4.1 occurs together with the glutamate
transporters GLT-1 and GLAST in astroglial cells (135,
593). Knockdown and knockout of the Kir4.1 gene resulted in impairment of glutamate uptake by astrocytes
(135, 398). Astrocytic glutamate transport, when actively
working, depolarizes the membrane. Since functional expression of Kir4.1 would hyperpolarize the membrane,
Physiol Rev • VOL
341
this could facilitate glutamate influx from the extracellular space via the transporter.
In early postnatal stages, the spinal cord expresses
Kir4.1 in both gray and white matter (132, 562). Because
the Kir4.1 channel is not expressed in neurons, this observation indicates that Kir4.1 occurs in oligodendrocytes
as well as in astrocytes. Kir4.1 labeling becomes weaker
in white matter and predominantly confined to gray matter around 10 –20 days after birth (132, 348), suggesting
that the expression of Kir4.1 shifts from both astrocytes
and oligodendrocytes to astrocytes during postnatal development. Kir4.1 expression may therefore be necessary
for early development of oligodendrocytes (see also sect.
VB6 and phenotypes of Kir4.1-null mice, Ref. 562). A possible role of Kir4.1 during development would be to hyperpolarize the membrane and form the driving force for
Ca2⫹ influx via Ca2⫹-permeable channels as the case in
the linkage between Kir2.1 and differentiation of myoblasts (387) (see sect. IID4). Kir4.1 has been found in
oligodendrocytes of adult animals, in the optic nerve and
brain where Kir4.1 is detected in the oligodentrocyte cell
bodies and rarely in their processes (myelin sheaths)
(352, 624). This pattern of distribution implies that Kir4.1
in adult oligodendrocytes is not involved in K⫹ buffering
(see sect. VB6).
As described in section VB3, the Kir4.1 homomer is
expressed on the myelin sheath membrane of satellite
cells wrapping ganglion neurons. Like in astroglial cells,
Kir4.1 in this region is expected to absorb excess extracellular K⫹ induced by excitation of ganglions (“K⫹ buffering”) (256). Silencing of Kir4.1 by RNAi technology clarified the physiological role of Kir4.1 in satellite cells of rat
trigeminal ganglions (808) where Kir4.1 disruption decreased the nociceptive threshold and led to spontaneous
and mechanically evoked facial painlike behavior in freely
moving rats. A possible mechanism is that attenuation of
K⫹ buffering by satellite cells increases K⫹ concentration
in the extracellular space of the ganglions and enhances
of their excitability.
B) KIR4.2. A recent study has demonstrated that Kir4.2
is involved in integrin-dependent migration of mouse embryonic fibrocytes and human microvascular endothelial
cells (124). This effect seems to depend on the intracellular polyamine content that is controlled by interaction
between ␣9-integrin and spermidine/spermine acetyltransferase (SSAT). The interaction causes catabolism of
spermidine and spermine, which enhances K⫹ efflux
through Kir4.2 by reducing inward rectification. The increase of K⫹ efflux facilitates the formation of the leading
edge of migrating cells by an as yet unidentified mechanism. A mutant ␣9-integrin which lacks the SSAT binding
site, overexpression of a nonfunctional mutant of SSAT,
and block of Kir4.2 by Ba2⫹ or siRNA techniques, all
suppress integrin-dependent migration of these cells
(124).
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Kir4.2 has also been identified in mouse liver (609)
where its function is unknown.
5. Pharmacology
Ba2⫹ and Cs⫹ block Kir channels that contain Kir4.1
and Kir4.2 (388, 763, 765) (see sect. IE). In addition to
these nonselective Kir channel blockers, tricyclic antidepressants (TCAs) such as nortriptyline, amitriptyline, desipramine, and imipramine block Kir4.1 channels (Fig. 18)
(747). The inhibition of Kir4.1 by nortriptyline depends on
the voltage difference from EK, with greater potency
against outwardly flowing currents. Selective serotonin
reuptake inhibitors (SSRIs), such as fluoxetine, sertraline,
and fluvoxamine, also block Kir4.1 current, in a voltageindependent manner (585) (Fig. 18B). Interestingly, these
drugs have little effect on the K⫹ current carried by the
neuronal Kir2.1 channel and by Kir1.1 channel (Figs. 18A).
The block of astroglial Kir channels by TCAs and SSRIs
may contribute to the therapeutic and/or adverse actions
of these compounds. Our recent study has further demonstrated that these antidepressants may interact with
T128 and E158 residues facing the central cavity of the
pore of Kir4.1 (199).
6. Diseases
The phenotypes of Kir4.1 knockout mice suggest that
Kir4.1 channels are relevant to a variety of pathophysiological conditions. First, Kir4.1 knockout mice showed
loss of the slow PIII response in the light-evoked ERG
(381). Second, targeted disruption of Kir4.1, which is
abundantly expressed in oligodendrocytes during early
postnatal development (348), induced marked motor impairment in mice (562). The cellular basis of this phenotype appears to be hypomyelination in the spinal cord
accompanied by severe spongiform vacuolation, axonal
swelling, and degeneration (562). Third, Kir4.1 knockout
mice are deaf because of a lack of the endocochlear
potential and loss of endolymphatic K⫹ (500). In addition,
anion transporter SLC26A4-null mice, which are a model
for Pendred syndrome with thyroid goiter and deafness,
have severe hearing loss probably due to loss of Kir4.1
channels from the cochlear stria vascularis (822).
It has been recently suggested that abnormality of
Kir4.1 function in the brain could induce epilepsy. Examination of the membranes of glial cells obtained from
patients with intractable epilepsies sometimes demonstrates a near-complete lack of Kir conductance (58),
reduction of the inward rectification (267), and an impaired ability for K⫹ clearance (200, 331). Two early
studies reported that mutations of the Kir4.1 gene might
be involved in epilepsy and seizure. The missense variation (T262S) was found to be a candidate for causing the
susceptibility to seizure of DBA/2 mice (170). Linkage
analysis identified a mutation in the human Kir4.1 gene
R271C that might be associated with generalized seizures
in humans (72) (Table 2). But equivalent mutations of
Kir4.1 expressed alone or with Kir5.1 in a heterologous
2⫹
FIG. 18. Effects of antidepressants on Kir4.1 currents. A, top panel: response of Kir4.1 currents to nortriptyline (100 ␮M) and Ba
(3 mM).
Whole cells currents of HEK293T cells transfected with Kir4.1-cDNA were voltage-clamped at EK (⫺40 mV) and stepped to EK ⫾ 70 mV for 300 ms
each. Arrowheads indicate basal current level at EK. Macroscopic current traces during control (a), application of nortriptyline (b), washout (c), and
application of Ba2⫹ (d) are shown. Bottom panel: response of Kir2.1 currents to nortriptyline (100 ␮M) and Ba2⫹ (3 mM) recorded under identical
conditions. In both experiments, the cells were bathed in extracellular solution which contained 30 mM [K⫹]. [From Su et al. (747).] B: a comparison
of the effects of various antidepressants on Kir4.1 currents. The response to the drugs (100 ␮M each, except for 30 ␮M sertraline) was evaluated
as the current ratio (IDrug/IControl) where the current was evoked by a voltage step to ⫺110 mV. Extracellular solution contained 30 mM K⫹. [Data
derived from Ohno et al. (585) and Su et al. (747).]
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STRUCTURE AND PHYSIOLOGICAL FUNCTION OF Kir CHANNELS
system elicited K⫹ currents with properties almost identical to those of wild-type channels (704). A recent genetic
analysis identified several mutations in the Kir4.1 gene of
patients afflicted by seizures, sensorineural deafness,
ataxia, mental retardation, and electrolyte imbalance
(SeSAME syndrome) (52, 693) (Table 2). The organs exhibiting the phenotypes (brain, ear, and kidney) correspond to the distribution of Kir4.1. In vitro electrophysiological assay has revealed that some mutations found in
the disease, R65P and G77R, result in large reduction of
Kir4.1 current (52). R65P is expected to involve the
PtdIns(4,5)P2 binding site and therefore cause loss of
channel activity (693). Conditional knockout mice that
lack Kir4.1 in astroglial cells display severe ataxia, and
stress-induced seizures (135) and conventional ablation
of the Kir4.1 gene cause deafness in mice (500), which
supports the idea of the pathophysiological relationship
of Kir4.1 channel impairment with epilepsy and hearing
disorders.
A physiological experiment demonstrated that Kir4.1
could be involved in protecting astroglial cells from severe damage during brain injury (132). In the spinal cord,
the application of a 30% hypotonic solution causes swelling of the astroglial cell body, whereas no swelling is
detected in their end feet. Hypotonic swelling of astrocyte
end feet was detected in Kir4.1-knockout mice and also
when Ba2⫹ was applied. The mechanism by which Kir4.1
prevents the swelling of astrocytic process is unknown.
Spinal cord injury sometimes causes a hypotonic condition that results in fluid accumulation and thus edema.
Also, astroglial Kir channel expression is downregulated
after ischemia of either the rat retina (603), which may
occur in the CNS as well. Impaired K⫹ buffering by Kir4.1
during these pathological conditions may be involved in
the formation of edema.
Finally, the Kir4.2 gene is located close to the locus of
the Down syndrome chromosome region-1 (DCR1) (see
sect. VB1), the chromosome 21 trisomy that causes dysmorphic features, hypotonia, and psychomotor delay
(214). This suggests a possible linkage between dysfunction of Kir4.2 and the disease, although the amount of
Kir4.2 protein expressed in the brain of Down syndrome
patients was comparable to that in the normal human
brain (169).
C. Kir7.1
1. Historical view and molecular diversity
Kir7.1 was independently identified by three groups
(140, 393, 604). Its sequence is quite different from those
of other types of Kir channel and shares only ⬃38% homology with its closest relative, Kir4.2. Only one isoform
has been isolated so far. Kir7.1 is expected to function as
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343
a tetramer. No reports support heteromeric assembly of
Kir7.1 with other subunits.
2. Pore function and structure
Kir7.1 expressed in heterologous expression systems
elicits inwardly rectifying K⫹ currents with some unique
properties. First, the single-channel conductance of Kir7.1
is extremely small, ⬃50 fS (393). Second, the sensitivity of
the channel to Ba2⫹ and Cs⫹ is very low with IC50 values
of ⬃1 and ⬃10 mM, respectively. These IC50 values are
⬃10 times greater than those for other types of Kir channel (393). Third, inward rectification of Kir7.1 is independent of [K⫹]o (140, 393). The residue responsible for these
unusual characteristics in the pore region is M125 (Arg in
all other Kir subunits). Replacement of this amino acid
with the otherwise conserved Arg dramatically increased
the single channel conductance of Kir7.1 ⬃20 fold and
Ba2⫹ sensitivity ⬃10 fold as well as re-establishing a
rectification profile similar to other Kir channels (140,
393). The equivalent residue in Kir2.1 (R148) has been
reported to be involved in determining single channel
conductance and sensitivity to blockers (670). The residue in this position may be important for the pore structure of Kir channels.
Like other Kir channel subunits, Kir7.1 is activated by
PtdIns(4,5)P2. However, its binding affinity is weaker than
Kir2.1 and other subunits (661). Kir7.1 can also be activated by PtdIns(3,4,5)P3 but not by PtdIns(3,4)P2 (661).
Kir7.1 expressed in native bovine retinal pigmental
epithelia (RPE) (see sect. VC4) and in Xenopus oocytes is
sensitive to pHi (291, 864). The response is bell-shaped:
maximum activity of the channel is observed at pHi 7.0,
and the current is attenuated by either acidification or
alkalinization (291). Substitution of His at position 26 in
the cytoplasmic NH2 terminus with Ala or Arg results in
continuous activation of Kir7.1 at alkaline pHi (⬎pH 8)
and in increase of the sensitivity to proton-induced inhibition. Of note, only Kir2.1 has His at the same position
where it is involved in PtdIns(4,5)P2 binding (474); H26 in
Kir7.1 does not share this character (291).
3. Intracellular localization
Kir7.1 is expressed on the membrane of various types
of epithelial cells (see sect. VC4). However, there is little
information concerning the trafficking from intracellular
organelle to membrane surface. In MDCK cells, truncation of up to 37 amino acid residues from the COOH
terminus of Kir7.1 has no effect on its trafficking to the
membrane, but further deletions resulted in retention of
the channel in the cytosol; residues 309 –323 in the COOH
terminus may also be involved in membrane targeting
(770).
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4. Physiological functions in cells and organs
Kir7.1 protein is found in epithelial cells in the choroid plexus (140, 245, 552) and RPE (418, 714). In these
cells Kir7.1 is expressed in their apical membrane (418,
552, 714, 864). On the other hand, Kir7.1 immunoreactivity
has been detected in the basolateral membrane of thyroid
follicular cells and renal epithelia in the distal convoluted
tubule, proximal tubule, and collecting duct (129, 552,
596). The physiological functions of Kir7.1 are largely
unknown, although their localization in diverse epithelial
cells suggests a role in cellular ion transport mechanisms.
The epithelial cells of the choroid plexus and RPE have
an unusual polarity. They express the Na⫹-K⫹-ATPase on
their apical membrane (74, 133, 159, 505, 506, 588). Thyroid
follicular cells and renal epithelial cells are more regular and
harbor the pump on their basolateral membrane (247, 552).
The pattern of Kir7.1 localization follows the Na⫹-K⫹-ATPase
in each of these epithelial cells, suggesting a function that
resembles the “K⫹-recycling” action of Kir1.1 and Kir4.1containing channels (see sect. V, A4 and B4).
An irregular pigment pattern is observed in jaguar/
ovelix zebrafish due to mutations in the Kir7.1 gene (329).
The mutations occur in either the pore region (T128M and
L130F) or in the TM2 helix (F168L). All of these mutations
result in the suppression of Kir7.1 currents in heterologous expression systems. Kir7.1 is expressed in melanophores. Cells expressing mutated Kir7.1 could not respond correctly to the melanosome dispersion signal derived from neurons.
5. Pharmacology
There are neither specific blockers nor activators for
Kir7.1. Kir7.1 is unusually insensitive to block by Ba2⫹ and
Cs⫹ (see sect. VC2).
6. Diseases
Snowflake vitreoretinal degeneration (SVD) is an autosomal-dominant pathology characterized as a developmental and progressive eye disease that causes fibrillar
degeneration of the vitreous humor, early-onset cataract,
minute crystalline deposits in the neurosensory retina,
and retinal detachment (269, 657). Genomic analysis of
SVD patients revealed a heterozygous mutation (484C ⬎
T, R162W) in the Kir7.1 gene, KCNJ13 (250) (Table 2).
Modeling of Kir7.1 based on the structure of KirBac1.1
suggested that R162 was localized in the short polypeptide chain between the TM2 ␣-helix and the COOH terminus composed of ␤-sheets and which may be involved in
channel activation by PtdIns(4,5)P2 (see sect. ID2). The
R162W mutant Kir7.1 channel carries a nonselective cation current that depolarized transfected cells and increased their fragility. Functionally, this is the equivalent
of the weaver mutation in Kir3.2 (see sect. III, B5 and F2).
Physiol Rev • VOL
Since Kir7.1 is diffusely expressed in various regions of
the retina, including the retinal pigmental epithelia and
the internal limiting membrane (250, 418), the degeneration of cells in these areas may cause the abnormalities
observed in the retina of SVD patients. The relevance of
this mutation to disorders of the vitreous humor and
cornea remains to be determined.
VI. CONCLUSION
We have come far, from the identification of an
“anomalous” K⫹ current to the three-dimensional structure of the KATP channel heterooligomer in the plasma
membrane. Classical voltage-clamp and then single-cell
electrophysiological techniques described Kir channels
and suggested roles in the physiological function of a
variety of tissues. The development and application of molecular biology techniques isolated 15 Kir channel subunits
and provided insights into channel assembly, function, and
structure. In particular, Kir channels were found to be made
up of homomeric and heteromeric assembly of the subunits
which multiplied ion channel characteristics. This divergence expanded the roles of Kir channels in tissues and
organs. Biochemical assays identified interactions between
Kir subunits and anchoring proteins including PDZ-proteins
that are crucial for subcellular localization. Kir channels
have been found to be dynamically controlled by posttranslational modification including phosphorylation. The identification of the crucial involvement of PtdIns(4,5)P2 in contributing to the activity of Kir channels has clarified a number of points underlying their functionality and physiological
regulation. Gene-targeting techniques and the human genome project have greatly contributed to understanding the
contributions of Kir channels to organ physiology and disease. Phenotypes of Kir knockout animals and the identification of loss-of-function and gain-of-function mutations
in Kir genes not only reveal the functional significance of
Kir channels in cells and tissues but also disclose their
pathophysiological relevance. The evidence accumulated
by these assays ensures that Kir channels are key elements that set Eres, control cell excitability, regulate hormone release, and drive epithelial transport. The last 10
years have seen enormous advances in structural biology
that have clarified not only structure-based mechanisms
underlying Kir channel activity but also the relationships
between channel architecture, binding of diverse small
substances, and regulation of the channel’s function.
These experimental approaches are now being effectively
combined to advance understanding of Kir channels.
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence: Y.
Kurachi, Dept. of Pharmacology, Graduate School of Medicine,
Osaka University, 2–2 Yamada-oka, Suita, Osaka 565-0871, Japan
(e-mail: ykurachi@pharma2.med.osaka-u.ac.jp).
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STRUCTURE AND PHYSIOLOGICAL FUNCTION OF Kir CHANNELS
GRANTS
H. Hibino and Y. Kurachi are supported by the following
research grants and funds: Grant-in-Aid for Scientific Research
A20249012 (to Y. Kurachi), Scientific Research on Priority Areas
17081012 (to H. Hibino), the Global COE Program “in silico
medicine” at Osaka University (to H. Hibino and Y. Kurachi), and
a grant for “Research and Development of Next-Generation
Integrated Life Simulation Software” (to Y. Kurachi) from the
Ministry of Education, Culture, Sport, Science, and Technology
of Japan.
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