Physiol Rev 95: 179 –217, 2015
doi:10.1152/physrev.00016.2014
MOLECULAR ASPECTS OF STRUCTURE, GATING,
AND PHYSIOLOGY OF pH-SENSITIVE
BACKGROUND K2P AND Kir Kⴙ-TRANSPORT
CHANNELS
Francisco V. Sepúlveda, L. Pablo Cid, Jacques Teulon, and María Isabel Niemeyer
Centro de Estudios Científicos, Valdivia, Chile; UPMC Université Paris 06, Team 3, Paris, France; and Institut
National de la Santé et de la Recherche Médicale, UMR_S 1138, Paris, France
Sepúlveda FV, Cid LP, Teulon J, Niemeyer MI. Molecular Aspects of Structure, Gating,
and Physiology of pH-Sensitive Background K2P and Kir K⫹-Transport Channels. Physiol Rev
95: 179 –217, 2015; doi:10.1152/physrev.00016.2014.—K⫹ channels fulfill roles
spanning from the control of excitability to the regulation of transepithelial transport. Here
we review two groups of K⫹ channels, pH-regulated K2P channels and the transport group
of Kir channels. After considering advances in the molecular aspects of their gating based on structural
and functional studies, we examine their participation in certain chosen physiological and pathophysiological scenarios. Crystal structures of K2P and Kir channels reveal rather unique features with
important consequences for the gating mechanisms. Important tasks of these channels are discussed
in kidney physiology and disease, K⫹ homeostasis in the brain by Kir channel-equipped glia, and central
functions in the hearing mechanism in the inner ear and in acid secretion by parietal cells in the
stomach. K2P channels fulfill a crucial part in central chemoreception probably by virtue of their pH
sensitivity and are central to adrenal secretion of aldosterone. Finally, some unorthodox behaviors of
the selectivity filters of K2P channels might explain their normal and pathological functions. Although a
great deal has been learned about structure, molecular details of gating, and physiological functions of
K2P and Kir K⫹-transport channels, this has been only scratching at the surface. More molecular and
animal studies are clearly needed to deepen our knowledge.
L
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
X.
INTRODUCTION
MOLECULAR ASPECTS OF...
FUNCTIONAL ROLES OF Kir AND K2P...
Kir4.1 IN PARIETAL CELLS OF THE...
Kir4.1 IS THE DOMINANT Kⴙ...
Kir4.1 GENETIC ABLATION IN MICE...
Kir AND K2P CHANNELS IN THE...
ROLE OF POTASSIUM CHANNELS IN...
UNEXPLORED AND SURPRISING...
CONCLUSIONS
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I. INTRODUCTION
The inwardly rectifying Kir K⫹ channels are the simplest
from the point of view of topology within the ion channel
superfamily. As will be discussed below, they form a K⫹selective pore by assembling as tetramers of two transmembrane domains with a membrane reentrant loop that contains the elements for ion selectivity. K2P channels are like
two Kir pore structures linked together and assemble in
pairs in a pseudotetrameric arrangement. Both types of
channels are central in the control of the membrane potential of cells, but recent work has brought to light a much
more sophisticated contribution to cell and organ function.
The present review will look at two subsets of Kir and K2P
channels: the so-called K⫹-transport channels among the
Kir channels and those of the K2P channels that are prominently regulated by changes in pH. In addition to sharing
structural simplicity with K2P channels, Kir K⫹-transport
channels are crucially dependent on pH for their function.
As will be seen below, their apparent simplicity in basic
blueprint hides sophisticated regulation, originally unsuspected structural complexity, and central importance in
physiological processes.
This work is not exhaustive and need not be, as excellent
reviews touching on different aspects of the subject matter
have been published recently and will be cited below. It is also
biased by the penchant of the authors for certain physiological
functions adjudged more thoroughly investigated, controversial, or both. Others, like the conspicuous absence of an analysis of the function of K2P channels in the central nervous
system (CNS), are arbitrarily not touched upon, but the reader
is directed to previous works covering this area.
A. Tandem Two-Pore Domain K2P Channels
Channels belonging to the K2P family of proteins underlie
the background conductance essential to the maintenance
of the interior negative membrane potential of all cells. Leak
0031-9333/15 Copyright © 2015 the American Physiological Society
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SEPÚLVEDA ET AL.
currents carried by K⫹ ions had long been known to be key
players in setting the membrane potential in excitable cells
(158 –160), but the nature of these membrane leaks remained mysterious until relatively recently, and their identification with bona fide ion channels had to await the discovery of their molecular counterparts. In 1995, a K⫹ channel was identified in yeast with a predicted topology of eight
transmembrane domains and two pore domains in tandem
(191). Shortly afterwards, the first K2P channels (see FIGURE
1A) were obtained: human TWIK-1 (219), Drosophila
melanogaster ORK1, later called KCNK0 (120) and mouse
TREK-1 (99). These channels differed from those cloned
thus far in having two pore domains (P) in tandem, rather
than the usual single P-domain, and four transmembrane
domains (TM1-TM4). As we will discuss in more detail
below, they form homodimers, and sometimes heterodimers, where each monomer contributes two P-domains and transmembrane domains 2 and 4 to the conduction pore (FIGURE 1A). Functional study of TWIK-1 [K2P1 in
the International Union of Pharmacological Sciences nomenclature (118)] led the authors to postulate it as responsible for a leak or background conductance of a type present
in all cells (219). Indeed, TWIK-1 was found to be open at
A
Eight of the 15 mammalian K2P channels respond prominently to changes in extra- or intracellular H⫹ concentration (218). TWIK-related acid-sensitive K⫹ channel 1,
B
SID
P1
2
3
P2
4
3
2
1
4
3
2
2
4
1
3
Turret
N-term
SF
C-term
1
4
1
2
2
2
1
Outer
helix
2
Inner
helix
P
1
Pore helix
2
P
P
4
1
C-term
N-term
1
C
P2
1
3
rest and therefore is capable of driving the membrane potential towards the equilibrium potential for K⫹. TWIK-1
lacked time or voltage dependence and approached open
channel rectification properties. A small deviation from perfect Goldman-Hodgkin-Katz rectification of the TWIK-dependent current was given by a weak inward rectification at
depolarized voltages due to intracellular Mg2⫹ inhibition.
The name given to the channel is an acronym for tandem of
P-domains in a weak inwardly rectifying K⫹ channel.
TWIK-1 was the first in a family of proteins comprising 15
mammalian members all sharing the property of having
four transmembrane domains and two P-domains in tandem that rapidly emerged in the scientific literature and are
now termed K2P channels (219). The biology, physiology,
and biophysics of K2P channels have been widely reviewed
(119, 221, 236), including a recent in-depth comprehensive
review by Enyedi and Czirják (93) and one dealing specifically with pH-gated K2P channels, the focus of the present
work, by Lesage and Barhanin (218).
2
1
N-term
C-term
FIGURE 1. A: topological model for two-pore-domain potassium (K2P) channel subunit (top: shown in side
view with the membrane in gray). Each subunit is made of four transmembrane segments, TM1-TM4, and two
pore-forming domains, P1 and P2. P domains penetrate partially into the membrane. A thicker portion
represents the selectivity filter. The SID (self-interacting domain) is a large extracellular loop often making
contact with its partner in the other subunit of the channel through a disulfide bridge. Two subunits associate
to form a dimer with bilateral symmetry and pseudo-fourfold symmetry around the pore as shown in the bottom
diagrams (seen from the extracellular side looking into the membrane). Two alternative forms of association of
transmembrane domains are shown. The one on the left corresponds to that revealed by the first two X-ray
structures of K2P channels and the one on the right to what has been termed a domain-swapped configuration
(see text for further description of these) where outer helices are exchanged between protomers. P1 and P2
domains contribute to the pore with identical P domains facing each other diagonally across the permeation
pathway. B: topological model for inwardly rectifying K⫹ (Kir) channel subunit (top) with the organization of
tetramers illustrated at the bottom. Other details as in A. C: schematic depiction of K⫹ channel KcsA structure.
Two of the four subunits of this two-TM homotetramer pore are shown with the extracellular side on top. Each
subunit contains an outer helix (helices shown as boxes) close to the membrane, an inner helix close to the
pore, a short pore helix, and a selectivity filter (SF, thick line). K⫹ ions are shown as gray spheres along the
pore. Accompanying water molecules have been omitted. [Based on MacKinnon (245).]
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pH-GATED K2P AND Kir TRANSPORT CHANNELS
TASK-1 (K2P3), was identified in 1997 and so christened to
reflect its gating by extracellular pH (pHo) (90). TASK-1
was later joined by similarly pHo-gated TASK-3 (K2P9) as
the only functional channels in the TASK subfamily of K2P
channels (194, 326). TASK channels are rather indifferent
to changes in intracellular pH (pHi).
TASK-2 (K2P5) was discovered in 1998 by Reyes et al. (335)
and so-called despite the fact that it could be better described as an alkali-activated channel, being half-maximally activated at pHo 8.0. Together with more appropriately named TALK-1 (K2P16) and TALK-2 (K2P17), Twikrelated alkaline pH-activated K⫹ channels that require high
pHo values well above conceivable physiological levels for
activity (75, 113), they form the separate TALK branch of
pH-gated K2P channels. In addition to their sensitivity to
extracellular pH, TASK-2 and TALK-2 are also gated open
by increases in pHi (281). TASK-2 is also activated by increases in cell volume (280) and inhibited by a direct effect
of G protein ␤␥ subunit (1).
TWIK-1-related K⫹ channels TREK-1 (K2P2) (99) and
TREK-2 (K2P10) (17, 223) form together with TRAAK
(K2P4) (100), a further subgroup of K2P channels. These
channels have complex gating including dependence on free
polyunsaturated fatty acids and phospholipids, increases in
temperature, and plasma membrane mechanical stretch
(93, 218). TRAAK shows a shallow dependence on pHo
with an increase in activity with alkalinization within the
physiological range (193). TREK-1 and TREK-2 are, respectively, steeply inhibited and activated by extracellular
acidification with half-maximal effects at pHo 7.4 (63, 346).
The inhibition of TREK-2 by extracellular alkalinization tails
off at ⬃60% at pHo 8.5. pHi gates TREK-1 and TREK-2, both
of which are activated by acidification (163, 247). TRAAK, on
the other hand, is activated by pHi ⬎7.8 (193).
Extracellular pH affects TWIK-1, which is inhibited by
acidification (325). TWIK-1 and TWIK-2 are inhibited by
exposure to 100% CO2, which is expected to acidify the
intracellular milieu, but there is no evidence that the inhibition is due to a direct effect of pHi (50, 219).
These effects of extracellular pH on the gating behavior of
K2P channels seem likely to take place through changes in
the probability of the channels to be in the open state,
without any change in single-channel conductance or other
changes such as the number of channels expressed at the
surface membrane (63, 186, 235, 335).
B. Kir Kⴙ-Transport Channels
Kir channels have only two transmembrane segments and
form tetramers where P-domain and TM2 contribute to the
permeation pore structure (FIGURE 1B). The Kir potassium
channel family comprises 15 members in mammals. It
emerged in 1993 with the expression cloning of ROMK1
(Kir1.1) (157) and IRK1 (Kir2.1) (205). The general properties of Kir channels have been presented elsewhere in several reviews (145, 153, 278) and will not be dealt with in
detail here. Kir channels are formed by proteins possessing
two transmembrane domains with intracellular NH2 and
COOH termini connected by a reentrant P-domain loop
organized in a homo- or heterotetrameric arrangement (FIGURE 1B). The inward rectification results from an interaction between magnesium and polyamines with the pore
(153, 278). We should be reminded that inward rectification was discovered in the skeletal muscle by Bernard Katz
long before the cloning of Kir channels (188; see Ref. 153)
and was originally designated as anomalous rectification,
i.e., not following the Goldman-Hodgkin-Katz equation.
The elementary conductance of all Kir channels is dependent on extracellular K⫹ concentration over a large range
(344; see Ref. 153). Some of these channels are also slowly
activated (minutes) when exposed to increasing extracellular K⫹ concentration (86, 91, 92, 312, 343, 354). The membrane phospholipid phosphatidylinositol 4,5-bisphosphate
(PIP2) is necessary to sustain the activity of most Kir channels (169, 170, 224, 225, 238, 330, 355, 427). Kir channels
are readily blocked by low concentrations of Ba2⫹ and Cs⫹
(153), a feature that distinguishes them from K2P channels.
Kir7.1 is an exception as it requires millimolar concentrations of Ba2⫹ and Cs⫹ for blockade (204).
The Kir K⫹ channel family has been divided into four distinct subsets according to their biophysical properties and
physiological roles (153): classical Kir (Kir2.x), G proteingated Kir (Kir3.x), KATP channels (Kir6.x/SURx), and K⫹transport channels (Kir1.1, Kir4.x, Kir5.1, and Kir7.1). The
present review focuses on the latter subgroup, composed of
channels involved mainly in epithelial transport or K⫹ homeostasis rather than simply controlling excitability.
Kir 1.1 was identified by expression cloning using a library
from the renal outer medulla of the rat (hence the original
name of this channel, ROMK) (157). Several isoforms have
been identified, three in the rat, which are distinguished by
short truncations at the NH2 terminal of the protein (409).
Kir1.1 is expressed mainly in the kidney. Kir4.1 and Kir4.2
were cloned independently by several groups (35, 123, 312,
362, 377). Kir5.1 was cloned by Bond et al. (35) together
with Kir4.1 and Kir4.2. Kir4.1 is expressed in the brain,
sensory organs, kidney, and stomach (35, 104, 260, 362);
Kir4.2 in the brain, liver, pancreas, kidney, and lung (35,
315, 362, 381); and Kir5.1 in the kidney, sensory organs,
and brain (151, 176, 315, 381, 394). The homotetrameric
Kir5.1 channel is not functional in heterologous expression
systems (35, 315, 316, 381), but surprisingly it can be found
alone in different parts of the cochlea (151). It has also been
reported that Kir5.1 can be driven to the plasma membrane
in the presence of PSD95 anchoring protein (380). Kir5.1 is
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SEPÚLVEDA ET AL.
presently considered as an associated, modulatory subunit
of Kir4.1 and Kir4.2. Kir7.1, identified in 1998 (87, 204,
305), differs markedly in sequence from other Kir channels
and has not been reported to form heteromers. Functional
properties of Kir7.1 include low sensitivity to Ba2⫹ and Cs⫹
compared with other family members as well as a very low
single-channel conductance (204). Also unusual for a Kir
channel, Kir7.1 inward rectification properties are independent of K⫹ concentration (87, 204), and its activity is PIP2dependent though showing rather low affinity for the phospholipid (306, 339). The unusual pore properties of Kir7.1
appear to be due to the presence of a methionine residue at
position 125, which is occupied by an arginine in other Kir
channels (204).
All these channels are sensitive to pHi as shown in FIGURE 2.
Kir1.1, Kir4.1, and Kir4.2 are inhibited by intracellular
acidification with pKa values of 6.5, 6.0, and 7.1, respectively (94, 216, 256, 315, 422, 423, 427). The association
of Kir4.1 or Kir4.2 with Kir5.1 modifies the pKa of the
heterotetramers, making these channels highly sensitive to
pHi (Kir4.1-Kir5.1: 7.5; Kir4.2/Kir5.1: 7.6) (315, 421, 423,
427). At pH 7.3, a plausible figure for physiological pHi, the
open probability (Po) of Kir1.1, Kir4.1, and Kir4.2 is nearly
maximal; these homotetramers will be more sensitive to
intracellular acidification than to alkalinization. The Po of
the heterotetramers Kir4.1/Kir5.1 and Kir4.2/Kir5.1 is
largely lower, around 0.3– 0.4, implying high sensitivity to
1.0
Kir4.1
Kir4.2
Kir7.1
Kir1.1
Kir4.1/Kir5.1
Kir4.2/Kir5.1
Open probability
0.8
0.6
0.4
0.2
0.0
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
Intracellular pH
FIGURE 2. Sensitivity of the inwardly rectifying K⫹ channels to
intracellular pH. The dependence on [H⫹] is variable from channel to
channel. A vertical gray line is drawn at pH 7.3 to illustrate the
values of the open probability (Po) for the different channel types at a
likely value for normal intracellular pH. Note that the sensitivity of the
complex Kir4.1/Kir5.1 is markedly different from that of Kir4.1. It
is also apparent that Kir4.1 and Kir1.1 have an open probability
close to one at pH 7.3, whereas that of Kir4.1/Kir5.1, Kir4.2, and
Kir4.2/Kir5.1 is lower. Kir7.1 shows high activity over the whole
physiological range of pH, but no measurements of Po are available,
therefore relative activity values are reported. The data for drawing
the curves are taken from the following publications: 94, 216, 256,
315, 421, 423, 427. The pKa values and slopes for the different
channels were, respectively, as follows: Kir4.1 (6.0, 2.3), Kir4.1/
Kir5.1 (7.4, 1.5), Kir4.2 (6.9, 1.4), Kir4.2/Kir5.1 (7.6, 1.2), and
Kir1.1 (6.9, 3.5).
182
pH changes in both directions. Kir7.1 exhibits a bell-shaped
dependence on pH (171) and appears rather insensitive to
pH changes around pH 7.3. It is important to underscore
that there are multiple interactions between sensitivity to
pH and other regulatory processes (153, 169, 409, 420).
C. Gating Processes of Kⴙ Channels
The process by which ion channels fluctuate between open
and closed states has been termed gating. As the name suggests, the pore of channels is regulated through the action of
gates that can be opened to reach an active state or closed in
a deactivated or inactivated state (154). The gating processes
of K⫹ channels have long been explored in functional experiments using ion channels in native cells or in heterologous
expression systems utilizing cloned ion channels. The vision of
channel morphology and the possible location of these gates
garnered from early functional experiments has received reassuring confirmation with the more recent availability of highresolution structures of K⫹ channels (6, 429).
A key element in what were later to become known as K⫹
channels was the description by Hodgkin and Keynes of the
passive permeation of K⫹ across the squid axon membrane
as though “in a system in which ions move through the
membrane in single file” (161). These findings presented a
view of permeation through structures made of narrow
tubes crossing at least part of the membrane thickness.
Other experiments addressing the function of voltage-dependent K⫹ channels in squid giant axons exploited the
effect of intracellular injection of tetraethylamonium (TEA)
to block the currents. It was observed that fast blockade of
K⫹ channels by TEA required the channels to be opened by
depolarization and that gate closure could actually trap the
blocker in what was deduced to be a pore cavity accessed
from the intracellular space. These pioneering experiments
by C. M. Armstrong provided the first, as it turned out
remarkably accurate, idea of K⫹ channel structure and its
main gating process, as he concluded that “a K⫹ pore has
two distinct parts: a wide inner mouth that can accept a
hydrated K⫹ ion or a TEA⫹-like ion, and a narrower portion that can accept a dehydrated or partially dehydrated
K⫹ ion, but not TEA⫹” (4). The channel was therefore
envisioned as a (proteinaceous) structure spanning the
membrane and constituted of a narrow tube affording single file migration of at least partially dehydrated ions and an
intracellular large vestibule accepting hydrated ions and
larger molecules such as TEA derivatives. Gating was proposed to occur via the action of a sort of flap that would
close or open the intracellular entrance of the vestibule (5).
Soon after cloning the nicotinic acetylcholine receptor, the
first ion channel to be thus isolated, the same group led by
Shosaku Numa cloned the Na⫹ channel of Electrophorus
electricus electroplax responsible for the depolarization of
excitable membranes (288). The channel was a membrane
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pH-GATED K2P AND Kir TRANSPORT CHANNELS
protein with four repeats of six transmembrane ␣-helices
with several positively charged residues in helix number
four, which immediately was suggested to play a role in
sensing membrane potential. Cloning of K⫹ channels followed soon, the first being reported in 1987 and corresponding to a channel that when mutated in Drosophila
flies gives rise to the phenotype known as Shaker (184, 302,
387). The Shaker K⫹ channel was predicted to form a protein with a single six-transmembrane domain structure with
no repeats, and was later suggested to organize as a tetramer to
form a K⫹ channel pore. Subsequently cloned K⫹ channels
uncovered a high conservation in the linker joining transmembrane segments 5 and 6 (S5 and S6), suggesting that it might
contribute to the formation of a selective pore, a concept that
was supported by mutagenesis followed by functional expression in heterologous systems. Examples of these advances are
papers that demonstrated that point-mutations in the S5-S6
linker affected the action of inhibitors known to interact with
the pore (430) or producing marked changes in ion selectivity
(431). It was also shown that exchanging the S5-S6 segment
between channels with differing permeation properties also
exchanged those properties (143). These advances identified
the segment between putative transmembrane domains 5 and
6 as the pore-forming region, or P-domain, that confers its
conductance and selectivity properties to this six transmembrane domain protein (373).
The identification of Shaker K⫹ channel brought about a
flurry of activity in the field of molecular identification of
K⫹ channels that now constitute a large superfamily of 88
human membrane proteins that share the highly conserved
P-domain (434). They include voltage-gated KV1–9 channels that share the characteristic six transmembrane domain one P-domain structure of the Shaker K⫹ channel,
Ca2⫹-activated KCa channels of similar general topology,
and CNG/HCN channels that together with KV10 –12
channels are cyclic nucleotide gated. The K⫹ channels we
are concerned with in the present review belong to two
different clades of the K⫹ channel superfamily: the inwardly
rectifying Kir channels and K2P channels. The general topology of Kir and K2P channels is shown in cartoon form in
FIGURE 1. Neither of these channel types, which have been
the object of recent detailed and authoritative reviews (93,
153), possesses the equivalent of the transmembrane domain carrying charged residues characteristic of voltagedependent K⫹ channels, and they are not voltage dependent
in the accustomed sense of this description. They are nevertheless under gating control by a number of physiological
stimuli among which are the extra- and pHi.
D. Kⴙ Channel Gating in the Era of Atomic
Scale Structural Studies
The publication of the first atomic structure of a K⫹ channel
obtained by the laboratory of Roderick MacKinnon initiated a new era in the understanding of ion channel function
influencing the views about how these proteins select and
conduct ions, how the pore is gated, and how the gating is
coupled to relevant signals such as membrane voltage, second messengers, and the interaction with lipid or protein
cell constituents. The emerging structural data came to confirm many of the general principles about channel structure
deduced from early functional studies (245). The first K⫹
channel structure to emerge was that of a Streptomyces
lividans KcsA channel which has only two transmembrane
helices in addition to the P-domain (88). The protein was
seen to organize as four identical subunits surrounding the
permeation pore in a fourfold symmetrical way. As shown
schematically in FIGURE 1C, the NH2 terminus of KcsA is
intracellular, with TM1 as an outer helix that becomes a
turret domain as it emerges extracellularly. A so-called pore
helix enters about one-third of the membrane, and turns
outwardly to form the selectivity filter (SF) with its highly
conserved TVGYG signature sequence. TM2 is a pore-lining, inner helix that forms the COOH terminus of the protein at its intracellular end. The SF at the outer end of the
pore ensures selectivity and conductance at rates approaching the diffusion limit. The section of the pore towards the
intracellular end of the permeation pathway is wider with a
large water-filled cavity lined by the TM2 transmembrane
helices. The TM2 helices come together to form a packed
bundle at their intracellular ends. This packing would become interesting in the context of the gating behavior of K⫹
channels.
Three different types of gating processes had been identified
in functional studies of K⫹ channels. A first gating process
was associated with the opening of voltage-gated channels
selective for K⫹, but also for channels selective to other
ions, brought about by changes in membrane potential and
corresponding to the operation of what has been termed the
activation gate. A great deal is known, and debated, about
the voltage-sensing mechanism (27, 228), but this will not
occupy us here as conventional voltage dependence does
not appear to exist in the Kir and K2P channel families we
are discussing. Although described in voltage-gated channels, such as for example the Na⫹ and K⫹ channels involved
in the generation of the action potential in excitable cells,
the mechanisms of channel opening and closing are thought
to apply to gating processes obeying other stimuli such as
membrane tension, phospholipid signaling, pH, intracellular Ca2⫹, etc.
A clue as to what must be the mechanism that opens and
closes K⫹ channels arose from the discovery of the structure
of another prokaryotic channel, the MthK K⫹ channel
(180, 181). Comparison of KcsA and MthK structures revealed that whilst inner helices in the first bundle together at
the intracellular COOH-terminal end, they were splayed
apart in MthK. Evident in the MthK structure, there was a
bend of ⬃30° in the inner helices at what was called a
“gating hinge” so the inner helices instead of bundling to-
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SEPÚLVEDA ET AL.
gether are separate, leaving a wide intracellular entry pathway of ⬃12 Å. The gating hinge corresponds to a glycine
residue located roughly halfway down the helix, which is
highly conserved throughout the K⫹ channel superfamily.
The gating suggested by the structures of KcsA and MthK
beautifully explains experimental results using the Shaker
K⫹ channel showing that the inner helix bundle coincides
with a point where the pore becomes inaccessible to thiolreactive compounds and metal ions applied at the intracellular side of closed channels (78, 79). Conservation of the
glycine hinge in both Kir and K2P channels would suggest
that this type of activation or inner gating exists in these
channel families, a point that will be discussed in more
detail below.
A second type of gating process characterized in many K⫹
channels takes place at the SF and corresponds to what has
been termed C-type inactivation (166). The possible structural counterpart of C-type inactivation was again first suggested by structural information obtained with the KcsA
channel crystallized in low K⫹ concentrations (443). This
type of gating appears to close the channel by a deformation
of the SF as it loses some of its coordinated K⫹ ions essential
for the structure and partially collapses. This is consistent
with the findings that C-type inactivation is dependent on
the extracellular concentration of K⫹ or other permeant
cations or TEA and is sensitive to mutation of residues in
the SF region (56, 195, 231). Although C-type inactivation
gating can act independently, there is evidence that it is
often coupled to the activation gate whose opening is followed by closure of the SF gate (21, 301). More recent
structural work using the KcsA channel reports channels at
different degrees of activation gate opening. Strikingly there
is correlation between the degree of gate opening and presence of ions in the SF, where the wider this opening the
lower the occupancy of the SF by permeant ions and therefore its capacity to conduct (66).
A third mode of gating known as ball-and-chain or N-type
inactivation, which unlike C-type inactivation occurs with a
fast time course, involves an autoinhibitory open pore block
by an NH2-terminal portion of the channel protein (165).
This type of gating has not been reported for the Kir and K2P
channels that occupy us here.
II. MOLECULAR ASPECTS OF
pH-DEPENDENT K2P AND Kir
CHANNEL GATING
A. K2P Channel Structure
The study of the relationship between K2P channel structure
and function took advantage of the high degree of homology
between these channels and those for which structures were
available to produce homology models to guide experimental
work or simply to gain mechanistic insights into function
184
(202, 284, 319, 374, 435). Recently, the field of K2P channels
received with delight the publication of the first two X-ray
structures of K2P channels, those of TRAAK and TWIK-1 (42,
259). The structures were remarkably similar, and the features
commented upon here about TWIK-1 generally apply to
TRAAK. FIGURE 3 illustrates the general structure of TWIK-1
where the salient features are identified. As predicted, the selectivity filter of these dimeric channels has a quasi fourfold
symmetry coordinating K⫹ ions as in their tetrameric relatives
despite their differences in amino acid sequences, particularly
in TWIK-1. Examination of inner helices shows that the equivalent of the activation gate is wide open. G141 and G256 are
positioned in the inner helices at a location equivalent to those
residues that form the gating glycine hinge in other channels
and are conserved in K2P family. M2 helix is notable for being
longer and bending at a proline residue halfway down the
membrane (P143, FIGURE 3), while M4 is shorter and straight.
A remarkable feature of TWIK-1 and TRAAK structures is
the presence of a helical cap that sits at the extracellular side
of the channel apparently obstructing access to the pore
mouth normal to the membrane. This cap corresponds to
the large extracellular loop known as self interacting domain (SID) that is stabilized by a disulfide bridge (C69,
FIGURE 3) essential for function in TWIK-1 (222) but not in
every K2P channel (282). The extracellular cap is formed by
two ␣-helices from each subunit that are connected to the
pore-forming portion of the channel at bend-forming residues G89 and P47 (FIGURE 3), which are conserved among
K2P channels. An amphipathic helix located near the membrane/cytosol interface, the C helix (FIGURE 3), is speculated
to serve a gating purpose (259). This structure is not conserved in TRAAK, where instead an amphipathic helix at
the intracellular end of the first inner helix (TM2) is seen to
be positioned to interact with both the hydrophobic tails
and charged heads of membrane phospholipids (42).
Access to the external mouth of the pore is provided by
long, tunnel-like entrances of molecular dimensions, the
extracellular ion pathways (EIP). This type of arrangement
explains the resistance of K2P channels to inorganic and
toxin pore blockers (100, 219). The presence of the EIP also
offers the possibility for interaction between ions affecting
the state of the SF in a confined space where electrostatic
interactions might develop less hindered by solvent screening than in free solution. Both K2P channel structures have
unusual prominent fenestrations connecting the inner part
of the pore and the lipidic milieu of the membrane. This
type of arrangement, which has also been seen in the voltage-gated sodium channel structure, might help in supporting the effect of lipidic signals that affect the gating of several K2P channels (e.g., Refs. 51, 289). Future X-ray work
will surely reveal more secrets of the K2P channel structurefunction relationship. Already a new, higher resolution
TRAAK structure has been generated showing a domainswapped chain connectivity exchanging outer helices 180°
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pH-GATED K2P AND Kir TRANSPORT CHANNELS
A
B
Extracellular
cap
C69
E2
E1
Out
Out
G89
P47
M4
M3
In
M1
G141
P143
G256
90°
M2
In
C helix
FIGURE 3. Overall structure of TWIK-1 derived from its 3.4-Å-resolution crystal structure. A: tertiary
structure of TWIK-1 shown in a ribbon representation viewed from the side. One subunit is colored blue-to-red
from the NH2 to the COOH terminus. The other subunit is gray. K⫹ ions are represented as green spheres.
Approximate boundaries of the lipid membrane are shown as horizontal lines. The intersubunit disulfide bond
(at C69) at the apex of the extracellular cap is in green. The location of P143 and hinge glycines G141 and
G256 is also shown. E1 and E2 are extracellular cap helices. Bends in the structure at the base of the
extracellular cap are afforded by G89 and P47. C helix is stretch of ␣-helix running parallel to the membrane
at the COOH terminus of the channel. Loop regions not included in the final model are indicated as dashed lines.
The central cavity is located below the K⫹ ions along the axis of symmetry (behind G141). The intramembrane
fenestration is visible above the C-helix. B: a view after a 90° rotation around the axis of symmetry. The
extracellular ion pathway is seen above the outermost K⫹ ion. [From Miller and Long (259). Reprinted with
permission from AAAS.]
around the channel, plus an unrelated conformational change
of an inner helix sealing a fenestration to the bilayer and associated structure changes around the SF with possible functional consequences (41). Interestingly, the domain-swapped
type organization can be observed in the crystal structure of
human TREK-2 (K2P10.1) recently deposited by Carpenter et
al. (PDB ID: 4BW5). It might turn out that this form of organization could apply to the K2P family at large.
B. Gating of K2P Channels by Extracellular
pH Takes Place at the Selectivity Filter
As discussed above, a type of gating occurring in various K⫹
channels and thought to take place at the SF might involve
a profound change in its structure (429) and corresponds to
what has been termed C-type inactivation (166). Gating at
the SF is characterized by its dependence on extracellular
K⫹ concentration and is particularly sensitive to mutations
near or at the external opening of the conduction pore.
Opening and closing of various K2P channels by changes in
extracellular pH has been demonstrated to entail extracellular K⫹-dependent C-type inactivation. Other forms of
gating, including that modulated by changes in pHi, have
been attributed to changes in the bundle crossing or internal
gate (see Refs. 64, 253 for reviews). But recent evidence, to
be discussed here, has very seriously upset this view.
First inklings for a prominent role of gating at the selectivity
filter in K2P channels came from work with the Drosophila
KCNK0 channel. KCNK0 channels gate by switching between open and closed states driven by protein kinases C, A,
and G acting at the COOH-terminal end of the protein
(445). That the gating process occurs through changes at
the SF was suggested by the effect of extracellular Zn2⫹ that
inhibits KCNK0 in a state of activation-dependent manner
and requires the presence of a histidine residue in P1, demonstrating that gating affects the conformation of the outer
side of the pore even if phosphorylation occurs at the intracellular COOH terminus (444). Additional evidence pointing to a C-type inactivation process was that the effects of
the external permeant ion, extracellular TEA, and mutagenesis of residues near the SF affecting KCNK0 were as predicted for the analogous phenomenon in voltage-dependent
K⫹ channels (444).
TASK-1 and -3 as well as TWIK-1 are gated closed by
extracellular H⫹. The sensor for this effect is a histidine
residue located at the mouth of the SF at a position occupied
by negatively charged glutamic acid in most K⫹ channels
(194, 230, 325, 326). It is therefore easy to conceive that the
action mediated by the histidine sensor, presumably neutral
at physiological pHo, will be to interfere with SF permeant
ion occupancy leading to C-type inactivation upon acquiring a H⫹ with acidification.
The conductance of TASK-1 was found very early on to be
dependent on extracellular K⫹ concentration (90). In addition to this dependence, extracellular K⫹ also affected
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SEPÚLVEDA ET AL.
TASK-1 gating modulation by pHo, a result that is expected
from an effect taking place at the SF (229, 230). An idea of
the possible structure of TASK-1 was obtained by homology modeling using the crystal structure of KcsA as a template by Yuill et al. (435). This work suggested that histidine-98 (H98) pHo sensor was located behind the SF. Protonation of H98 at acidic pHo was proposed to distort the
SF leading to a nonconducting state. Site-directed mutagenesis affecting ion selectivity brought about modifications in
agreement with pHo-gating taking place at the SF (435).
Later, molecular dynamics studies showed that protonation
of H98 is accompanied by its removal from behind the SF
and by loss of a coordinated water molecule that contributes to the structural stability of the filter. The flipping away
of H98 from behind the selectivity filter is also proposed to
create an electropositive barrier to K⫹ ions at the outer
mouth of the pore (370).
Although closely related, there are differences in the pHo
responses between TASK-1 and TASK-3. These include
differences in pHo sensitivity and in the cooperative nature of pHo effect and the effect of the permeant ion
(122). Some of these differences may be due to an effect
of the TM1-P1 extracellular loop on the pHo sensors.
Clarke et al. (60) exchanged TM1-P1 domains between
TASK-1 and TASK-3 and demonstrated the importance
of this loop in pHo dependence and Zn2⫹ blockade. The
effect of pHo on TASK-3 gating is cooperative occurring
with a Hill number nH of 2 (194). The same holds true for
TASK-1 but only at high extracellular K⫹ whilst the effect of pHo occurs with an nH of 1 at physiological K⫹
(230). A study in TASK-3 determined that H98 pHo sensors in both subunits of TASK-3 channel need to be neutral for the channel to open, with neutralization of H98
in one subunit enhancing the propensity of H98 in the
second to become neutral. The permeant ion plays a central role in this change of acidity of the histidine sensor,
an effect that is enhanced by the electrostatic effect of
glutamate E70 (122). This glutamate is part of the wall
the EIP of K2P channels (see above), is known to be close
to the H98 sensors (60), and is proposed to influence the
electrostatic potential in EIP, presumably becoming a
major determinant of EIP K⫹ concentration and affecting
the occupancy of the outermost SF K⫹ binding sites
(122). A similar mechanism might take place in TASK-1
where E70 replaced by a lysine and the K⫹ effect is only
manifest at high concentrations. The importance of electrostatics of the EIP is also highlighted by the effect of
TASK-3 blockers Zn2⫹ and ruthenium red which depend
on the presence of the E70 residue (59, 60, 69, 265),
requires a neutral H98 (59, 60), is cooperative, and is
strongly impeded by increasing extracellular K⫹ concentration (122).
TWIK-1 pHo gating is a process in which the occurrence of
changes at the SF becomes manifest through major altera-
186
tions in ion selectivity, with TWIK-1 changing from being
K⫹ selective to a generally monovalent cation-permeable
channel (49, 244). The study of TWIK-1 has been difficult
because of a generally low plasma membrane expression.
The low currents observed for TWIK-1 in heterologous expression systems have been interpreted as TWIK-1 K⫹
channels having a very low Po in the cell surface owing to
silencing by sumoylation at residue K247 in the intracellular COOH terminus (325). Desumoylation with SUMO
isopeptidase is reported to open silent TWIK-1 channels
reversibly in isolated membrane patches (321). This
mechanism has also been proposed to regulate TWIK-1
activity in cerebellar granule neurons (322). The concept
of TWIK-1 regulation by sumoylation has been directly
challenged (95), and the alternative view has been advanced that TWIK-1 silencing is due to its internalization
to the recycling endosomal compartment by a dynamindependent mechanism (96). This mechanism, dependent
on a COOH-terminal di-isoleucine motif, is thought to
be responsible for the localization of TWIK-1 in native
renal epithelial cells and nonpolarized cells in the subapical and pericentriolar recycling compartments, respectively. Independently of the mechanism involved, use of
mutated TWIK-1 channels has allowed its detailed functional study in heterologous systems. The change in ion
selectivity upon acidification is unique among pH-sensitive K2P channels with significant increase in the permeability to Na⫹ relative to that to K⫹ upon acidification to
pH 6. Paradoxically, there is a transient increase in current upon acidification before inhibition takes place. Histidine-122 acts as H⫹ sensor and is necessary for both
phases of the effect, but the results suggest that additional, as yet unidentified sites contribute to inhibition
by acidification. TWIK-1 differs significantly from
other K2P channels in the P1 and P2 domains, the pore
helix of the first P1 domain among other changes, which
might account for its markedly different functional behavior (49).
TASK-2 is activated by alkalinization with a pK1/2 of
around 8 and no evidence for cooperativity (284, 335).
Decreasing pHo to 6.0 abolishes TASK-2 channel activity. A group of five charged residues in the TM1-P1 extracellular loop affects pHo-gating sensitivity of TASK-2
(261, 283), but coupling of TASK-2 activity to extracellular alkalinization is mediated by neutralization of R224
near the second pore domain (284). In the protonated
form, R224 might decrease occupancy of the SF by K⫹
leading to a blocked state. Analysis of free-energy profiles
delineating ion permeation in the SF studied in silico
(446) suggests that protonated R224 electrostatically increases the height of energy barriers between binding
sites impeding ion movement. The pK1/2 of the pHo effect
on TASK-2 measured under quasi-physiological K⫹ concentration gradient ([K⫹]i/[K⫹]o ⬃140/5 mM) decreases
with depolarization (281, 335). Voltage dependence of
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pH-GATED K2P AND Kir TRANSPORT CHANNELS
the effect by charged compounds on ion channels can be
interpreted on the basis of a model where H⫹ interacts
with a site within the electric field of the membrane (417),
as has been proposed for H⫹ effect on TASK-1 channels
(230). This does not seem to be the case for TASK-2,
however, as there is no voltage dependence of pHo gating
measured in symmetrical high K⫹ concentrations ([K⫹]i/
[K⫹]o 140/140 mM) (58). The effect has been interpreted
to occur by changes in SF, or EIP, occupancy by K⫹ ions
electrostatically favoring deprotonation of charged R224
sensing residues. At low [K⫹]o a low occupancy is assumed at the outermost coordination sites, a situation
reversed by depolarization-promoted flux of K⫹ from the
intracellular K⫹-rich compartment. These external sites
would already be highly occupied when high K⫹ concentration is used at both sides of the membrane, hence the
lack of voltage dependence (58, 281).
Extracellular acidification inhibits TREK-1 but activates
TREK-2 within the same pH range. The mechanism for this
difference has been explored by Sandoz et al. (346). The
proton sensor in TREK-1 and -2, conserved histidines H126
and H151, respectively, is located in the TM1-P1 loop preceding P1 domain. Molecular modeling and mutagenesis
indicate that the differential effect of acidification in
TREK-1 and TREK-2 resides in differences in residues in
the also extracellular P2-TM4 loop leading to attraction or
repulsion between the protonated sensing histidine and
closely located negatively or positively charged residues impacting the SF gate of the channels. This is supported by
effects of extracellular K⫹ concentration on the pHo dependence of these channels (63, 346).
C. Gating of K2P Channels by pHi
pHi prominently affects the gating of TREK-1 and -2 with
activity increasing with decreases in pHi (163, 247). The
gating of these channels is complex, but at the same time it
has been extensively studied (see specific reviews in Refs.
77, 162, 289). TREK-1 is activated by a variety of chemical
and mechanical stimuli such as arachidonic acid, stretch,
and phosphorylation. Internal acidification converts lowactivity TREK channels into high-activity channels. Glutamate E306 behaves as the intracellular proton sensor in this
activation by acidification process, and its neutralization by
mutation converts TREK-1 into a permanently active K⫹
channel. Membrane phospholipids open TREK-1 channels,
and a cluster of positively charged residues is the phospholipid-sensing domain. E306 is found within this stretch of
positively charged residues, and its negative charge counteracts their ability to interact with the membrane. Protonation of E306 by acidification or neutralization by mutation
is thought markedly to enhance channel-phospholipid interaction thus favoring TREK-1 opening. Although revealing a series of molecular details, these studies did not disclose which gate was put into action to mediate the effect of
pHi in modulating opening and closing of TREK-1 channel.
Nevertheless, cartoon illustrations of the phenomenon
(e.g., FIGURE 3 in Ref. 162) left one with the impression that
it might correspond to the activation, bundle crossing gate.
TASK-type K2P K⫹ channels are insensitive to pHi (93), but
TALK-subfamily K2P K⫹ channels TASK-2 and TALK-2
are activated by increases in pHi (281). The pHi dependence
occurs in the same pH range as the pHo dependence but is
unrelated as pHo-insensitive TASK-2-R224A is not altered
in its pHi dependence. A truncation and directed mutagenesis approach identified COOH-terminal K245 as pHi sensor. It was proposed that separate gates mediate pHo and
pHi gating of TASK-2, as pHi gating lacks voltage and
[K⫹]o dependence, both typical of pHo gating (281). Based
on this separateness and on the location of the pHi sensor, it
was argued that the gate affected by pHi might correspond
to the bundle crossing gate (281).
The existence of separate inner helices bundle crossing
opening/closing gating mechanism in K2P channels has been
approached in an examination of the voltage dependence of
TASK-3. This study advocated the existence of an activation gate that could be interfered with by mutation of putative hinge glycine residues in TM2 and TM4 (9). Ben-Abu
et al. (25) used roughly the TM1-P1-TM2 transmembrane
portion of the KCNK0 structure transplanted into a Shaker
KV to supplant the whole of its TM5-P-TM6 region. This
structure possessed an activation gate that could be opened
by depolarization. This demonstrated that the bundle crossing machinery can be put into action using a K2P channel
conducting structure and the voltage-sensing portions of a
KV channel, albeit under artificial conditions including the
use of a single set of KCNK0 TM-P-TM units in a tetrameric arrangement.
A direct examination of this problem was more recently
carried out after the discovery by Piechotta et al. (319) that
hydrophobic quaternary ammonium ions are rather efficient
intracellular blockers of K2P channels TRESK, TREK-1, and
TASK-3. Experiments exploring the access of inhibitors to the
inner vestibule of TREK-1 channel suggest that gating by
pHi and pressure do not involve gating transitions taking
place at an inner helix bundle-crossing location, as inhibitors reach their site of action freely even when the channel is closed (319, 331). This very clear set of experiments
indicates that the primary activation mechanism in
TREK-1 channels is close to or at the SF and that the
activation gate is locked permanently open. Thermaland pHi-sensing elements are found at the intracellular
COOH terminus of TREK-1, but this sensing is probably
translated into changes at the SF. This separates the sensing elements from remotely located action elements of the
gating process in K2P channels (10, 11). In retrospect, this
idea was already implicit in the work of Zilberberg and
co-workers (444, 445) who demonstrated that phosphor-
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SEPÚLVEDA ET AL.
ylation of the COOH terminus of KCNK0 channel was
translated into marked changes in activity with the hallmark of C-type inactivation at the SF. Nevertheless, even
for TREK-1, observations pointing to separate gates for
pHi or AKAP150 and pHo (346) will have to be incorporated into an integrated view of channel gating. Whether
the hypothesis that the main gating process in TREK-1 is
located at the SF, with the inner gate being locked open,
can be extended to the rest of the family members will
have to await further work.
In view of the paucity of K2P inhibitors, the pharmacological tools introduced by Piechotta et al. (319) should become
useful in probing not only the gating of other K2P channels
but also their function in native cells and tissues.
D. Structure-Function Studies in Kir
Channel Gating
The inwardly rectifying K⫹ channels known as Kir are important regulators of excitability but also, and particularly
with the group of channels we are concerned with in this
paper, they support a variety of transport processes particularly in epithelia. The Kir family of proteins has been comprehensively reviewed (153), and therefore, we will center
here on specific aspects relating to their gating by pH and on
some new aspects of how molecular structure relates to
function. As illustrated in FIGURE 1, Kir channels are made
of only two transmembrane domains and lack a voltage
sensor. There is, however, evidence for both C-type inactivation gating at the SF and for activation gating that opens
the intracellular access to the inner vestibule of the channel
by splaying apart/turning the TM2 pore lining transmembrane domains.
None of the Kir channel types we discuss in the present
work has been crystallized. As for other classes of K⫹ channels, their prokaryotic counterparts were the first to prove
amenable to successful exploration using X-ray diffraction
methods. The first of the Kir channels to disclose its structure was KirBac1.1 (206). The structure of KirBac1.1
showed a transmembrane portion (TMD) in general quite
reminiscent of that of KcsA, with the selectivity filter at the
extracellular entrance connecting to an intramembrane cavity. Flexible linkers connect the TMD with a large cytoplasmic domain (CTD) made of numerous ␤-sheets and enclosing a large intracellular pore. A similar structure emerged
from the analysis of a chimera between the TMD of bacterial KirBac3.1 and mammalian Kir3.1 CTD (287) and later
for full-length eukaryotic Kir channels, examples of which
are Kir2.2 and Kir3.2 (386, 411). These main organizational features are illustrated in FIGURE 4A that reports the
structure of Kir3.2 (GIRK2), a Kir channel gated by G protein (411). The CTD is interesting as it prolongs the length
of the pore in an unusual way with respect to channels from
other subfamilies. The intracellular reaches of the pore are
188
contained within the CTD and harbor the sites responsible
for interaction with particles, such as Mg2⫹ or polyamines,
responsible for the inward rectification phenomenon. The
cytoplasmic domain is also the structure through which the
gating of Kir channels can be regulated by a variety of
stimuli such as ATP, Na⫹, pH, and signaling proteins or
lipids (29, 153). Early structures such as that of KirBac1.1
presented a closed channel as the intracellular exit pathway
of the transmembrane pore was occluded by the bundling of
TM2 helices (61, 206, 287, 386). In more recent structural
studies, Kir channels in various states of activation by physiological modulators such as G protein subunits and PIP2,
or by mutation, have provided a picture of pore opening
(22, 142, 411, 412). PIP2 binding to Kir2.2 was seen to
induce a transition as though there was an upward movement of the CTD to make contact with the TMD producing
a preopen PIP2-bound channel (142). Whorton and MacKinnon (411) resolved the molecular structures of wild-type
(WT) G protein-gated K⫹ channel Kir3.2 and a single point
mutant (R201A) that is constitutively active, both in the
absence and presence of the physiological activator PIP2.
The resulting structures were used to deduce the changes
taking place in the channel in the transition from the closed
to the open state. Unlike other K⫹ channels, GIRK channels
possess two activation gates, the conventional inner helix
gate and a G-loop gate (FIGURE 4). The channel was found
to present the inner helix gate in the closed state in all but in
the R201A mutant in the presence of PIP2, where it appears
wide enough to allow conduction of a hydrated K⫹ ion
(FIGURE 4B). Mutation R201A is viewed as mimicking the
action of G proteins in opening the G loop gate. Complete
opening with inner helices splaying open requires the presence of PIP2. Channel opening occurs through a conformational change in the CTD acting allosterically to control the
gates in the pore. Later work has confirmed this view and
associates a twisting motion of the CTD in the opening of
Kir gates by action of G␤␥ subunits and PIP2 (412). These
structural studies suggest that an inner or activation gate is
functional in Kir channels. In addition, analysis of the structure reveals a mechanism for channel gating in which TM2
bending at the glycine hinge and bundle crossing splaying is
coupled to a twist of the CTD. Channel activation coupled
to a CTD conformational change of the nature described
could account nicely for the action of various intracellular
signals that modulate Kir channels. It is not known whether
changes in pHi exert their regulatory effects on Kir channels
through a similar mechanism.
E. Molecular Mechanism of the Regulation
of Kir1.1 by pHi
All transport type channels of the Kir superfamily are pHi
sensitive. Among those inhibited by intracellular acidification are Kir1.1 (ROMK), Kir4.1, and Kir4.2, with Kir5.1
acquiring pHi sensitivity when expressed together with either Kir4.1 or Kir4.2. Kir7.1 has a biphasic, shallow depen-
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pH-GATED K2P AND Kir TRANSPORT CHANNELS
A
B
extracellular
turret
SF
outer helix
inner helix
Inner helix
gate
Inner helix
gate
interfacial helix
TM-CTD
linker
G loop
gate
G loop
gate
intracellular
N-terminus
WT apo
hydrophobic
R201A + PIP2
hydrophilic
C-terminus
FIGURE 4. Structure of the Kir3.2 channel and changes taking place upon opening. A: cartoon diagram of
the Kir3.2 structure showing a ribbon representation from the side. Each subunit of the homotetramer is
identified by using a different color. Undetermined structures in the turret and NH2-terminal linker are shown
as dashed lines. The approximate location of the lipid bilayer is indicated by horizontal lines with the extracellular
side on top. B: mutation R201A in the presence of PIP2 creates a continuous cavity along the conduction axis
of Kir3.2 affording enough space for a hydrated K⫹ to migrate unhindered from the cytoplasm to the selectivity
filter. Inner surface representations of the pore of the wild-type apo (left) and a fourfold symmetrical model of
mutant R201A plus PIP2 (right). The colors of the surfaces of the cavities show the hydrophobicity of the
surrounding side chains according to a relative scale given at the bottom of the figure. Relevant positions along
the pore are identified by the legends on the right. [From Whorton and MacKinnon (411), reprinted with
permission from Elsevier].
dence on pHi with a peak of activity at around physiological
pHi and partial inhibition with either acidification or alkalinization. The molecular mechanism of Kir channel pH
regulation has been intensively studied but remains rather
mysterious.
The gating of ROMK1 channels, the first Kir channel to be
molecularly identified (157), by protons has been extensively scrutinized, a body of work whose basis has been ably
summarized in recent reviews (145, 153) and will not be
rehearsed here. ROMK1 or Kir1.1 channels are closed by
intracellular acidification with a pK1/2 of 6.5, and the mechanism for this regulation was ascribed to a trio of basic
residues R41, K80, and R311. The TM1 lysine residue was
identified as the sensor itself, i.e., the residue whose side
chain that become protonated at neutral pH drove the gating of ROMK (353). As lysine ⑀-amino group pKa in solution is 10.5, a marked shift in titration is necessary for K80
to act as the pHi sensor of Kir1.1 channels, and purportedly
accomplished by NH2 and COOH terminus R41 and R311
closeness in the tertiary structure of the protein affecting
K80 pKa electrostatically. More recent studies, however,
cast doubt on the role of K80 as a bona fide pHi sensor. In
2006, Rapedius et al. (329) examined the issue of K80 as the
pHi sensor of ROMK channels by looking at its conservation across species, finding that it is generally highly conserved in vertebrates, but a neutral hydrophobic residue
was present at the equivalent location in zebrafish and
Fugu. Despite this, Fugu Kir1.1 exhibits a healthy pHi dependence (329). Homology modeling of Kir1.1 implies indeed that K80 occurs in a lipidic environment compatible
with it being permanently protonated and that R41 and
R311 are too far to affect K80 electrostatically. Instead, it is
considered that K80 participates in a TM1-TM2 H bonding
at the bundle crossing influencing the pHi sensitivity of
Kir1.1 by stabilization of the closed state (328, 329). An
independent study by Leng et al. (217) also probed the
validity of the K80 pHi-sensor hypothesis upon noticing
that residues scattered along the NH2 and COOH termini
of Kir1.1 modify its pHi sensitivity. They proposed that salt
bridges forming subunit interactions at the NH2 and
COOH termini of the channel, and crucial for stabilizing
the open state, are affected by proton concentration and
therefore account for pHi gating.
Challenging of the hypothesis that K80 acts as a proton
sensor in Kir1.1 begs the question of what is the main sensor
prompting the closing of Kir1.1 and other transport Kir
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SEPÚLVEDA ET AL.
channels. A systematic search for such sensor(s) has been
done using randomly mutagenized Kir1.1 expressed in a
K⫹-auxotrophic strain of S. cerevisae in a high-throughput
assay (311). This unbiased approach failed to identify any
pHi-sensor candidate residue in Kir1.1. Instead, the idea
was put forward that pH modulation takes place by an
effect on an intrinsic gating mechanism common to all
Kir channels with pH sensitivity obeying the titration of
a number of intracellular domain salt-bridges stabilizing
the channel in the open state. Differences in Kir channel
pH sensitivity can therefore arise by altering the relative
stability of the open and closed states rather than the
presence of additional pH sensors. The alanine scanning
mutagenesis approach coupled to electrophysiological
and homology modeling analysis of Kir 1.1 has also been
used to study the sensitivity to pHi (34). The results show
that a large number of mutations destabilize the open
state and that pHi gating may operate at the transition
between the preopen and closed conformations previously identified in crystallographic analyses of Kir channel gating (22, 142, 411, 411).
III. FUNCTIONAL ROLES OF Kir AND K2P
IN RENAL EPITHELIAL CELLS
Many K⫹ channels have been found to be expressed in the
kidneys. Here, we will concentrate on the data on Kir1.1,
Kir4.1, Kir4.2, Kir5.1, and TASK-2 and refer to other channels only when sound data on the native tissue is available.
Indeed, some K⫹ channels have been described using the
patch-clamp technique, but there is no clue about their molecular identity. Conversely, others have been detected in
parts of the renal tubule using in situ hybridization, RTPCR, or immunohistochemistry, but there is no functional
data available, for example, TWIK-1 (62, 220, 279) and
KCNA10 (428) in the proximal tubule as well as TWIK-1
(279, 295), TASK-2 (335), and KCNQ1 (KVLQT1) (80) in
the distal nephron. Kir7.1 has been shown to be present in
the basolateral membranes of rat distal convoluted tubule,
connecting tubule, and cortical collecting duct (except for
the intercalated cells), and at much lower intensity in the
thick ascending limb and outer medullary collecting duct
(294); in contrast, Derst et al. (81) found that Kir7.1 was
expressed in the basolateral membranes of the guinea pig
and human proximal tubule and thick ascending limb.
A. Basolateral Kⴙ Channels: Recycling of
Potassium
One chief function of the kidneys concerns sodium balance.
The stock of sodium in the organism controls extracellular
volume and volemia, and thereby regulates the arterial pressure on the long term according to Guyton’s hypothesis
(140). The fine regulation of sodium balance is achieved by
the kidneys according to the following steps: glomerular
190
filtration of an enormous quantity of sodium (⬃25 mol/
day), bulk reabsorption in the proximal tubule and thick
ascending limb, and finely tuned reabsorption in the socalled aldosterone-sensitive distal nephron, which comprises the distal convoluted tubule, connecting tubule, and
collecting duct (97, 128, 372). The various mechanisms
governing sodium reabsorption in different parts of the renal tubule are shown in FIGURE 5 and can be described as
follows: entry at the apical membrane via a cohort of coupled transport systems (Na⫹/H⫹ exchanger, Na⫹-K⫹-2Cl⫺
cotransporter, Na⫹-Cl⫺ cotransporter, etc.) or channels
(ENaC) and exit at the basolateral membrane via the Na⫹K⫹-ATPase. A bicarbonate/sodium cotransporter participates in this process in the proximal tubule.
Within this context, basolateral K⫹ channels have two main
functions: they maintain a negative membrane potential
(around ⫺70 mV in renal cells), which is important for
electrogenic transport of substances, and recirculate the potassium entered into the cells via the Na⫹-K⫹-ATPase. This
second function is crucial in epithelia in general, and in
renal cells in particular, where high-rate transepithelial Na⫹
absorption occurs. The “recycling” of potassium was proposed as early as in the 1950s when Koefoed-Johnsen and
Ussing (196) produced their seminal scheme of Na⫹ absorption in frog skin. Several studies have documented a
possible coupling between the pump and the potassium
channels in the basolateral membranes of the renal tubule
(203, 266, 393, 407). It should be emphasized however
that the apical K⫹ channels, in addition to their specific
function of secreting potassium (see next section), may
participate in K⫹ recycling together with basolateral K⫹
channels. Indeed, this phenomenon can be understood as
the necessity of balancing K⫹ influxes and effluxes across
the cell membranes. The apical or basolateral location of
efflux may then be considered as secondary. However,
the basolateral K⫹ conductance is generally higher than
the apical one and plays a dominant role, at least in the
proximal tubule (212).
In the past, several groups using microelectrode recording
techniques have described the properties of basolateral K⫹
conductance in most renal segments of the amphibian, rabbit, rat, and mouse kidneys. In most cases, a large K⫹ conductance highly sensitive to barium was detected (320, 347,
349, 371, 432, 433), anticipating the discovery that Kir
channels had a major importance in renal function. It was
also observed that intracellular acidification depolarizes the
cell membrane in the proximal tubule, likely by inhibiting
K⫹ conductance (24, 28) (see also Ref. 212). With the availability of the patch-clamp technique, a wealth of information on the basolateral channels was then obtained showing
that many distinct K⫹ channels were present in the renal
tubule (23, 155, 156, 210, 237, 241, 254, 255, 310, 336,
337, 385; see also Ref. 145).
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pH-GATED K2P AND Kir TRANSPORT CHANNELS
A
B
Proximal
tubule
Glucose
Amino acids
Phosphate
Na+
2 Cl–
K+
3Na+
2K+
Na+
K+
?
C
Thick
ascending
limb
Kir6.1
Kir4.2 ?
Kir5.1 ?
TASK2 ?
K+
Kir1.1
80pS
K+
Cl–
D
Distal
convoluted
tubule
Cl–
Kir4.1/Kir5.1
KCa4.1
Collecting
tubule/
collecting
duct
3Na+
Cl–
3Na+
2K+
Na+
2K+
3Na+
2K+
Kir4.1/Kir5.1
Na+
K+
K+
Cl–
Kir1.1
KCa1.1
K+
Kir4.1/Kir5.1
20 & 80pS
FIGURE 5. Mechanisms of sodium reabsorption in the mammalian renal tubule. A: proximal tubule. Many
distinct Na⫹-coupled transport systems are present on the apical membrane of the proximal tubule. On the
⫺
basolateral side, together with the Na⫹/HCO3
cotransporter, many facilitated transport systems (not represented) allow the exit of glucose, phosphate, etc. The K⫹ channels have not been identified with certainty,
except for Kir6.1, an ATP-sensitive K⫹ channel. B: thick ascending limb. The Na⫹-K⫹-2Cl⫺ cotransporter
NKCC2 is the main actor of NaCl reabsorption in the apical membrane. KCa4.1 (also named slo2.2) is a
high-conductance Na⫹ and Cl⫺-sensitive channel; 80pS represents an unidentified channel type. C: distal
convoluted tubule. The Na⫹ and Cl⫺ cotransporter NCC is present on the apical membrane. The chloride
channels in the thick ascending limb and the distal convoluted tubule are ClC-K1 and ClC-K2 (ClC-Ka and ClC-Kb
in humans) associated with the regulatory subunit Barttin. D: connecting tubule/collecting duct. Sodium
enters the cell from the apical side via the epithelial Na⫹ channel, ENaC. In this renal segment, part of sodium
is exchanged for potassium, while some chloride is absorbed through the intercalated cells or the paracellular
pathway. KCa1.1 (also named Slo1, maxi K or BK) is a high-conductance calcium-activated channel; 20 & 80pS
represent as yet unidentified channel types.
B. The Population of Basolateral Kⴙ
Channels in the Proximal Tubule Includes
Kir Channels and the K2P TASK-2
Channel
The proximal tubule was one of the first segments to be
searched for K⫹ channels. In the amphibian proximal tubule, whole cell recording studies reported a dominant K⫹
conductance that was barium sensitive and inwardly rectifying (172, 336, 357). At the single-channel level, a 25- to
30-pS, inwardly rectifying channel was described (172,
190, 254, 255), the properties of which were studied in
great detail by Mauerer et al. (254): the channel was inhib-
ited by ATP (⬎2 mM), blocked by glibenclamide, a classical
inhibitor of KATP channels, but only at very high concentrations (0.5 mM), and activated by the channel opener diazoxide. The channel also required ATP for maintaining its
activity in cell-free patches (254, 255). In the rabbit proximal tubule, several studies also reported the properties of an
inwardly rectifying K⫹ channel with a conductance of
40 –50 pS (23, 116, 173, 303) that was sensitive to ATP (23,
173). The group of Lapointe proposed that this channel
belonged to the KATP family (40). In contrast, there is no
precise description of K⫹ channels in the mouse or the rat
proximal tubule. Likewise, their molecular identity remains
undetermined. Kir4.1 is clearly not present in the proximal
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191
SEPÚLVEDA ET AL.
tubule (33, 178, 210, 334), and the presence of Kir5.1 is
controversial (82, 210, 237, 394). Kir4.2, detected in human kidney (123, 362), associated or not with Kir5.1,
might constitute a pHi-dependent, inwardly rectifying potassium channel in the proximal tubule.
The expression of TASK-2 is more firmly established: using
TASK-2 knockout (KO) mice in which the LacZ marker
gene was inserted, Barrière et al. (19) observed TASK-2
promoter-driven staining by ␤-galactosidase all along the
proximal tubules. In addition, recordings of whole cell currents from proximal cells in primary culture demonstrated
that cell swelling-activated K⫹ currents, inhibited by quinidine and clofilium, were present in WT but not in TASK-2
KO cells. A follow-up study on primary cultures of proximal cells further demonstrated that TASK-2 currents were
activated during HCO3⫺ transport provided that extracellular pH is increased (209, 406). These interesting results led
Warth et al. (406) to hypothesize that TASK-2 activity
might be linked to extracellular pH variations and help
maintaining membrane polarization during HCO3⫺ reabsorption. However, it is not known whether bulk reabsorption of Na⫹, HCO3⫺, and water is associated with a measurable local increase in pH at the basolateral side of the
proximal tubule cells. In addition, the expected presence of
TASK-2 K⫹ channels in the basolateral membrane has not
yet been ascertained. Likewise, no patch-clamp study has
identified TASK-2 in the native proximal tubule. Nevertheless, the fact that TASK-2 KO mice appeared to have a
reduced maximum capacity for HCO3⫺ transport in vivo
and exhibited slight acidosis are a strong argument in favor
of Warth et al.’s hypothesis (406).
C. Kir4.1/Kir5.1 Supports a Major
Basolateral Kⴙ Conductance in the Distal
Nephron
The distal nephron comprises the thick ascending limb,
early and late distal convoluted tubule, connecting tubule,
and collecting duct; of note, the two latter segments are
composed of different cell types, i.e., the principal and intercalated cells. As in the proximal tubule, the basolateral
K⫹ conductance in the distal nephron is dominated by a
Ba2⫹-sensitive conductance (127, 347, 349, 432, 433) that
was shown to be inhibited at acid pH in the rat collecting
duct (348). In addition to the Kir4.1/Kir5.1 dominant type
described below, several additional K⫹ channels have been
described: a Na⫹-dependent high-conductance K⫹ channel
of the slo2.2 type (KCa4.1) in the mouse thick ascending
limb (309) and various channels in the rat and mouse collecting ducts [Ca2⫹-inhibited, 200-pS channel (155); 85-pS
channel (402); 20-pS channel stimulated by cGMP-dependent protein kinase (403, 407) probably underlain by
Kir2.3 (258, 405, 408)].
192
However, the predominant channel along the distal nephron
is an inwardly rectifying channel, of ⬃50 pS which was
initially reported in the rabbit thick ascending limb and
distal convoluted tubule (174, 385). More recent studies
have concentrated on the distal nephron of the mouse where
a major channel with an inward conductance of 40 – 45 pS
was described in the thick ascending limb, distal convoluted
tubule, connecting tubule, and cortical collecting duct (210,
237, 308, 310). This channel is the only one detected in the
distal convoluted tubule (237). Inward rectification with a
Gin-to-Gout ratio of 3–5 was due to intracellular Mg2⫹ (210,
237, 310). As is the case for other K⫹ channels, the inward
unit conductance was dependent on external K⫹ concentration over a large range of concentrations (Km ⫽ 65 mM,
Gmax ⫽ 48 pS) and the channel was inhibited by spermine,
which reduced the Po (210, 237, 310). In outside-out
patches, Ba2⫹ at 0.1 mM blocked the channel by 70%. In
addition, following excision, channel activity ran down
progressively in the presence of Mg2⫹ (237). The channel
exhibited great sensitivity to pH (pKa ⫽ 7.2–7.6) but was
not inhibited by ATP (210, 237, 310). All these properties
compare well with the general properties of Kir K⫹ channels and more particularly with those of Kir4/Kir5, prompting the proposal that they are Kir4.1/Kir5.1 heterotetramers (210, 237). The renal localization of Kir4.1 and Kir5.1
channels, examined using immunofluorescence in the rat,
mouse, and human kidneys (33, 178, 210, 237, 315, 334),
is coherent with this hypothesis. The general pattern shows
that Kir4.1 and Kir5.1 are present in the basolateral membranes of the distal convoluted tubule and principal cells of
the connecting tubule and cortical collecting duct in mouse,
rat, and human. Kir4.1/Kir5.1 is also present in the thick
ascending limb of humans and CD1 mice (210, 334) but not
in the rat (178) or in the C57BL6 mouse strain (33, 334). In
addition, it was found that Kir4.1 and Kir5.1 coimmunoprecipitate in rat renal tissue (381). According to these data,
it is probable that the two channels exist as heterotetramers
in kidney cells. Using Kir5.1 KO mice, Paulais et al. (308)
showed that in the absence of Kir5.1, the only K⫹ channel
present in the distal convoluted tubule had a conductance of
⬃25 pS (instead of 45 pS in WT mice) and was independent
of pH in the range 6.8 –7.8. This is typical of the properties
of Kir4.1 described in heterologous expression systems and
further documents that the K⫹ channel in the mouse distal
convoluted tubule is composed of Kir4.1/Kir5.1. It should
also be noted that the number of K⫹ channels was not
reduced in the absence of Kir5.1 (308), making evident that
Kir5.1 is not necessary for targeting Kir4.1 to the membrane.
Kir4.1/Kir5.1 is therefore a major player in the control of
basolateral membrane potential and in recycling potassium
entered into the cell via the Na⫹-K⫹-ATPase. This could
hold particularly true for the distal convoluted tubule compared with other renal segments because of Kir4.1/Kir5.1
predominance in this segment: this is the only K⫹ channel
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pH-GATED K2P AND Kir TRANSPORT CHANNELS
described so far in the basolateral membrane of this segment (237).
In conclusion, Kir5.1 provides Kir4.1 channel with great
sensitivity to pHi. Small intracellular acidification due to
lowering of external pH in perturbed acid-base condition
(26) could then result in a large decrease of K⫹ conductance
and possibly a reduction in renal reabsorption of NaCl. It
should be borne in mind that pH sensitivity can be modulated by PIP2 (328, 330) and via phosphorylation-dephosphorylation (340, 436, 438). Thus channel regulation
might not proceed directly via pHi variations but indirectly
by additional factors modulating the sensitivity to pHi. In
addition, Kir4.1 interacts with the CaSR calcium receptor
(48, 168) and several PDZ-domain proteins (164, 378,
382). All these agents are possible physiological modulators
of Kir4.1-Kir5.1 activity.
D. Mutations Affecting Kir4.1 and Renal
SeSAME-EAST Disease
Several laboratories have recently reported SeSAME-EAST
syndrome, a new multiorgan syndrome, due to mutations in
the KCNJ10 gene encoding Kir4.1 K⫹ channel (33, 350,
351). Patients display tonic-clonic seizures in infancy,
speech and motor delay, and ataxia. Sensorineural hearing
loss and mental retardation are frequent. The central (33) or
peripheral (350) origin of ataxia is a matter of debate (15,
351). About 10 disease-related mutations have been reported, which increase sensitivity to protons or reduce
channel surface expression (304, 334, 345, 379, 384, 414).
Coexpression of mutant Kir4.1 with WT Kir5.1 does not
restore impaired Kir4.1 function, while coexpression with
WT Kir4.1 does (334, 345, 384; for a review, see Ref. 15).
Recently, a mutation in the KCNJ10 gene has been associated with juvenile-onset spinocerebellar ataxia in terrier
dogs, but the functional effect of this predicted single point
mutation at the COOH terminus of the channel has not
been studied (112).
In addition to their symptoms in the nervous system,
SeSAME-EAST patients develop a renal disease characterized by sodium wasting, secondary hypokalemia, and metabolic alkalosis associated with hypomagnesemia and hypocalciuria. It was suggested that Kir4.1 dysfunction induced partial inhibition of NaCl reabsorption in the distal
convoluted tubule, likely because of an impaired K⫹ recycling across the basolateral membrane (15, 33, 334, 350).
According to Paulais et al. (308), Kir5.1 KO mice develop a
partially converse disease with acidosis, hypokalemia, and
hypercalciuria. A functional assay in vivo using thiazide
inhibition of distal convoluted tubule transport demonstrated that Kir5.1 KO mice displayed an increased transport activity in the distal convoluted tubule, likely due to an
increased K⫹ conductance. Taken together, these data indicate that modifications in Kir4.1/Kir5.1 channel activity
result in altered transport capacity in the distal convoluted
tubule, and establish a functional link between Na⫹-K⫹ATPase and Kir4.1/Kir5.1 channel activity in this segment.
It can be speculated that modifying Kir4.1/Kir5.1 has less
effect on other renal segments because the latter are endowed with additional potassium channels.
E. Apparent Absence of Kir Kⴙ Channels at
the Apical Membrane of Proximal Tubules
Most of the studies devoted to the apical K⫹ channels in the
proximal tubule were done in the beginnings of the patchclamp technique and provided little information on channel
molecular identity (115). The general view is that apical K⫹
channels in the proximal tubule should be able to counteract membrane depolarization induced by the coupled transport of substrates such as glucose, amino acids, or phosphate together with Na⫹ (145, 212, 269, 396). Notice that
the proximal tubule being a leaky epithelium, this role can
equally be achieved by basolateral channels. In possible
agreement with this requirement, KCNQ1 (396) and regulatory subunit KCNE1 (375), which form a depolarizationactivated K⫹ channel, were found in the apical membrane
by immunohistochemistry. However, KCNQ1 KO mice
have no renal phenotype (397) in contrast to KCNE1 KO
mice, which exhibit a moderate increase in NaCl and water
excretion (269, 396). Neal et al. (269) supported the idea
therefore that KCNE1 regulated a K⫹ channel distinct from
KCNQ1 in the proximal tubule on the basis of pharmacological inhibition. As far as we currently know, the inwardly rectifying K⫹ channels do not play any role at the
apical side of the proximal tubule. Indeed, studies published
so far discard the presence of the following channels at the
apical side of the proximal tubule: Kir 1.1 (145, 200, 409),
Kir4.1 (33, 178, 210, 334), and Kir7.1 (81, 294).
F. Kir1.1 Is Necessary for Potassium
Recycling in the Thick Ascending Limb of
the Henle Loop
A small-conductance K⫹ channel, rapidly ascribed to
Kir1.1, was detected in the apical membrane of the thick
ascending limb of the rabbit, rat, and mouse, together with
a second 80-pS conductance K⫹ channel (32, 239 –241).
The identity of the small-conductance K⫹ channel has been
definitely demonstrated by its absence in Kir1.1 KO mice
(240). That of the 80-pS K⫹ channel has not been elucidated
yet, but it has been proposed that Kir1.1 participates to its
formation since it is no longer observed in Kir1.1 KO mice
(240). The 80-pS K⫹ channel, therefore, is not expected to
compensate for the loss of Kir1.1. The pioneering work of
Rainer Greger’s laboratory in the 1980s described the
scheme for NaCl reabsorption in the thick ascending limb
(129 –133) based on experiments using the then novel technique of isolated microperfused tubules (201). The model
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SEPÚLVEDA ET AL.
introduced in a paper by Greger et al. (130) proposes that
recycling of potassium across the apical membrane was
strictly necessary to the process of reabsorption. At the
time, this observation was linked to the necessity of maintaining a sufficiently high K⫹ concentration in the lumen of
the renal tubule to keep the Na⫹-K⫹-2Cl⫺ cotransporter
working (105). However, it has been contended that the
luminal K⫹ concentration at the beginning of the distal
convoluted tubule was increased to ⬃8 mM in Kir1.1 KO
mice (12), and could not therefore be considered limiting
for NaCl reabsorption via the Na⫹-K⫹-2Cl⫺ cotransporter
(129). Likely, Na⫹ absorption in the thick ascending limb
of Kir1.1 KO mice is reduced, not only because recirculation towards the tubule lumen is reduced, but because K⫹
recycling at both membranes, via basolateral (Kir4.1/
Kir5.1, KCa4.1) and apical (80 pS, Kir1.1) K⫹ channels, is
necessary to maintaining steady-state [K⫹]i and normal reabsorption of NaCl in this segment. The thick ascending
limb is the only renal segment where NaCl reabsorption is
accompanied by massive K⫹ entry both at the apical (via the
Na⫹-K⫹-2Cl⫺ cotransporter) and basolateral (via the Na⫹K⫹-ATPase) sides. In summary, it could be concluded that
the loss of Kir1.1, by decreasing global K⫹ conductance,
critically impairs the overall capacity of the TAL cells to
counterbalance the K⫹ influx experienced during the process of NaCl absorption.
G. Kir1.1 Is a Key Player in Kⴙ Secretion in
the Connecting Tubule and Collecting
Duct
The control of kalemia is another crucial function of the
kidneys, achieved according to the following scheme. Following glomerular filtration, most of the potassium is reabsorbed passively in the proximal tubule, thick ascending
limb, and inner collecting duct. In the usual state of a potassium-rich diet, the final balance of potassium is achieved
in the distal nephron via apically located K⫹ channels (111,
404). Frindt and Palmer (102) were the first to identify a
small-conductance inwardly rectifying K⫹ channel in the
apical membrane of the rat collecting duct, which was further studied by Wang et al. (405). Many properties of this
channel in rat and mouse collecting duct have been described, including high sensitivity to pHi, activation by
PKA, and inhibition by PKC (111, 404). After the cloning of
Kir1.1, it was rapidly accepted that the so-called small secretory potassium channel was underlain by Kir1.1, due to
similar conductance and rectification, as well as cation selectivity sequence and sensitivity to pHi (299). This was
confirmed by immunofluorescence studies showing the location at the apical membrane of the collecting duct of rat
and mouse, but also of the late distal convoluted tubule,
connecting tubule, and thick ascending limb (103, 200,
404, 409). Furthermore, patch-clamp studies provided
compelling evidence that Kir1.1 was active in the apical
membranes of the connecting tubule (103). Aldosterone
194
and kalemia are responsible for the modulation of potassium absorption/secretion in the distal nephron (111, 200,
300, 404, 409). In particular, changes in external K⫹ concentration are by themselves powerful modulators of Kir1.1
channels, the mechanisms of which have been studied intensively and have been reviewed elsewhere (111, 404).
Although Kir1.1 is the major channel in potassium secretion, evidence has progressively emerged that the high-conductance, calcium-activated K⫹ channel (KCa1.1, slo1.1) is
also involved in potassium secretion (12), probably at the
level of the CNT (103). In a very elegant study, Bayley et al.
(12) established that K⫹ secretion was still present in Kir1.1
KO mice and supported by KCa1.1. This channel had been
previously detected in the intercalated cells of the collecting
duct (298). Other K⫹ channels have been postulated at this
site, where their functional role remains elusive. This includes TWIK-1, reported by Millar et al. (257) as a Ba2⫹insensitive, quinidine-sensitive, outwardly rectifying K⫹
current in principal cells of the collecting duct, which were
absent in TWIK-1 KO mice. In agreement with the recent
findings on TWIK-1 cation selectivity, this conductance did
not have a high K⫹-to-Na⫹ selectivity ratio.
In conclusion, Kir1.1 plays a crucial role in the renal tubule,
both for K⫹ recycling (in the thick ascending limb) and K⫹
secretion (in the collecting duct). On the short term, acidemia reduces while alkalemia increases K⫹ secretion in the
distal nephron (7). This effect is probably transmitted via
variations of pHi inhibiting the Na⫹-K⫹-ATPase and the
apical K⫹ channels (7). Thus sensitivity to pHi might be a
physiological modulator of Kir1.1 activity. It should be
noted that the Po of Kir1.1 is very high at pH 7.4, implying
that intracellular acidification rather than alkalinization
would modulate channel activity. However, Kir1.1 as other
Kir channels is sensitive to a myriad of factors; as noted
above for Kir4.1/Kir5.1 channel, the activity of Kir1.1
might be modulated by shifts of the pHi sensitivity curve
possibly via cAMP-dependent phosphorylation (214, 216).
In addition, as far as Kir1.1 is concerned, channel trafficking to the membrane is held to be the most important physiological regulator (409).
H. Kir1.1 in Bartter Syndrome and Effect of
Its Inactivation in the Mouse
Bartter syndrome is a hereditary renal disease with autosomal recessive transmission, resulting from mutations in
genes encoding ion transporting proteins in the thick ascending limb. There are four variants all characterized by
salt wasting, normal to low blood pressure, hypokalemic
alkalosis, and secondary hyperaldosteronism (for reviews,
see Refs. 39, 318, 360, 395). Bartter syndrome type II resulting from mutations in the KCNJ1 gene encoding Kir1.1
(364) has a severe, antenatal presentation, the major symptoms including prematurity, polyuria, hyperprostaglandin-
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pH-GATED K2P AND Kir TRANSPORT CHANNELS
uria, hypercalciuria, and frequent nephrocalcinosis. A large
number of KCNJ1 mutations have been described, affecting
protein abundance, targeting to the apical membrane, or
channel regulation (317, 353, 356, 364).
tion by parietal cells is shown on FIGURE 6. The main actor
in the secretory process, the H⫹-K⫹-ATPase, is inserted in
the apical membrane upon gland stimulation by fusion of
tubulovesicles containing the H⫹-K⫹-ATPase, which
largely expand secretory canaliculi of the apical membrane.
Stimulation also activates Cl⫺ and K⫹ conductances in the
luminal membrane, which are silent in resting cells. The
initial secretion of K⫹ and Cl⫺ ions drives secondarily
the onset of the H⫹-K⫹-ATPase, which needs exchanging
K⫹ for H⫹ for its functioning. The protons originate from
the hydration of CO2 within the cell in the presence of
carbonic anhydrase producing H⫹ and HCO3⫺. The HCO3⫺
anion exits the cell at the opposite, basolateral membrane
side via HCO3⫺/Cl⫺ exchangers. Also present in the basolateral membrane, Na⫹-K⫹-2Cl⫺ cotransporter NKCC1
supplies the cell with chloride together with the anion exchanger. As in most epithelia, the Na⫹-K⫹-ATPase is present in the basolateral membrane where it coexists with K⫹
channels. Several K⫹ channels have been described in the
basolateral membrane using the patch-clamp technique on
isolated parietal cells from different species, but their molecular identity has not been elucidated (148, 149).
Lorenz et al. (233) created a Kir1.1 KO mouse model for
Bartter syndrome type II. The phenotype is very severe, with
95% of the mice dying within 21 days. A disorganization of
the medulla, indicative of hydronephrosis, was apparent in
young and adult mice. Adult Kir1.1 KO mice exhibited
polyuria, polydipsia, volume depletion, low blood pressure,
and huge urinary concentrating defect, as expected for salt
wasting disease targeting the thick ascending limb (46,
233). Unexpectedly however, homozygous mice were acidotic and showed normal kalemia. As glomerular filtration
rate was reduced by a factor of 10, a likely consequence of
a reduced number of nephrons (233), renal insufficiency
may explain metabolic acidosis (179). Lu et al. (240) analyzed in detail inbred Kir1.1 KO mice having a high survival
rate and showed that they recapitulate most of the features
of Bartter syndrome type II, except for their normal kalemia.
The channels on the apical membrane cannot be studied
directly by electrophysiological methods. A number of studies have tried identifying K⫹ channels in the apical membrane by biochemical, immunofluorescence, and physiological assays. A large body of evidence favors the implication
of KCNQ1 (148, 149, 189); the channel and accessory subunit are found at the apical membrane and intracellular
tubulovesicles but not in the basolateral membrane, and a
IV. Kir4.1 IN PARIETAL CELLS OF THE
STOMACH AS A COMPONENT OF THE
ACID SECRETION MACHINERY
The parietal cells in the oxyntic glands of the stomach are
able to secrete protons at high rate when stimulated by
hormones and mediators that include gastrin, histamine,
and acetylcholine (148, 149). The scheme of proton secre-
A
B
S
3Na+
2K+
K+
K+
Na+
2 Cl–
K+
P
H+
K+
Cl–
HCO3–
Cl–
Kir4.1
KCNQ1/KCNE2
Kir4.2 ?
Kir2.1 ?
FIGURE 6. Proton secretion in parietal
cells of the stomach. A: histology of mouse
gastric mucosa. Cells producing mucous (S)
cover the surface of the mucosa. Cells secreting acid are located within gastric glands
(dark cells, P). B: cell model for acid secretion
in gastric parietal cells. Protons are secreted
by the H⫹-K⫹-ATPase in exchange for potassium, which recycles via K⫹ channels. Cl⫺
⫺
enters the cell via Cl⫺/HCO3
exchanger and
Na⫹-K⫹-2Cl⫺ cotransporter NKCC1. Cl⫺ is
secreted by an apical Cl⫺ channel. [From
Heitzmann and Warth (149).]
50 µm
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195
SEPÚLVEDA ET AL.
chromanol inhibitor of KCNQ1 channel led to partial or
total inhibition of acid secretion in mice, rats, and dogs (8,
126, 189). Moreover, KCNQ1 KO mice that were created
for investigating arrhythmias and deafness caused by mutations in this channel gene unexpectedly showed stomach
enlargement, a defect in stomach acidity and elevated gastrin levels (215). KCNQ1 is apparently associated with
KCNE2, which modifies the conductive and regulatory
properties of KCNQ1, rendering it voltage independent and
most importantly insensitive to external pH in contrast to
KCNQ1 alone (147). It was recently shown that KCNE2
KO mice showed impaired acid secretion, hypergastrinemia, and glandular hyperplasia (338).
Kir channels are also present in the apical membrane of
parietal cells. One early finding (332) was that proton secretion could be inhibited by application of barium, a nonspecific blocker of inwardly rectifying K⫹ channels. More
recently, the presence of Kir4.1, Kir4.2, and Kir7.1 was
detected in rat parietal cells (104, 189). According to Fujita
et al. (104), Kir4.1 was present solely at the apical membrane but not in the intracellular tubulovesicles or at the
basolateral membrane. In contrast, work from the group of
Seidler and colleagues (189) showed that Kir4.1 and
KCNQ1 colocalized with the H⫹-K⫹-ATPase, although in
distinct tubulovesicular pools. Such localization suggested
that Kir4.1 might participate in the process of gastric acid
secretion. Indeed, it was shown that blocking KCNQ1 completely did not totally inhibit acid secretion but rather delayed it (189). In addition, Kir4.1 fully translocated to the
apical membrane upon stimulation, whereas KCNQ1 remained mostly cytoplasmic. The same workers succeeded in
examining the gastric acid secretion in Kir4.1 KO mice,
which die very early (366). They report that, unexpectedly,
rather than being impaired acid secretion is faster in KO
mice than WT animals. This might be due to the contrasting
morphologies of the parietal cells in the two types of mice:
in the resting state, membranes of the tubulovesicular system are clearly detected close to the canaliculi in WT mice
but are absent in KO mice. In addition, the microvilli of the
canaliculi were more elaborate (without the lumen being
expanded). This was explained by the absence of Kir4.1
impairing the endocytic machinery. As a result, there was a
permanent high expression of the H⫹-K⫹-ATPase in the
apical membrane, resulting in a rapid onset of acid secretion. In summary, KCNQ1/KCNE2 and Kir4.1 K⫹ channels appear to have complementary roles in gastric acid
secretion. Kir4.1 is well suited for participating in the process of proton secretion since its activity is independent of
extracellular pH (104) and does not change much around
the expected physiological pHi (see FIGURE 2). It is likely
that additional K⫹ channels participate in the proton secretion process since there are reports that Kir4.2 and Kir2.1
channels are present on the apical membrane of parietal
cells (144, 249).
196
V. Kir4.1 IS THE DOMINANT Kⴙ
CHANNEL OF GLIAL CELLS: ROLE IN
Kⴙ BUFFERING
A. Müller Cells and Kⴙ Siphoning in the
Retina
Müller cells, the major glial cells in the retina, extend
through the whole thickness of the tissue from proximal
aspect, where endfoot processes envelop blood vessels and
the vitreal endfoot is adjacent to the vitreous body, to the
distal portion which forms villi adjacent to the pigment
epithelium. As in other neuronal tissues, K⫹ ion tends to
accumulate during cell activity within the confined external
space and needs to be removed. Eric Newman was the first
to recognize that Müller cells had an essential function in
this respect; he elaborated the so-called “K⫹ siphoning”
concept also recognized for glial cells of the CNS (K⫹ clearance) (272). This paradigm proposes that K⫹ excess is not
removed by simple extracellular diffusion but involves longitudinal K⫹ transport within the Müller cells. As demonstrated in the nonvascularized retina of the salamander
(272), Müller cells take up K⫹ where its concentration is
elevated and release it for clearance at the endfeet, close to
the large reservoir of vitreous humor, via K⫹ channels. Accordingly, the K⫹ conductance along the Müller cell was
highest at the endfeet close to vitreous body and declined
steadily in the soma and the distal end, where it was very
low (274). Rather similar profiles were found in other nonvascularized retinas (274). Newman (275, 276) also
showed that the major part of the K⫹ conductance in the
amphibian Müller cell was inwardly rectifying, depended
on external K⫹, and was blocked by Ba2⫹. The single-channel variant of the patch-clamp technique allowed describing
an inwardly rectifying K⫹ channel with a conductance of 28
pS present in all parts of the Müller cell but with highest
density at the endfeet (38, 276). Additional types of K⫹
channels have also been recorded at low to very low frequency in various species (38, 273, 274, 285, 333, 365).
The K⫹ conductance shows a different pattern in vascularized retinas, being maximal at the endfeet (close to the vitreous body) and blood vessels, but not negligible at the
distal end (close to the pigment epithelium), while showing
a secondary peak in the soma close to capillaries (274, 333).
Newman (274) concluded that “in vascularized retinas, excess of K⫹ is transferred into retinal capillaries as well as
into the vitreous.” One type of inwardly rectifying potassium channel has been described in rabbit and rat Müller
cells (177, 208, 376), and it seems to correspond to the
activity of Kir4.1: the properties of the native channel were
quite similar to those of Kir4.1 homotetramer (177, 376);
immunofluorescence established the presence of Kir4.1 protein in the plasma membrane of Müller cells but not that of
neurons (176, 197); and Kofuji et al. (197) indirectly demonstrated that channel activity disappeared in Kir4.1 KO
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pH-GATED K2P AND Kir TRANSPORT CHANNELS
mice. As expected, the distribution of the channel along the
Müller cell was highest close to the capillaries and endfeet
(176, 197).
Overall, the major role of Kir4.1 in the second step of K⫹
clearance, that of evacuating K⫹ in excess from the Müller
cells towards capillaries or vitreous body, is well documented (45, 198, 199, 293). The mechanism of K⫹ flux into
Müller cells for removing K⫹ from the regions where external K⫹ concentration is most elevated is far less well established. The influx may theoretically be due to two contrasting mechanisms, active uptake via Na⫹-K⫹-ATPase and
Na⫹-K⫹-2Cl⫺ cotransporter on the one hand, and K⫹
channels on the other. The latter mechanism can only operate if K⫹ accumulation in a limited extracellular region
between neurons and Müller cells locally reverses the K⫹
driving force while membrane potential remains comparatively stable. This is generally considered as plausible in the
setting of the retina (45, 198, 199, 293). The localization of
Kir4.1 is not incompatible with this channel mediating influx as well as efflux: Kir4.1 is also present in the inner and
outer plexiform layers where external K⫹ is dramatically
increased by light (177).
It should also be mentioned that Kir5.1 has been detected in
the cell body and distal parts of the Müller cells where it
could form heterotetrameric channels with Kir4.1 (176)
while homomeric channels would be concentrated adjacent
to capillaries and vitreous body (176, 177). Ishii et al. (176)
proposed that pH sensitivity of Kir4.1-Kir5.1 channels
might be important in K⫹ uptake: an increase of external
K⫹ would depolarize the membrane and stimulate HCO3⫺
uptake by the electrogenic Na⫹-HCO3⫺ cotransporter, inducing alkalinization and promoting positive feedback for
the activity of the channel. Additional K⫹ channels including Kir2.1 are thought to mediate K⫹ influx into Müller
cells (see Refs. 198, 293).
B. Kⴙ Spatial Buffering: A Role for
Astrocytes in the CNS
Astrocytes help stabilize the extracellular environment
around neuronal cells. In particular, they are involved in
removing K⫹ accumulated in the narrow CNS extracellular
space and glutamate released at the synapses (198, 199,
293). According to the K⫹ spatial buffering mechanism postulated by Orkand et al. (296), glial cells transfer K⫹ ions
from high to low K⫹ regions adjacent to capillaries by
means of K⫹ channels. This mechanism is very similar to the
one described above for Müller cells, considering that astrocytes form a syncytium network communicating through
gap junctions and maintaining an ensemble membrane potential. In regions with elevated K⫹, the locally less negative
K⫹ Nernst potential will reverse the driving force for K⫹,
allowing inward K⫹ current (via Kir K⫹ channels), K⫹ flow
along the glial network, and K⫹ exit in regions of low ex-
ternal K⫹ (also via Kir K⫹ channels). Nevertheless, as further discussed below, whether the uptake of K⫹ depends on
Kir K⫹ channels or proceeds by means of other K⫹ transport systems (Na⫹-K⫹-ATPase, Na⫹-K⫹-2Cl⫺ cotransporter) remains a matter of debate.
The electrophysiological profile of astrocytes is heterogeneous in terms of resting membrane potential and K⫹ currents (73), including the so-called complex and passive, or
mature astrocytes. Only the passive astrocytes seem to be
connected through gap junctions. However, it is largely
accepted that Ba2⫹-sensitive inwardly rectifying currents
predominate at least in a subpopulation of astrocytes (13,
292, 327; see also reviews in Refs. 199, 293). Other types of
glial K⫹ channels, nevertheless, have been described in different CNS regions (45, 109, 114, 141, 367, 439), including
TWIK-1 and TREK-1 K2P channels (401, 442). Kir4.1,
which is highly expressed in glial cells (35, 45, 183, 377)
while its presence in neurons is restricted to specific brain
regions (418, 424), is emerging as a key component of glial
K⫹ conductance (see Refs. 198, 199, 293). At the singlechannel level, Zhang and Verkman (439) reported a Kir4.1like inwardly rectifying K⫹ channel displaying weak inward
rectification, 20-pS conductance, high Po, and inhibition by
external Ba2⫹ in hippocampus glial cells. In line with this
report, it is generally considered that Kir4.1 homotetramers
underlie K⫹ conductance in most glial cells even if Kir4.1/
Kir5.1 heterotetramers might be functional in specific brain
regions (151). Taking advantage of the existence of total
and glia-specific Kir4.1 KO mice, two groups showed that
the resting membrane potential was less negative and input
resistance increased in Kir4.1 KO glial cells (83, 270). The
same observation was done in spinal cord astrocytes where
Kir4.1 expression was knocked down with shRNA (292).
In addition, inwardly rectifying K⫹ currents were no longer
visible when Kir4.1 was absent, verifying that these currents
were supported by Kir4.1 (83, 270, 292, 384).
The possible implication of Kir4.1 channels in K⫹ spatial
buffering has been evaluated using glia-specific Kir4.1 KO
mice. Trains of electrical stimulation in the hippocampus
induced a comparable fast increase in external K⫹ in WT
and KO mice, which was followed by exponential decay.
Although slower, this decay was still present in glia-specific
Kir4.1 KO mice (55), which implies that Kir4.1 is not
strictly necessary for K⫹ clearance. After repetitive longlasting stimulations, a phenomenon called [K⫹]o undershoot is observed, which is attributed to stimulated activity
of the Na⫹-K⫹-ATPase: [K⫹]o reaches a plateau that is
lower than the one before stimulation (72). The undershoot
was magnified in glia-specific Kir4.1 KO mice (55). This
observation is in accordance with the hypothesis of
D’ambrosio et al. (72) that Kir channels might be crucial for
K⫹ recirculation towards the extracellular compartment
following K⫹ uptake by the Na⫹-K⫹-ATPase. Similar results were obtained by Neusch et al. (270) in the ventral
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SEPÚLVEDA ET AL.
respiratory group and by Haj-Yaein et al. (139) in the hippocampus. As a whole, these studies disclose that Kir4.1 is
not strictly necessary for K⫹ clearance, but rather that it
plays a major part in setting the membrane potential of glial
cells and the level of resting [K⫹]o. Compensation via alternative mechanisms to K⫹ spatial buffering probably occurs.
Such an alternative pathway involves K⫹ uptake through
the Na⫹-K⫹-ATPase and Na⫹-K⫹-2Cl⫺ cotransporter (13,
72, 246; see also Refs. 199, 293).
Tong et al. (390) studying mouse models of Huntington’s
disease (HD) have provided evidence that alteration in astrocyte maintenance of extracellular K⫹ due to downregulation of Kir 4.1 channels correlates with depolarization of
striatal astrocytes and the onset of motor symptoms. HD is
an autosomal dominant disease and characterized by chorea, cognitive, and neuropsychiatric defects caused by polyglutamine expansion at the NH2 terminus of the protein
huntingtin. The mechanism by which the mutated huntingtin (mHTT) leads to degeneration of striatal medium spiny
neurons (MSN) is not completely understood. Striatal but
not hippocampal astrocytes were depolarized by 5 mV and
exhibited lower membrane conductances by up to 20% at
age P60 – 80. These findings were associated with electrophysiological and Western blot analysis evidence of loss of
Kir4.1 channels in striatal astrocytes. The spatial K⫹ buffering impairment elicited a twofold increase in extracellular
K⫹ in the striatum in vivo, and its relationship with MSN
hyperexcitability was established in vitro. Viral-based delivery of Kir4.1 channels to striatal astrocytes rescued the
electrophysiological defects, ameliorated the motor deficits,
and extended the survival of the mutant mice (390). This
interesting study provides a new explanation for MSN hyperexcitability in current mouse models of HD and a potential therapeutic approach. How mHTT impairs astrocyte
Kir4.1 function is not known.
Additionally, Kir4.1 expression in glial cells is developmentally regulated. Bordey and Sontheimer (36, 37) observed
that the percentage of astrocytes showing Kir currents increased from day 5 after birth to day 20 in rat hippocampal
astrocytes in situ. Kir4.1 immunoreactivity was also developmentally regulated in fine processes of astrocytes from
the optic nerve or ventral respiratory group (183, 271).
Likewise, several studies demonstrated that Kir4.1 expression and current increased when progenitor oligodendrocyte cells differentiated into mature oligodendrocytes (248,
271). A similar observation had also been made for Kir
currents in Schwann cells (415). The increasing expression
of Kir4.1 after birth correlates quite well with the increasingly tighter regulation of [K⫹]o, which becomes optimal
around day 20 –30 after birth when Kir4.1 expression is
maximal (293). It can be concluded that Kir4.1 dysfunction
might prevent normal maturation of astrocytes, oligodendrocytes, and Schwann cells, a feature indeed observed in
198
Kir4.1 KO mice and implying that the presence of Kir4.1 is
essential for motoneuron myelination (271).
C. Heterotetrameric Kir4.1/Kir5.1 Channel
in Glial Cells
K⫹ clearance should be quite independent of pHi variations
in all regions where the homotetramer Kir4.1 is the predominant K⫹ channel, i.e., in most glial cells. However, there is
a clear suggestion that Kir5.1 is also present in glial cells.
Hibino et al. (151), investigating the expression of Kir 5.1 in
the mouse brain by immunolabeling, showed that this channel was expressed in astrocytes but not in neurons of the
neocortex and olfactory bulb, where their expression overlapped with that of Kir4.1. The authors contend that heteromeric Kir4.1/Kir5.1 channels were functional in processes around blood vessels and pia mater. In agreement
with these findings, pH-sensitive K⫹ currents that could
possibly be attributed to Kir4.1/Kir5.1 were reported in the
astrocytes of the retrotrapezoid nucleus (410). Other parts
of the mouse brain, such as the hypothalamus and thalamus,
only expressed homomeric Kir4.1 channels (151). Kir5.1 has
also been detected in the cell body and distal parts of the
Müller cells where it could form heterotetrameric channels
with Kir4.1 while homomeric channels would be concentrated
adjacent to capillaries and vitreous body (176, 177). Thus
Kir4.1/Kir5.1 channels seem to be located at the site of K⫹
efflux in astrocytes while they are at the site of K⫹ uptake in
Müller cells. Whatever the exact meaning of these distinct
locations, the presence of Kir5.1 in astrocytes in several parts
of the brain and in Müller cells obviously makes the Kir4.1dominated K⫹ conductance highly sensitive to pH. As mentioned above, the pH sensitivity of Kir4.1/Kir5.1 channels
might contribute to spatial K⫹ buffering through their positive
feedback resulting from the electrogenic activation of the
Na⫹-HCO3⫺ cotransporter (151, 176).
VI. Kir4.1 GENETIC ABLATION IN MICE
REVEALS ITS ROLE IN INNER EAR
FUNCTION AND DISEASE-CAUSING
MUTATIONS SUGGEST NOVEL
PHYSIOLOGICAL ROLES
Two Kir4.1-deficient mouse lineages have been generated,
the first using a standard gene-targeting approach for total
ablation and the second using the Cre/loxP system for specific deletion in glial cells. At birth, total and glia-specific
Kir4.1 KO mice showed no obvious difference with WT
animals, but both rapidly stopped gaining weight around
10 –15 days after birth and could subsequently be easily
identified by their small size. They displayed severe defects
in posture, balance, and movement control and were unable
to perform some simple motor tasks. There was 100% mortality at 4 wk age (83, 197, 271). In addition, Kir4.1 KO
mice had a dehydrated appearance. The investigation of
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pH-GATED K2P AND Kir TRANSPORT CHANNELS
renal function revealed polyuria, renal salt loss, and reduced calcium excretion, as observed in patients with
SeSAME-EAST syndrome (33) (see sect. IIID).
Kir4.1 expression is predominant, seemed normal despite
large membrane depolarization and high input resistance
(197). Kofuji et al. (197) observed that a component of the
electroretinogram, the so-called slow PIII response, was absent in Kir4.1 KO mice, indicating that this response was
due specifically to Kir4.1. Overall, despite the proposed role
of Kir4.1 in the regulation of [K⫹]o in the retina, Kir4.1 KO
mice do not seem to present any important visual alteration.
Analyzing the consequences of the deletion of Kir4.1 in glial
cells, Djukic and co-workers (55, 83) observed that spontaneous neuronal activity was reduced and synaptic potentiation enhanced in hippocampus slices, possibly because of
impaired glutamate uptake and K⫹ clearance by astrocytes.
Neusch et al. (271) described hypomyelination, spongiform
vacuolation, and degeneration events in the spinal cord in
Kir4.1 KO mice, which they considered as possible causes
of motor dysfunction. Kir4.1 is present in oligodendrocytes
and oligodendrocyte precursors where it is important for
membrane potential setting (67, 183, 248, 271, 323).
Kir4.1 appears to be necessary for maturation of cultured
oligodendrocytes as only a small proportion of cultured
Kir4.1 KO oligodendrocytes produces myelin compared
with cells of WT origin (271).
In contrast, the impact of Kir4.1 deletion on hearing is
dramatic. Kir4.1 is mainly expressed in the satellite glial
cells of cochlear ganglia (152) and in the intermediate cells
of the stria vascularis (2), underscoring two distinct functions for Kir4.1 at this site: [K⫹]o control around ganglion
neurons and generation of endocochlear potential (see Ref.
52). K⫹ accumulation within the endolymph (up to 140
mM) and generation of endocochlear potential (up to about
⫹80 mV) are due to the stria vascularis (see FIGURE 7). The
stria vascularis in the inner ear is an elaborate structure
formed by fibrocytes, basal cells, and intermediate cells connected by gap junctions on the perilymphatic side, and by
marginal cells on the endolymphatic side. The fibrocytes
take up K⫹ from the perilymphatic compartment via the
Na⫹-K⫹-ATPase and Na⫹-K⫹-2Cl⫺ cotransporter while
intermediate cells secrete it into the astrial compartment via
Patients affected by EAST syndrome (see sect. IIID) showed
altered electroretinogram response to light flashes (389).
The retina function has been investigated at weeks 2–3 after
birth in Kir4.1 KO mice. The general organization of the
retina as well as the morphology of Müller cells, in which
Perilymph
Intrastiel fluid
Blood
vessel
3Na+
Endolymph
3Na+
2K+
2K+
K+
Na+
2 Cl–
K+
Na+
2 Cl–
K+
K+
Kir4.1
Cl–
KCNQ1/
KCNE1
Cl–
Fibrocytes
Basal
cells
Intermediate
cells
Marginal
cells
FIGURE 7. Mechanism of K⫹ accumulation in endolymph. The stria vascularis in the inner ear is formed on
the perilymphatic side by fibrocytes, basal cells, and intermediate cells connected by gap junctions and on the
endolymphatic side by marginal cells. The fibrocytes take up K⫹ from the perilymphatic compartment via the
Na⫹-K⫹-ATPase and Na⫹-K⫹-2Cl⫺ cotransporter. K⫹ is secreted via Kir4.1 K⫹ channel into the atrial compartment and taken by the Na⫹-K⫹-ATPase and Na⫹-K⫹-2Cl⫺ present on the marginal cells. K⫹ is finally
secreted into the endolymph by KCNQ1/KCNE1 K⫹ channel. The process is only possible because of the
polarized organization of K⫹ channels. The largely positive (around ⫹80 mV) endolymphatic potential is
necessary to the transduction of sound. [Adapted from Nin et al. (286). Original figure copyright (2008)
National Academy of Sciences, USA.]
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SEPÚLVEDA ET AL.
Kir4.1 channels. Marginal cells then remove K⫹ by the operation of Na⫹-K⫹-ATPase and Na⫹-K⫹-2Cl⫺ cotransporter and finally secrete K⫹ into the endolymph through
KCNQ1/KCNE1 K⫹ channel (437). Marcus et al. (251)
observed that Kir4.1 KO mice did not generate the highly
positive endocochlear potential (52) that is necessary to
hearing function and showed a reduced [K⫹]o in the endolymph. This interesting result highlights the major role of
Kir4.1 in the apical membrane of intermediate cells of the
stria vascularis. In addition, a large alteration of ear anatomy was apparent in Kir4.1 KO mice, including collapse of
the scala media, swelling of the tectorial membrane, as well
as their central processes (341). Whether the loss of endocochlear potential is due solely to the absence of Kir4.1 or it
is aggravated by the associated ear degeneration remains an
open question (242, 251).
A totally novel and unexpected implication of Kir4.1 in
pathology came to light recently: in the course of screening
serum IgG from patients with multiple sclerosis, Srivastava
et al. (369) observed that levels of antibodies to Kir4.1 were
higher in affected individuals. When injected into the cisternae magna of the mouse brain, human serum antibodies
specific for Kir4.1 decreased the expression of glial fibrillar
acid protein, a specific marker of glial cells; reduced the
abundance of Kir4.1 channel protein; and activated the
complement cascade in the cerebellum.
VII. Kir AND K2P CHANNELS IN THE
PROCESS OF CENTRAL
CHEMORECEPTION
Arterial PCO2 is tightly regulated through the control of
pulmonary ventilation, and CO2 in mammals is sensed primarily at central chemoreceptor neurons (101, 324). Central chemosensitive neurons responding to CO2/H⫹ levels
are located in defined brain stem areas such as the the retrotrapezoid nucleus (RTN), the nucleus of the solitary tract,
the locus coeruleus (LC), and caudal raphe nuclei (98, 268).
K2P and Kir channels have been examined as possible candidates to act as CO2/H⫹ sensors (263).
A. Are Kir Channels Involved in Central
Chemoreception?
Kir4.1 and Kir5.1, highly prominently expressed in several
brain stem nuclei involved in cardiorespiratory control
(418), associated as heterodimers are inhibited by hypercapnia-induced intracellular acidification (421). Jiang and
co-workers (67, 421, 427) have studied the pH sensitivity of
Kir4.1/Kir5.1 channels using CO2/HCO3⫺ mixtures and
were the first to suggest that owing to their high sensitivity
to pHi around the physiological level, these channels are
poised to play an important role in central CO2 chemoreception. The LC is a noradrenergic neuronal cluster con-
200
nected with most parts of the forebrain and involved in
several functions such as attention, cognitive behaviors,
arousal states, cardiovascular function, and control of
breathing (107). It is also documented that most neurons in
this region are sensitive to changes in extracellular pH (71,
107). Kir4.1 and Kir5.1 proteins are present in the mouse
LC, and their messengers have been detected in the rat (418,
441); however, immunohistochemistry has failed to discern
Kir4.1 and Kir5.1 coexpression in rat medulla oblongata
(424). D’Adamo et al. (71) demonstrated a reduced response of LC neurons to cytoplasmic alkalinization and
acidification in a Kir5.1 null mouse, concluding that the
channel is essential in the chemosensitivity process.
Rett syndrome, a genetic disease due to mutations in the
X-linked MeCP2 gene, is characterized by dramatic defects
in the development of the brain and respiratory abnormalities. It has been hypothesized that LC noradrenergic neurons are involved in these disturbances of Mecp2-null
mouse. Similar to humans affected by Rett syndrome, the
null mice breathing atmospheric air display irregular respiratory patterns, gasping, and apnea. Affected mice also had
a selective loss of respiratory response to 1–3% CO2 but
exhibited regular breathing in response to 6 –9% CO2
(441). Interestingly, LC neurons obtained from null mice
lacked sensitivity to mild hypercapnia (1–3% CO2), along
with an increase in Kir4.1 protein expression without
changes in Kir5.1. Electrophysiological experiments with
isolated neurons evidenced a reduced pHi sensitivity in the
Kir currents of Mecp2-null neurons. Similar type of experiments in HEK-293 cells cotransfected with Kir4.1 and
Kir5.1 in various ratios led to the conclusion that the decrease of pHi sensitivity observed in Mecp2-null cells was
due to an overexpression of Kir4.1 (441). Mice KO for
Kir4.1 present 100% mortality at P24 associated with
marked motor impairment secondary to dysmyelination
and axonal degeneration; nevertheless, there were no reports of respiratory abnormalities (67, 197). Neither peripheral nor central chemosensitivity is impaired by ablation of Kir5.1 (392). Kir5.1 inactivation nevertheless results
in a well-defined respiratory phenotype with reduced ventilatory responses to hypoxia or hypercapnia that is probably the consequence of an altered signal transmission from
carotid body to CNS. This could be due to a compensatory
modulation that develops in response to the profound metabolic acidosis shown by Kir5.1 KO mice (308, 392).
The hypothesis that Kir4.1 might influence chemoreception
by modulating astrocyte activity, rather than having a direct
influence on chemosensitive neurons, has also been examined. Kir4.1 is abundantly expressed in the astrocytes of the
ventral respiratory group, mostly in the processes surrounding capillaries and also in close proximity of the neurons.
But although Kir4.1 makes a major contribution to K⫹
conductance, it has no effect on the rhythmic bursting activity of the pre-Bötzinger complex (270). There is also
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pH-GATED K2P AND Kir TRANSPORT CHANNELS
convincing functional evidence for the presence of the
Kir4.1/Kir5.1 complex in astrocytes of the RTN. It has been
hypothesized that acidification-induced inhibition of
Kir4.1/Kir5.1 would induce ATP secretion, which in turn
would influence neuronal activity (410). Indeed, pH-dependent secretion of ATP by astrocytes in chemosensitive regions of the brain stem has been proposed as a possible
modulator of chemosensitive neurons (124, 125, 187).
Nevertheless, membrane depolarization of astrocytes is not
required for ATP secretion according to Trapp et al. (392).
The possible role of Kir5.1/Kir4.1 channels in the respiratory response to hypercapnia has been explored through
genetic inactivation of Kir5.1 in mice. Chemosensitive neurons of the LC from Kir5.1 KO animals become unresponsive to changes in cytoplasmic pH, hinting that Kir5.1 may
be involved in the response to hypercapnic acidosis (71).
However, experiments in the same animals show that
Kir5.1 channels are not essential for functional central and
peripheral respiratory chemosensitivity (392).
B. Role of the K2P Channels in Chemoreception
TASK subfamily K2P channels TASK-1 and TASK-3 as well
as TALK channel TASK-2 have been considered as part of
the intrinsic chemosensitivity of neurons owing to their high
responsiveness to extracellular pH changes. TASK-1 and
TASK-3 channels are sensitive to pHo in the physiological
range and are expressed in a number of different neuron types
in the respiratory network. Simultaneous deletion of TASK-1
and TASK-3 in the mouse abolishes the acid-sensitive background K⫹ current of raphe neurons, but leaves central ventilatory chemosensory response unaffected (264).
The involvement of TASK-1 or TASK-3 in the control of
ventilation by peripheral chemoreceptors is a matter of controversy. Oxygen-regulated K⫹ channels have been postulated to contribute importantly to chemosensory transduction in the carotid body (CB). Indeed, it is generally accepted that depolarization of glomus cells via inhibition of
K⫹ channels leading to Ca2⫹ influx and transmitter release
is at the heart of chemosensing in the CB (106, 232). More
recently, the attention has been focused on K⫹ channels of
the K2P family as candidates to underlie the chemosensitive
K⫹ conductance of the CB. Experiments using CB type 1
cells demonstrated a close similarity in biophysical and
pharmacological properties between O2-sensitive currents
and those generated in heterologous systems by K2P channel
expression. TASK-1 and TASK-3 were both postulated as
O2-sensing CB channels (44, 413) with the TASK-1/
TASK-3 heteromer better emulating the characteristics of
native O2-sensitive channels (192). The possible role of
TASK-1 and TASK-3 in peripheral chemoreception was addressed directly by examining the ventilatory response to
hypoxia and moderate normoxic hypercapnia in mice deficient in TASK-1, TASK-3, or both channels simultaneously
(391). The results of these experiments indicated the participation of TASK-1, but not TASK-3, in the peripheral control of ventilation. More recent experiments have used the
same animals to explore the function of these channels in
CB cells in vitro. TASK-1 was dispensable for O2/CO2 sensing in mouse CB cells, whilst TASK-3 channels, or its heteromer with TASK-1, could be involved in the depolarization of glomus cells under hypoxia. Catecholamine release
in response to O2/CO2 changes was, however, normal in
glomus cells from either TASK-1 or TASK-1/TASK-3 null
animals (297). Similar results were obtained more recently
in which Ca2⫹ responses to hypoxia and metabolic inhibition were unaltered in the absence of TASK-1 or TASK-3,
or the combined absence of TASK-1 and TASK-3 (43).
There might be other O2-sensitive K⫹ currents in the CB
that take over the role of the genetically removed TASK
channels in the KO animals. This possible redundancy of
sensor and effector mechanisms in the CB has been mentioned as an adaptation to the important function the CB
cells plays in the physiology of the animal (297).
TASK-2 expression in the brain is circumscribed to certain
RTN neurons and based on work with a KO mouse has
been proposed to be important in central CO2 and O2 chemosensitivity (110, 400). It is interesting that in addition to
its extracellular pH sensitivity, TASK-2 is regulated by pHi,
thus offering a possible way to couple channel activity to
CO2 levels (281). Intriguingly, TASK-2 has also been found
to be directly inhibited by CO2 without any need for
changes in pHi. The molecular determinants of this modulation have not been unveiled (313).
Gestreau et al. (110) reported that TASK-2 is expressed in a
scattered manner in brain stem nuclei that are involved in
central chemoreception, including the dorsal raphe, the
RTN, and the parafacial respiratory group. Transgenic expression in mice of a mutated Phox2b gene, coding for the
paired-like homeobox 2b transcription factor, led to disappearance of certain parafacial neurons, unresponsiveness to an
increase in CO2 and premature death from central apnea (89).
All expression of TASK-2 in the RTN is also lost under the
effect of this transgene. The respiratory effects of TASK-2 inactivation were studied by whole body plethysmography. The
acute response to hypoxia was normal, suggesting appropriate
O2 sensitivity of the carotid body chemoreception. Exposure
to a 20-h hypoxia did not elicit the normal ventilatory depression followed by acclimatization in the KO mice. This indicated that TASK-2 channels could be involved in the changes
in the CO2 set-point in RTN neurons. The use of an en bloc
preparation of neonatal brain stem-spinal cord allowed to determine that a 5-min anoxia provoked a reduction of the respiratory frequency in the WT preparation but not in that
derived from KO mice. The authors concluded that TASK-2
channels are involved in the stabilization of membrane potential of chemosensitive neurons of brain stem nuclei, including
the RTN region. TASK-2 might not be the sole chemosensory
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ion channel since KO animals had a definite ventilatory response to hypercapnia (110).
The role of TASK-2 in central chemosensing was initially
put in doubt because its ablation, in contrast to what is seen
after deletion of RTN neurons, does not produce the expected alteration in pH sensitivity of the neonate breathing
network in vitro. Additionally, the metabolic acidosis presented by the TASK-2 null mice secondary to the impaired
bicarbonate renal reabsorption by itself could have altered
the central response to CO2 (53, 138). A direct electrophysiological recording of RTN neurons was therefore needed
to test whether TASK-2 deficit leads to a modification of
their intrinsic pH sensitivity.
The answer to this criticism (400) came through the use of two
different genetically modified mice to study the contribution of
TASK-2 to the intrinsic pH sensitivity of the RTN chemoreceptor neurons: the ␤-galactosidase expressing TASK-2 KO
mouse (110) and a Phox2b-eGFP BAC transgenic line (213).
Patch-clamp recordings were made in brain stem slices from
RTN neurons identified by Phox2b promoter-driven expression of GFP or ␤-galactosidase. Nearly all RTN cells from WT
animals were pH sensitive, and only about one half of GFPexpressing RTN neurons from TASK-2 KO mice responded to
this description. No TASK-2 KO RTN neuron, identified by
the ability to express ␤-galactosidase and that made a subpopulation of GFP-expressing neurons, showed pH sensitivity.
RTN neurons from TASK-2 KO mice that had some pH sensitivity, but not those that were pH insensitive, had some kind
of background K⫹ current of unknown molecular identity
activated by alkalinization of reduced magnitude. The use of a
working heart-brain stem preparation disclosed that a respiratory alkalosis caused a blunted decrease in phrenic burst
amplitude in the KO preparation. The pH at which the phrenic
activity stops, the apneic threshold, was significantly higher in
the KO preparations (400). These results undoubtedly demonstrate the contribution of TASK-2 channel to the intrinsic
pH sensitivity of the major part of Phox2b expressing RTN
neurons. In those cells where the expression of TASK-2 is
higher, this conductance accounts fully for the pH-sensitive
background K⫹ current and pH-dependent effects on neuronal firing. However, there is a subset of RTN neurons where a
redundant or compensatory pH-sensitive background K⫹
conductance of unknown molecular identity becomes evident.
VIII. ROLE OF POTASSIUM CHANNELS IN
ADRENAL BIOSYNTHESIS OF
ALDOSTERONE
A. Modulation of Kⴙ Channels in the
Production of Aldosterone
Cells of the adrenal zona glomerulosa (ZG) are central in water and electrolyte homeostasis and consequently in the regu-
202
lation of blood pressure via aldosterone secretion. The most
relevant physiological stimuli for aldosterone secretion are the
plasma concentrations of K⫹ and angiotensin II (ANG II),
either acting independently or synergistically (368). Aldosterone is not accumulated in secretory granules, but its rate of
synthesis and secretion critically depends on Ca2⫹signaling at
several points of the steroidogenesis pathway. An example is Ca2⫹/calmodulin kinase I-dependent transcription
of CYP11B2, the aldosterone synthase that catalyzes the conversion of 11-deoxycorticosterone to aldosterone (20, 65).
The activation of the ANG II type I receptor (AT1) elicits
a transient intracellular store release of Ca2⫹ followed by
influx of the ion mainly through T-type Ca2⫹ channels
that sustain the aldosterone secretion (368). This last
phase requires depolarization of the plasma membrane
which is achieved through inhibition of K⫹ channels responsible for the resting membrane potential of ZG cells.
In fact, both increases in extracellular K⫹ and ANG II
depolarize the ZG cells (234). Patch-clamp experiments
have identified inwardly rectifying, outwardly delayed
rectifying, Ca2⫹-activated, and background K⫹ current
in different species (121, 135). The resting membrane
potential of rat ZG cells is close to the equilibrium potential for K⫹ at which only Kir and K2P channels may
give rise to conductances; however, in that situation only
background K⫹ currents have been recorded (68, 135). In
addition, ANG II and acidosis lead to inhibition of
cloned TASK-1 and TASK-3 and native background K⫹
channels in ZG cells, suggesting that these channels in
homo- or heterodimeric association give rise to the
background hyperpolarizing conductance of ZG cells
(68, 70).
An alternative model to explain sustained production
and secretion of aldosterone has been raised by Barrett
and co-workers (167). They found that ZG cells behave
as intrinsic oscillators which depend on Cav3.2 currents
(167). Under stimulation with ANG II or K⫹, the elicited
depolarization is small (4 –10 mV), insufficient to elicit a
significant Ca2⫹ influx. The voltage oscillations in the
plasma membrane will amplify small changes in the potential, allowing repeated cycles of Ca2⫹ entry into the
ZG cell through Cav3.2 channels. In this scenario, TASK
channels would play their role principally during the depolarization phase of the oscillatory cycle by affecting the
rate of depolarization between spikes. Delayed rectifiers
and Ca2⫹-activated K⫹ channels will enter into action
when the membrane is depolarized by controlling the rate
of repolarization (167). Modulation of K⫹ channel activity therefore would be a major way of controlling the
production of aldosterone.
Studies in patients with autonomous secretion of aldosterone (primary aldosteronism) and KO mouse models have
further highlighted the importance of K⫹ channels in glomerulosa cell physiology and pathophysiology.
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pH-GATED K2P AND Kir TRANSPORT CHANNELS
B. KCNJ5 Gene Mutations and AldosteroneProducing Adenomas
Primary aldosteronism is a main cause of secondary hypertension. Mutations in the human KCNJ5 gene encoding the G protein-activated inward rectifier K⫹ channel 4
(GIRK4) have been found in familial hyperaldosteronism
type III patients and ⬃40% of sporadic aldosterone-producing adenomas (APA). Choi et al. (57) identified somatic and germline tumor-causing mutations in the human KCNJ5 gene which are heterozygous and located at
or near the selectivity filter, G151R and L168R, respectively. Whole cell patch-clamp studies of the mutant
channels revealed loss of channel selectivity, with increased sodium conductance leading to membrane depolarization and activation of voltage-gated calcium channels. In the same study, the T158A mutation was found in
familial hyperaldosteronism type III, a Mendelian autosomal-dominant form of primary aldosteronism. T158
lies in the loop between the selectivity filter and the second transmembrane domain and also led to loss of K⫹
selectivity. After these seminal discoveries, families carrying G151R and G151E mutations and somatic KCNJ5
mutations surrounding the selectivity filter in APA have
been reported (121, 262). The authors postulate that
KCNJ5 mutations involved in inherited and acquired aldosteronism with cell autonomous proliferation is the
result of increased calcium influx (57, 352). The role of
GIRK4 channels in glomerulosa cells in normal physiology is unknown.
C. Aldosterone Production Studies Using
TASK-1 and TASK-3 KO Mice
TASK-1 is expressed in the whole cortex, and TASK-3 is
restricted to the ZG in rodents and human adrenal glands.
Their genetic ablation results in a range of phenotypes with
abnormal aldosterone production.
The main pathophysiological feature in TASK-1 null mice is
mislocalization of aldosterone synthase observed before puberty and in adult females but not in males (146). The
enzyme was absent from the zona glomerulosa, but abundant in the reticulofasciculata zone. The observed phenotype was severe hyperaldosteronism that is reversed by
ACTH inhibition and is independent of salt intake, hypokalemia, and low renin hypertension. The pathological
change in adrenocortical zonation and the phenotype could
be reproduced in KO males by castration. Moreover, administrations of testosterone to KO females provoked a
redirection of the aldosterone synthase to the zona glomerulosa and suppression of the phenotype. These results suggest an androgen compensatory mechanism, such as an increased expression of TASK-3 channels. Electrophysiological studies demonstrate that TASK-1 KO adrenal cells were
depolarized compared with WT cells (⫺68 vs. ⫺75 mV,
respectively). A significant depolarization by extracellular
acidic pH in TASK-1 KO cells indicated the presence of
other pH-sensitive K⫹ currents that again could correspond
to homomeric TASK-3 channels (14, 146).
Double TASK-1 and TASK-3 KO adrenal glands had normal cellular organization (74). The complete loss of TASKlike current in glomerulosa cells provoked an ⬃20-mV depolarization of the cells resulting in autonomous aldosterone overproduction. Consequently, the double KO mice
show elevated plasma levels of aldosterone with low levels
of renin, hypertension, and hypokalemia. Plasma aldosterone remains increased under a low-salt diet, and it is not
suppressed by high-salt diet. Finally, the aldosterone levels
were resistant to AT1 receptor blockage. All these features
are reminiscent of patients with idiopathic primary hyperaldosteronism (74). Guagliardo et al. (134) working with
this double KO mouse found that physiological inhibition
of TASK channels by H⫹ is not required to evoke an increase in aldosterone production and concluded that pHsensing by glomerulosa cells does not require TASK-1 or
TASK-3 channels.
The effect of deleting only TASK-3 on the control of
aldosterone production and blood pressure has also been
examined (136). The KO mice displayed mild hyperaldosteronism associated with low renin levels, increased aldosterone-renin ratio, and hypersensitivity to angiotensin II. Interestingly, this phenotype recalls the human lowrenin essential hypertension, and the loss of the TASK-3
channel activity might be part of the pathophysiological
mechanism of this hypertensive syndrome. A similar phenotype using another TASK-3 KO mouse was described by
Penton et al. (314) who measured Ca2⫹ transients and aldosterone secretion in ex vivo adrenal glands, finding that a
significant part of the aldosterone secretion is autonomous
and not due to angiotensin II hypersensitivity.
Bandulik et al. (16) investigated the age dependence of the
adrenal phenotype of TASK-3 KO mice. The newborn KO
mice displayed a severe adrenal phenotype with strongly
increased plasma levels of aldosterone, corticosterone, and
progesterone. They found an increase of the activity of the
local intra-adrenal renin-angiotensin system and hypothesized that abnormal local renin expression could also be
relevant for the development of primary hyperaldosteronism in a subset of human patients.
The KO models discussed are very suggestive that abnormalities in TASK-1 and/or TASK-3 in humans might be
involved in the pathogenesis of primary aldosteronism.
Nevertheless, sequencing of these genes in 22 patients
with APA by Choi et al. (57) did not identify any causative mutations. However, a study was designed to explore genetic variation in KCNK3 and KCNK9 genes
coding for TASK-1 and TASK-3, respectively, in relation
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to blood pressure and aldosterone production in healthy
young humans (182). The main finding was that multiple
single nucleotide polymorphisms in KCNK9 were associated with systolic blood pressure in African Americans,
whereas associations were primarily with aldosterone
production in European American individuals. These interesting preliminary results should be replicated in other
populations fully to establish a role for KCNK9 in causing blood pressure variability.
IX. UNEXPLORED AND SURPRISING
RECENT DEVELOPMENTS IN K2P AND
Kir CHANNEL PHYSIOLOGY AND
PATHOPHYSIOLOGY
A. Kir7.1 Follows the Naⴙ/Kⴙ Pump in
Subcellular Distribution
Kir7.1 is prominently expressed in epithelial tissues where it
follows the subcellular distribution of the Na⫹/K⫹ pump:
Kir7.1 is present at the apical membrane of retinal pigmented epithelium (RPE) and in the choroid plexus that
also express the pump apically (207, 267, 361, 425), whilst
it is located at the basolateral membrane of intestinal epithelial cells, thyroid follicular cells, and epithelial cells of
proximal and distal convoluted tubule, all of which present
the pump at the more usual basolateral membrane location
(81, 267, 294). Inwardly rectifying K⫹ currents probably
corresponding to the activity of native Kir7.1 are present in
RPE (306) and small intestinal villus enterocytes (358). The
remarkably faithful subcellular colocalization of Kir7.1 and
the Na⫹/K⫹ pump has prompted the hypothesis that this
channel might be responsible for K⫹ recycling, allowing
continuous pump function in epithelia to sustain high rates
of ion transport (153).
liver, and pancreas (35, 315, 362). The KCNJ15 gene coding for Kir4.2 is located close to the locus of the Down
syndrome chromosome region 1, a fact that initially suggested a possible link with this disease (123). Okamoto et
al. (291) recently identified KCNJ15 as a susceptibility gene
for lean type 2 diabetes, which is highly prevalent in Japan.
A synonymous SNP in exon 4 resulted in increased Kir4.2
protein abundance. Kir4.2 is present in ␤-cells of the endocrine pancreas, where its expression is stimulated by glucose. It is thought that increased expression of Kir4.2 stabilizes the resting membrane potential of ␤-cells, preventing
membrane depolarization, which is necessary to insulin secretion (291), as previously indicated by gain-of-function
mutations in the KCNJ11 gene encoding Kir6.2 channel
(252). No similar association was found in the European
population (291). Most interestingly, knock-down of
KCNJ15 increased insulin secretion in vivo and in vitro
(290).
C. K2P Channels in CNS Function
A mutation in KCNJ13, the gene encoding the Kir7.1, leading to replacement of methionine-162 by tryptophan, has
been associated with the retinal disease known as snowflake
vitreoretinal degeneration (SVD) (150). The mutated channel reaches the plasma membrane where it is inactive (440),
or mediates only weak nonselective cation current (307). In
both cases, coexpression of mutated channels reduced the
activity of the WT protein. Recessive mutations in KCNJ13
are also a cause of Leber congenital amaurosis, another
retinal degenerative disease (359). These mutations, R166X
and L241P, lead respectively to loss of COOH terminus and
probable failure of membrane surface trafficking, and perhaps loss of function by a COOH-terminal folding defect.
No formal functional evaluation of these mutants has yet
been done.
Transient changes in pHo in the brain occur in the extracellular space near neurons and glia during neurotransmitter
or hormone action or after changes in metabolic activity
and ion transport that might also change pHi (54). Local
change in pHo at endfeet of astrocytes might link neuronal
activity and blood flow (277). In the retina, pHo in the
extracellular space of photoreceptors responds to light
stimulus and to a circadian rhythm (84, 85). It is therefore
conceivable that K2P channels expressed in the CNS might
respond to these changes and regulate excitability. Evidence
that TASK-1, TASK-3, and TREK-1 are important for neuronal excitability has come from the study of the respective
KO mice and has been reviewed in detail recently (93, 218,
342). KO mice for TASK-1, TASK-3, and TREK-1 show
increased resistance to halothane whilst TASK-1 and
TASK-3 KO animals have altered motor performance. Cerebellar granule cells express both TASK-1 and TASK-3 and
are not too sensitive to inactivation of either individually.
TASK-3 KO mice have altered sleep patterns, probably due
to the alteration of intrinsic neural rhythms. Studies with
TREK-1 KO mice support a participation of this channel in
neuroprotection, depression, and pain perception (162,
342). In addition, TREK-1 KO mice have a defective acetylcholine- and bradykinin-dependent vasodilation in mesenteric arteries, an effect that is dependent on the endothelium (108). TREK-1 might be important in the endothelial
cells forming the mammalian blood-brain barrier (BBB),
and recent studies show altered expression of TREK-1 in
human BBB upon inflammation and BBB defects in TREK-1
KO mice (31; reviewed in Ref. 30).
B. Kir4.2 Channels
D. K2P Channels and Human Disease
Little is known about the physiological function of Kir4.2
despite its broad tissue distribution in kidneys, brain, lungs,
Inhibition of TASK-1 mediates the depolarizing effects of
hypoxia and endothelin-1 in human pulmonary artery
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pH-GATED K2P AND Kir TRANSPORT CHANNELS
smooth muscle cells (383), although this does not occur in
the mouse (250). This looks like a genuine species difference, and indeed, mutations affecting human TASK-1 have
been found in familial and idiopathic pulmonary arterial
hypertension (242). The effect of the pathological mutations assayed in a heterologous expression system resulted
in nonfunctional channels, but some activity could be recovered using a phospholipase inhibitor. Unfortunately, for
a possible therapeutic intervention, the increase in current
occurred at highly depolarized voltages well outside the
physiological range of a background conductance.
A missense mutation in the maternal copy of KCNK9, the
TASK-3 gene, has been associated with Birk-Barel syndrome, a maternally transmitted genomic-imprinting disorder leading to mental retardation, hypotonia, and elongated
face dysmorphism (18). The mutated TASK-3-G236R
channel was described as nonfunctional either in homomeric or in heterodimeric form with nonmutated TASK-3.
A more recent detailed study by Veale et al. (399) shows
that TASK3-G236R channels generate decreased but significant currents when compared with WT channels. The currents are mildly inwardly rectified and differentially sensitive to extracellular acidification and muscarinic activation.
Importantly, a partial recovery of the current magnitude
could be achieved through the use of flufenamic acid, opening a possible avenue for the development of drugs effective
in treating the Birk-Barel syndrome.
To our knowledge, the only other genetic human disease
involving K2P channels refers to a dominant-negative mutation affecting pH-independent TRESK channel linked to
familial migraine with aura (211). The mutation, a
F139WfsX24 frameshift, abolishes function and exerts a
dominant negative effect. Surprisingly, however, several
other TRESK channel missense variants in unrelated patients did not have functional effect, and one point mutation, C110R, leading to complete loss of TRESK function
when expressed in Xenopus oocytes was present in affected
and control individuals (3). Intriguingly, expression of the
C110R variant in small-diameter trigeminal ganglion neurons, unlike migraine-associated frameshift TRESK mutant, does not lead to hyperexcitability, indicating that it is
incapable of exerting a dominant negative in a physiological
context (137). As is often the case with human inherited
diseases, migraine with aura belongs to the group of genetically complex disorders, and future work will have to elucidate the role played in it by TRESK malfunction.
Although as yet unrelated to human disease, it has been
reported that TWIK-2 null mice present elevated systolic
blood pressure associated with an increased peripheral vascular resistance and normal cardiac function. Isolated aortic smooth muscle cells are depolarized, and isolated segments of aorta presented greater contractile responses
(226). Unexpectedly, endothelium-dependent relaxation
was enhanced in the aortae of KO mice, which could represent a compensatory mechanism to the increased vascular
tone (227).
E. Fickle Filters in K2P Channels: Possible
Physiological or Pathophysiological Roles
Examples of changes in ion selectivity of K2P channels of
possible physiological importance have multiplied recently.
Some of these findings will have to await confirmation, but
their accretion speaks of some sort of functional plasticity in
the SF of these channels.
A paradoxical depolarization in hypokalemia is observed in
some cardiomyocytes and has recently been linked to the
expression of TWIK-1 channels (243). Using the channel
activity-boosted TWIK-1-K274E mutant, Ma et al. (243)
reported that this phenomenon is probably due to the remarkable acquisition of high relative Na⫹ permeability of
TWIK-1 channels in response to low extracellular K⫹.
TWIK-1 has an unusual SF in which the TIGYG motif normally encountered in K⫹ channels is replaced by TTGYG in
P1 domain and TIGLG in P2. This variation in SF is partly
to blame as introducing the TTGYG motif into TASK-3
made these normally solidly K⫹-selective channels also Na⫹
permeable in low extracellular K⫹. Observing that many
K⫹ channels exposed to low-K⫹/high-Na⫹ media experience an SF collapse which often passes through a phase of
Na⫹ coordination, Goldstein (117) postulated that this normally brief intermediate state is stabilized in TWIK-1.
The loss of the first 56 NH2-terminal residues in TREK-1
channels through alternative translation initiation (ATI)
may lead to the permanent acquisition of Na⫹ permeability.
The full-length TREK-1 is highly K⫹ selective, whereas the
shorter channel allows Na⫹ permeation and mediates depolarization (388, 426). Both the long and short versions of
TREK-1 are present in the rat CNS (388), but there are as
yet no functional reports of a Na⫹-permeable form in native
cells. A recent paper by Veale et al. (398) reports an enhancement of TREK-1 by fenamates that affects both the
full-length form and the ATI short form of the channel. In
addition to establishing fenamate derivatives as potential
pharmacological agents to modulate TREK-1, the investigation failed to observe the proposed change in selectivity of
the shorter version of the channel. Both pieces of work use
macroscopic currents to measure selectivity, the first after
expression in Xenopus oocytes (388) and the second after
expression in mammalian cells (398). Single-channel selectivity studies might help to resolve this discrepancy.
Intriguingly, similar ATI in TREK-2 deleting 54 or 66
amino acids of the NH2 terminus alters single-channel conductance without any change in ion selectivity (363). Highand low-conductance channel activity that probably corresponds to the expression of different forms of TREK-2 is
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detected in cerebellar granule neurons (185). High- and
low-conductance forms of TREK-1 have been seen in cardiac myocytes and in heterologous expression systems, but
it is not clear if they correspond to species with alternative
sequences (419).
The anomalous SF sequence of TWIK-1 plus other crucial
sequence variations are thought to be responsible of the
remarkable change in selectivity experienced by TWIK-1
upon extracellular acidification (49). TWIK-1, despite sharing the same extracellular H⫹ sensor with TASK-1 and
TASK-3, responds quite differently to acidification, becoming highly Na⫹ permeable rather than simply closing at low
pHo (49, 244). Independently of the mechanism mediating
it, what might be the physiological impact of such a change
of selectivity if any? Chatelain et al. (49) have attempted to
respond to this query. According to their own previous
results, TWIK1 is detected mainly in the recycling endosomes of native cells or those in which it has been heterologously expressed (76), and this is dependent on a COOHterminal di-isoleucine motif (96). TWIK-1 outward currents first increase and then diminish as TWIK1 becomes
permeable to Na⫹ and mediates its influx. This switch in
selectivity develops slowly, requiring several minutes for
completion and tens of minutes for recovery (49, 244). Considering that the pH of intracellular compartments is quite
acidic, around 6 in the lumen of the trans-Golgi network
and the endoplasmic reticulum (47), any TWIK-1 briefly
visiting the plasma membrane would elicit a depolarizing
effect before being recycled by endocytosis that leads to
complete internalization in ⬍10 min (76). Such a scenario
helps to explain the observation that TWIK1 gene inactivation in mice causes a paradoxical hyperpolarization of kidney principal cells, pancreatic ␤-cells, and hippocampal astrocytes (49, 257, 401). According to this reasoning,
TWIK-1 would be mainly a monovalent cation-permeable
channel of intracellular compartments. Its more rare visits
to the plasma membrane could be important in the physiology of ␤-cells, but otherwise remains enigmatic.
A rather more extreme case of SF eccentricity in K2P channels is provided by Woo et al. (416) who report a direct
effect of G protein ␤␥-subunits on TREK-1. This effect
observed in astrocytes remarkably appeared to change
TREK-1 selectivity so that the channel became anion selective, supporting astrocytic glutamate release to affect neighboring neurons. According to Hwang et al. (175), TWIK-1
becomes functional in cortical astrocytes only when forming dimers with TREK-1 in the plasma membrane of cortical astrocytes. This TWIK-1/TREK-1 dimer is present at the
plasma membrane of astrocytes, is regulated by endocytosis
but not by sumoylation, and is stabilized by disulfide bond
cross-linking of the subunits at the extracellular cap or SID.
Upon activation of cannabinoid receptor 1, and release of
G␤␥ subunits, it switches from being a K⫹-selective channel
into an anion-selective conductance capable of mediating
206
the release of glutamate (175). These extraordinary and
exciting claims clamour for further probing, and it will be
interesting to follow up future work in this front. A possible
note of caution is that work on hippocampal astrocytes
shows that although they also express TWIK-1 and
TREK-1, TWIK-1 is present in intracellular compartments
only (401).
X. CONCLUSIONS
Recent years have seen remarkable progress in our understanding of the molecular aspects of gating of pH-regulated
K2P and Kir K⫹-transport channels and in the physiological
roles they play in various important cellular processes. Kir
K⫹-transport channels are essential to renal function, regulation of K⫹ homeostasis in the brain, and acid secretion
in the stomach. pH-dependent K2P channels are critical in
central chemoreception, regulation of aldosterone secretion
in the adrenal gland, and bicarbonate conservation. Although their pH dependence is significant in some of these
functions, its importance remains to be ascertained in many
others. Mouse models attempting to understand the functions of these channels are increasingly becoming available,
and it is predicted that judicious use of the advances in
structure-function knowledge will increase their sophistication to interrogate specific channel function in a very direct
way. Important diseases are associated with mutations in
the genes encoding Kir K⫹-transport channels. The discovery of human inherited defects affecting K2P channels is
starting for this most recently discovered group of K⫹ channels.
Very important advances into how form relates to function
has been made recently thanks to the discovery of new Kir
structures that give clues about the mechanisms of gating of
these channels. The reports of the first atomic structures of
K2P channels, on the other hand, reveal unique features that
are already spurring fresh experimental work to improve
our knowledge of the very special gating characteristics of
these channels. These new insights come at a time when
concepts about rather unorthodox gating behavior of K2P
channels has come to light.
Our examination of the recent literature on pH-regulated
K2P and Kir K⫹-transport channels also reveals some quite
unexplored areas that are ready to be investigated: KO animals that have never reportedly been attempted and
strange gating or ion handling behaviors awaiting confirmation and mechanistic explanation.
The evidence accumulated these years shows clearly that in
addition to being important in setting the resting potential,
Kir transport channels are crucial in the control of cell excitability, K⫹ homeostasis, and epithelial transport. The
same can be said about the K2P channels examined here:
from being the molecular substratum of the long sought,
Physiol Rev • VOL 95 • JANUARY 2015 • www.prv.org
pH-GATED K2P AND Kir TRANSPORT CHANNELS
rather inert leak K⫹ conductance present in all cells, they
have revealed themselves as a group of channels with a wide
range of functions and exquisite regulation. The many questions still open about these channels augur a future of intense and satisfying research on these proteins in years to
come.
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence:
F. V. Sepúlveda, Centro de Estudios Centíficos (CECs),
Avenida Arturo Prat 514, Valdivia 5110466, Chile (e-mail:
fsepulveda@cecs.cl).
GRANTS
The work of the authors is supported by the following research grants: Fondecyt 1140153 and 1120743, ANR
BLAN 2010-111201, and ECOS-Conicyt C10S03. The
Centro de Estudios Científicos is funded by Centers of Excellence Base Financing Program of Conicyt.
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DISCLOSURES
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