REVIEWS
PATHWAYS MODULATING NEURAL
KCNQ/M Kv7 POTASSIUM
CHANNELS
Patrick Delmas* and David A. Brown‡
Abstract | K+ channels play a crucial role in regulating the excitability of neurons. Many
K+ channels are, in turn, regulated by neurotransmitters. One of the first neurotransmitterregulated channels to be identified, some 25 years ago, was the M channel. This was
categorized as such because its activity was inhibited through stimulation of muscarinic
acetylcholine receptors. M channels are now known to be composed of subunits of the Kv7
(KCNQ) K+ channel family. However, until recently, the link between the receptors and the
channels has remained elusive. Here, we summarize recent developments that have begun
to clarify this link and discuss their implications for physiology and medicine.
PHASICALLY FIRING NEURON
A neuron that responds with a
transient discharge of action
potentials when subjected to a
long-lasting excitatory current.
TONICALLY FIRING NEURON
A neuron that responds with a
sustained train of action
potentials when subjected to a
long-lasting excitatory current.
*Laboratoire de
Neurophysiologie
Cellulaire, UMR 6150
CNRS, Faculté de
Médecine, IFR Jean Roche,
Bd. Pierre Dramard,
13916 Marseille Cedex 20,
France. ‡Department of
Pharmacology, University
College London,
Gower Street, London,
WC1E 6BT, UK.
e-mail: delmas.p@jeanroche.univ-mrs.fr;
d.a.brown@ucl.ac.uk
doi:10.1038/nrn1785
850 | NOVEMBER 2005
M channels are low-threshold K+ channels that were
originally identified (as a macroscopic membrane
current) in the early 1980s in frog1 and rat2 sympathetic neurons. The current shows a characteristic
time- and voltage-dependence that results in the
‘clamping’ of the membrane potential if the channel
is exposed to an excitatory stimulus (FIG. 1). Therefore,
the M channel functions as a ‘brake’ on repetitive
action potential discharges and, as such, has a key
role in regulating the excitability of various central
and peripheral neurons3,4, including sympathetic
neurons (FIG. 2), hippocampal pyramidal cells5–8 and
striatal neurons9.
Of equal importance is that the current is inhibited by stimulating specific receptors, the substrates
of which are common neurotransmitters, such as
acetylcholine, and some peptides, such as luteinizinghormone releasing hormone (LHRH), angiotensin,
substance P and bradykinin3. This has the effect of
releasing the brake and allowing undiminished action
potential firing — in effect, converting a cell, such
as a sympathetic neuron, from a strongly-adapting,
PHASICALLY FIRING NEURON into a TONICALLY FIRING NEURON
(FIG. 2). When these receptors are activated synaptically
— for example, by acetylcholine release from cholinergic
| VOLUME 6
fibres — M-current inhibition results in the slow depolarization and enhanced discharge that was originally
associated with the slow excitatory postsynaptic potential
(slow EPSP)10–12.
M-current inhibition by a slow transmitter also
facilitates the response to a fast transmitter13. This,
combined with the changes in electrical excitability,
might provide a mechanism for selective ‘attentiontuning’ in the nervous system. A classic example can
be seen in the visual cortex, where the responsiveness
of direction-sensitive neurons is selectively upregulated by parallel stimulation of the cholinergic afferent
system14 (although, of course, this might result from
actions other than solely the closure of M channels).
Such effects might underlie the arousal of cortical
neurons by cholinergic basal forebrain activity; conversely, their absence following degeneration of this
input probably contributes to the cognitive deficits
observed in Alzheimer’s disease15. Indeed, linopirdine
(an M-channel blocker) has acute cognition-enhancing
properties in some animal experiments6, whereas
longer-term suppression of M channels by dominantnegative gene expression eliminates a component of
hippocampal theta rhythm and impairs spatial learning
in mice5.
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SHAKER CHANNEL
Prototypical inactivating
K+ channel (Kv1) with six
transmembrane segments.
CELLATTACHED MEMBRANE
PATCH
A recording microelectrode is
sealed onto a cell and allows the
measurement of the current
flowing through the ion
channels embedded in the
electrically-isolated membrane
patch.
EFOLD
Expresses the voltage-sensitivity
of channel opening. The
mathematical constant e
(occasionally called Euler’s
number or Napier’s constant) is
the base of the natural
logarithm function and its
approximate value is
2.7182818284.
a
It is, therefore, important to understand precisely
how these channels are regulated. In this review we
summarize the current knowledge about the cellular
mechanisms by which neurotransmitters inhibit
M channels and/or their component subunits, how
the different pathways might be segregated or integrated, and how alterations in M-channel regulation
might occur in genetic and metabolic disease states.
Molecular composition of M channels
The molecular composition of M channels defied
identification for almost 20 years, but it is now known
that they are composed of subunits of the Kv7 (KCNQ)
family of K+ channels16–18 (for nomenclature, see REF.
19). The five members of this family (Kv7.1 to Kv7.5)
are homologous to SHAKER CHANNELS but are structurally distinct in that M channels have long intracellular
tails at the carboxyl (C) terminus (FIG. 3). Kv7 channels
are also noteworthy because mutations in the genes
for four of these subunits (Kv7.1–Kv7.4) give rise to
human genetic disorders18. Whereas Kv7.1 is restricted
to the heart and peripheral epithelial and smooth
muscle cells, the other four Kv7 channels are confined
to the nervous system, where they have been found in
various cell types, including hippocampal and cortical
neurons 20,21 and dorsal root ganglion neurons 22 .
Although Kv7 channels are classically associated with
regulation of synaptic integration in somatodendritic
Current
Conductance
100
80
VC –30 mV
VH –60 mV
nS
60
1 nA
40
20
0
0.5 s
–100
–80
–60
–40 –20
0
mV
b
–90 mV
–46 mV
0.5 nA
I
10 mV
V
0.5 s
Figure 1 | Some basic properties of the M current as originally observed in frog
sympathetic neurons using twin microelectrode voltage-clamp recording. a | The left
panel shows how the current is slowly activated (lower record) during a 1-s increase in
membrane potential (upper record) from –60 mV (VH) to –30 mV (VC). The right panel shows the
consequent increase in whole-cell conductance: it fits to a simple Boltzmann expression with a
slope factor of 10 mV per EFOLD increase in conductance and a half-maximal voltage of –35 mV.
b | The current ‘clamps’ the membrane potential. When 0.2 nA current steps are injected into
the cell at –90 mV (all M channels are shut), there is a large membrane voltage-excursion;
when the same currents are injected at –46 mV (~30% of M channels are open) the increased
opening or closing of the channels produces an additional outward or inward current that
opposes the injected current and largely prevents any voltage change. Panel a adapted, with
permission, from REF. 122 © (1982) The Physiological Society. Panel b adapted, with
permission, from REF. 3 © (1988) Plenum.
NATURE REVIEWS | NEUROSCIENCE
plasma membranes, recent studies have shown that
they also have functions in specialized subcellular
domains such as axon initial segments, nodes23 and
nerve cell terminals24.
Mutations in the genes for Kv7.2 or Kv7.3 generate a form of juvenile epilepsy called benign familial
neonatal convulsions (BNFC)18. These two subunits
were originally identified as being the components
of the classical (ganglionic) M channel16, probably
assembled as a two-plus-two (Kv7.2 + Kv7.3) heterotetramer18,25,26. However, it is important to recognize
that all Kv7 subunits, when associated as homomeric
channels (even the cardiac Kv7.1 channel), can form
M channels, as defined kinetically and pharmacologically. Therefore, all homomeric Kv7 currents show
a similar time- and voltage-dependent gating27, are
inhibited by the M-channel blocker linopirdine28 and,
importantly, are inhibited by stimulation of G-proteincoupled receptors (GPCRs) such as the M1 muscarinic
acetylcholine receptor27,29. This is a crucial factor
when investigating their role in regulatory mechanisms
(see below).
Receptor–channel transduction
Because many types of receptor can close M channels,
it always seemed likely that the closure mechanism
was indirect and that the different receptors involved
in channel closure might use one (or more) common
intermediary second messenger(s). Research on sympathetic neurons during the two decades following
the discovery of M channels established that receptors that could close M channels are GPCRs, and, in
particular, belong to the subclass that activates the
G proteins G q and/or G11. M-channel closure was
shown to require G-protein activation30–32, and a combination of approaches (antibody injection, antisense
expression, Gαq-deficient mice and activated subunit
overexpression) led to identification of the dominant
G protein as the α-subunit of Gq33–35 (with contributions in some instances from G11REF. 35). In addition,
channels that were isolated in a CELLATTACHED MEMBRANE
PATCH could be closed by stimulating receptors outside
the patch36,37, which supported the proposed indirect
nature of the closure mechanism (FIG. 2c). Muscarinic
agonists have a similar effect on the N-type (Cav2.2)
Ca2+ current in the same cells, and the two mechanisms
were thought to be analagous38,39.
However, the nature of the common second
messenger(s) that lead to closure of M channels
remained elusive. Because the principal effect of
stimulating Gq (or G11) is the activation of membraneassociated phospholipase-Cβ (PLCβ), which thereby
leads to hydrolysis of membrane phosphatidylinositol4,5-bisphosphate (PtdIns(4,5)P2), it seemed most
likely that the messenger(s) would be one or more of
the products of this reaction. These include inositol1,4,5-trisphosphate (Ins(1,4,5)P3), Ca2+ ions (released
by Ins(1,4,5)P3), and diacylglycerol (DAG), which
activates protein kinase C (PKC) (FIG. 4). Considerable
evidence accrued both for and against the involvement of each of these messengers in channel closure
VOLUME 6 | NOVEMBER 2005 | 851
REVIEWS
b M-channels closed (muscarine)
a Control
Voltage clamp
1 nA
I
10 mV
V
0.2 s
0.2 s
Current clamp
I
1 nA
50 mV
V
c
Control
Pipette
Oxo-M (1 µM)
Kv7/M
–
Wash
Oxo-M
1 pA
Gq PLCβ
M1
0.2 s
Figure 2 | Regulation of neuronal firing by the M current, as originally observed in a
rat sympathetic neuron using single-microelectrode voltage-clamp recordings.
a | When M channels are functional (open), depolarizing the neuron from its holding (resting)
potential of –50 mV to –40 mV produces an outward M current (I) (voltage clamp), as in
FIG. 1a. Under this condition, the neuron shows strong spike adaptation so that, when the
clamp is released (current clamp), injection of a depolarizing current only produces a single
spike: the outward current resists the membrane potential changes and the increased
conductance raises the threshold for spike generation, so that subsequent action potentials
are suppressed. b | When the channels are closed by stimulating the muscarinic receptors
with the drug muscarine, the M current is largely suppressed and a depolarizing current
injection can generate a train of action potentials. c | Shows remote signalling by muscarinic
acetylcholine receptors. Stimulating muscarine receptors with the muscarinic agonist
oxotremorine-methiodide (Oxo-M) outside the patch closes M channels inside the patch in
sympathetic neurons (P. Delmas, unpublished observations; see also REF. 36). Channel
activity recovers fully after Oxo-M is washed out. These findings indicate that inhibition of
M-channel activity involves a molecule that is capable of diffusing into (or out of) the
membrane region circumscribed by the patch electrode. Panels a and b adapted, with
permission, from REF. 123 © (1983) Elsevier Science.
(and for some others, such as cyclic ADP-ribose) such
that, by 1997, the situation remained confusing (see
REF. 4). Assisted by the subsequent identification of
the molecular composition of M channels, recent
work has introduced considerable clarification of this
second messenger problem.
Gating of Kv7/M channels by PtdIns(4,5)P2
A breakthrough in our understanding of the regulation of Kv7/M channels came with the realization that
— in common with several other ion channels and
852 | NOVEMBER 2005
| VOLUME 6
membrane transport proteins39–41 — Kv7 channels
require a certain level of PtdIns(4,5)P2 in the cell
membrane to open. It was therefore proposed that
channel inhibition by G q-coupled receptors might
result from the depletion of membrane PtdIns(4,5)P2
as a result of its hydrolysis, rather than from the action
of a product of this hydrolysis.
Initial evidence in favour of this idea stemmed
from two complementary experimental approaches.
First, Suh and Hille42 reported that the compound
wortmannin (which, at certain concentrations,
inhibits the PtdIns(4)P-synthesizing enzyme phosphoinositide 4-kinase (PI4K); see BOX 1) reduced the
M current and greatly slowed the recovery of both
the ganglionic M current (as measured in the whole
cell) and that generated by recombinant Kv7.2/Kv7.3
channels as a result of inhibition produced by stimulating muscarinic acetylcholine receptors. This,
coupled with a similar requirement for hydrolysable
ATP, indicated (at the very least) that resynthesis
of PtdIns(4)P, and therefore of PtdIns(4,5)P2, was
needed for restoration of M-channel activity. Similar
effects were observed for the inhibition of the
M current in frog ganglia by nucleotides 43 or by
LHRH44. Second, Zhang et al.45 provided direct evidence that M channels require PtdIns(4,5)P2 to open.
Therefore, when membrane patches from oocytes
expressing Kv7.2 or Kv7.2/Kv7.3 were excised into
the inside-out configuration, currents showed a rapid
‘run-down’ , which could be reversed by the addition
of PtdIns(4,5)P2 or an analogue, to the inside face of
the membrane. This could then be suppressed again
by adding an antibody to PtdIns(4,5)P2 or a basic
peptide (polylysine) that binds to, and sequesters,
membrane PtdIns(4,5)P2. Importantly, this cycle of
restoration and current suppression extends to all
members of the Kv7 family, albeit with varying subunit
sensitivity 46 (see below). This includes Kv7.1 REF. 47,
but with the notable difference in this case that
PtdIns(4,5)P2 regulates the channel’s voltage-sensitivity47
rather than its maximum open probability46.
Two further pieces of evidence suggest that it is
the fall in membrane [PtdIns(4,5)P2], rather than the
accumulation of its hydrolysis products, that constitutes the primary mechanism for receptor-induced
M-channel closure. First, a mutation in KCNQ2
(H328C) that reduced (by threefold) the ability of an
exogenous PtdIns(4,5)P2 analogue (1,2-dioctanoylglycerol (diC8)-PtdIns(4,5)P2) to activate expressed
KCNQ2/3 channels in oocytes enhanced the sensitivity
of the current to the activation of co-expressed bradykinin receptors (see below for further consideration of
bradykinin action). Second, in sympathetic neurons
a threefold elevation in resting PtdIns(4,5)P2 levels
that was produced by overexpressing the synthetic
enzyme phosphoinositide 5-kinase (PI5K) reduced
M-current inhibition by a muscarinic agonist 48 .
Neither of these effects would be expected were inhibition to be induced by a product of PtdIns(4,5)P2
hydrolysis: both are in better agreement with the
PtdIns(4,5)P2-depletion hypothesis.
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a
Kv7.3
Kv7.2
P loop
S1
S2
S3
S4
S5
Kv7.2
S6
Kv7.2
Kv7.3
NH2
Trp 236
A
PtdIns(4,5)P2
b
310
G
321
328
H
COOH
497
341
I Q
372
R
CaMI
499
523
501
C T
530
529
S
ES
CaMII
P
AKAP
P
PKC
Figure 3 | Structure of Kv7 channels: interaction sites on the carboxy-terminal tail of
Kv7.2. a | Kv7/KCNQ channel subunits have a conventional Shaker-like K+ channel structure,
with 6 transmembrane domains (S1–S6), a single pore (P)-loop that forms the selectivity filter of
the pore, a positively-charged fourth transmembrane domain (S4) that acts as a voltage sensor
and a long intracellular carboxy-terminal tail. Four such subunits make up a functional Kv7
channel. All five (Kv7.1–Kv7.5) Kv7 channel subunits can form homomeric channels, whereas
the formation of heteromers is restricted to certain combinations18. The carboxyl terminus
contains a conserved domain (A domain) that determines the subunit specificity of Kv7 channel
assembly124,125. In the case of Kv7.2–Kv7.3 heteromers, two Kv7.2 and two Kv7.3 subunits
assemble to form a functional tetrameric channel. One residue of Kv7.2, tryptophan 236,
confers sensitivity to retigabine88. b | The carboxy-terminal tail of Kv7.2 subunits has binding
sites for several potential regulatory molecules, as depicted. CaMI and CaMII represent the two
binding sites for calmodulin (CaM). C497 is the Kv7.2 homologue of C519 in human Kv7.4,
which is required for N-ethylmaleimide (NEM)-induced enhancement of Kv7 currents57.
AKAP, A-kinase anchoring protein; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate;
S523, S530, serine residues phosphorylated by protein kinase C (PKC). Numbering of amino
acids according to the human KCNQ2 sequence.
Quantitative aspects of PtdIns(4,5)P2 depletion
PtdIns(4,5)P 2 comprises only ~1% of membrane
phospholipids, and is mostly confined to the inner
membrane leaflet 49 . How can one relate changes
in PtdIns(4,5)P 2 concentrations to changes in
M-channel activity? Biochemical measurements
indicate that stimulation of muscarinic or bradykinin receptors can produce substantial decreases
(of ~75% and ~35%, respectively) in the amounts
of PtdIns(4,5)P 2 in neuroblastoma cells, within
30–60 s 50,51. However, technical limitations mean
that such measurements cannot be made on single
neurons. An alternative method that can be used
in neurons simultaneously with membrane current
recording is to follow the translocation of the fluorescent PtdIns(4,5)P2-binding peptide green fluorescent protein (GFP)–PLCδ–pleckstrin homology
(PH) domain from the membrane into the cytosol as
PtdIns(4,5)P2 is hydrolysed52,53 (FIG. 5). Tests with this
probe have shown a good correlation with muscarinic
NATURE REVIEWS | NEUROSCIENCE
inhibition of both expressed Kv7 channels54,55 and
native M channels in sympathetic neurons48.
This probe measures PtdIns(4,5)P 2 hydrolysis
rather than directly measuring PtdIns(4,5)P2 depletion, because it also binds to the Ins(1,4,5)P3 formed
from PtdIns(4,5)P2. However, this property can itself
be used to estimate PtdIns(4,5)P2 levels, by competitive displacement of the membrane-bound probe with
set intracellular concentrations of Ins(1,4,5)P3 REF. 48.
From this, changes in membrane [PtdIns(4,5)P2] produced by increasing concentrations of a muscarinic
agonist (oxotremorine-methiodide) could be calculated and transcribed into fractional inhibition of the
M current expected from the PtdIns(4,5)P2–Kv7.2/Kv7.3
gating data of Zhang et al.45. The resultant prediction agreed closely with the observed data (FIG. 6a),
with a maximal decrease of ~90% of membrane
[PtdIns(4,5)P2]. Suh et al.54 used a different approach,
working with a virtual cell model (like that used by
Xu et al.51) to provide a comprehensive kinetic analysis
of the entire receptor–G protein–PLC–PtdIns(4,5)P2
system, as applied to the muscarinic inhibition of
expressed recombinant Kv7.2/Kv7.3 channels in a
human cell line. The model satisfactorily reproduced
the responses of these cells, and again predicted
a large (>95%) and rapid (half-time 2.7 s) fall in
[PtdIns(4,5)P2] after maximal muscarinic receptor
stimulation (FIG. 6b). Therefore, in both these studies,
Kv7/M-current inhibition could be quantitatively
accounted for by PtdIns(4,5)P2 depletion.
Recent experiments on expressed recombinant
Kv7 channels report that the affinity of different subunits for PtdIns(4,5)P2 varies widely, in the order Kv7.3
> Kv7.2 > Kv7.4, with EC50 values for diC8PtdIns(4,5)P2
ranging from 3 µM to several hundred micromolar,
and with that for co-expressed Kv7.2/Kv7.3 channels
being intermediate between those for Kv7.2 and Kv7.3
REF. 46. This is important for two reasons. First, it helps
to explain why different Kv7 channels show different
values for their maximum open probabilities (Popen)
when expressed in the same cell and recorded with
cell-attached patch electrodes56,57. Presumably, the
resting concentration of PtdIns(4,5)P2 in the native
cell membranes is sufficiently high to give the nearunity Popen that is observed with the high-affinity
Kv7.3 channels, but well below that needed for full
activation of the low-affinity Kv7.2 channel (Popen of
0.15–0.17 REFS 56,57). Second, M channels in different
neural loci can be composed of different Kv7 subunits
(either homomeric or heteromeric, see above), which
implies that the reduction in M-channel activity (and
thereby the increase in neural excitability) produced
by neurotransmitters that promote the hydrolysis of
PtdIns(4,5)P2 could vary considerably at different sites
within the nervous system.
Other influences on M-channel activity
Notwithstanding the evidence for PtdIns(4,5)P2 having
a major role in M-channel gating, and for its depletion
following hydrolysis being instrumental in the classical
inhibition of M channels after muscarinic receptor
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REVIEWS
Agonist
ACh
Plasma membrane receptor
M1
Gαq βγ
Heterotrimeric G-protein
PtdIns(4,5)P2
Enzyme PLCβ
Second messengers DAG
Ins(1,4,5)P3
Ca2+
PKC
Inhibition
Kv7/M
Figure 4 | Schematic diagram of the phospholipase
C-coupled pathway that links M1 muscarinic
acetylcholine receptors to Kv7 channels. The so-called
‘mysterious’ signal that mediates muscarinic receptor (M1)
modulation of Kv7/M channel activity involves the pertussistoxin-insensitive heterotrimeric G-protein, and specifically the
Gq/11 subunit, and phospholipase-Cβ (PLCβ). Modulation of
Kv7 channels seems to involve an unidentified diffusible
second messenger that inhibits Kv7 channel activity, either
directly or indirectly (via additional downstream molecules).
The breakdown products of phosphatidylinositol-(4,5)bisphosphate (PtdIns(4,5)P2)— such as diacylglycerol (DAG)
and inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) — and
subsequent downstream signals have been excluded as
major participants in the slow muscarinic inhibitory pathway.
Recent research suggests that membrane PtdIns(4,5)P2 is a
crucial determinant of modulation and might act as a major
signalling molecule in this pathway. Ca2+ and protein kinase C
(PKC) may serve to modify the interaction of the channels
with PtdIns(4,5)P2. ACh, acetylcholine.
stimulation, there remain several other potential
messengers involved in transmitter-induced closure.
How important are these, and how do they fit into the
overall picture of M-channel regulation?
Calcium. The concentration of Ca2+ in cells is normally
maintained at ~0.1 µM, but is frequently raised as a
result of Gq-linked receptor stimulation, because the
principal effect of one of the products of PtdIns(4,5)P2
hydrolysis — Ins(1,4,5)P3 — is to release calcium from
intracellular stores. It is hardly surprising, therefore,
that investigators looked first to Ca2+ as the most likely
second messenger for M-channel closure (see REF. 4).
M channels are certainly sensitive to Ca2+: native
M channels in inside-out membrane patches excised
from sympathetic neurons are inhibited by Ca2+ with an
IC50 of ~100 nM58 — only fractionally above the normal
resting concentration of 70–80 nM in these neurons.
This is a ‘direct’ effect in that it does not require
ATP, and so does not result from phosphorylation.
Expressed Kv7.2/Kv7.3 channels show an equivalent
sensitivity59; these experiments have also shown that
inhibition results from an interaction with endogenous calmodulin59. This molecule is associated with
854 | NOVEMBER 2005
| VOLUME 6
Kv7/M channels through two binding sites on the
C-terminal tail60,61 (FIG 3b). Inhibition by Ca2+/CaM
is subunit-specific: it is seen in channels that express
Kv7.2, Kv7.4 and Kv7.5 subunits, but not in those with
Kv7.1 or Kv7.3 subunits62.
Does this contribute to receptor-mediated inhibition? The answer is yes — at least for the action of
one hormone (bradykinin) on sympathetic neurons.
Therefore, the effect of bradykinin on these native
M channels is prevented by intracellular Ca2+-buffering
or by depletion of Ca2+ stores63. Although the observed
global rise is small (100–200 nM), it is compatible with
the high sensitivity of the channels to Ca2+ REF. 58, and
submembrane Ca2+ concentrations might be enhanced
beyond global cytoplasmic concentrations through
the close coupling of the bradykinin and Ins(1,4,5)P3
receptors (IP3R)64,65.
But if bradykinin releases Ca2+ through PtdIns(4,5)P2
hydrolysis, why does this hydrolysis itself not result in
channel closure through PtdIns(4,5)P2 depletion, as
proposed for the muscarinic receptor system? The
answer could be that the release of Ca2+ might simultaneously stimulate PtdIns(4,5)P2 synthesis, preventing
depletion — possibly through activation of the neuronal Ca2+ sensor protein (NCS1), which binds Ca2+ and
(among other effects) activates PI4K66,67. Stimulation
of PtdIns(4,5)P2 synthesis by bradykinin has been
observed in neuroblastoma cells51, and overexpression
of NCS1 further reduces the inhibitory action of bradykinin48. In addition, bradykinin does not inhibit either
G-protein-gated inward rectifier (GIRK; Kir3.1/Kir3.2)
channels or Ca2+ channels in sympathetic neurons, both
of which are gated by PtdIns(4,5)P2 and inhibited by
M1 muscarinic acetylcholine receptors, but neither of
which is sensitive to intracellular Ca2+. Indeed, in some
elegant experiments, Gamper et al.68 have shown that
bradykinin can inhibit Ca2+ channels, but only if the
effect of NCS1 activation is prevented by overexpressing a dominant-negative (non-Ca2+-binding) NCS1
construct or by impairing PtdIns(4,5)P2 synthesis with
wortmannin — in other words, if PtdIns(4,5)P2 levels
are allowed to fall below those required to maintain
Ca2+ channel activity. Normally, this does not occur;
presumably the same is true for M-channel gating, that
is, PtdIns(4,5)P2 levels do not fall far enough, in the
face of accelerated synthesis, to cause the channels to
close, thereby leaving Ca2+ to do the job.
The next obvious question is: why do muscarinic
receptors not work in the same way? The answer
— for sympathetic neurons, at least — is that stimulating these receptors does not produce an increase in
intracellular Ca2+ sufficient to accelerate PtdIns(4,5)P2
synthesis. The reason is that — unlike bradykinin
receptors — muscarinic receptors are too distant from
the IP3Rs for the transient increase in Ins(1,4,5)P3
to release Ca2+ from the endoplasmic reticulum in
the face of diffusional gradients and metabolism64.
By contrast, muscarinic receptor stimulation readily
produces large Ca2+ increases in some other cells
(but rarely primary neurons) that express Kv7/M
currents69, including the Kv7.2/Kv7.3-expressing tsA
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Box 1 | Schematic diagram of the phosphoinositide–phospholipase C cycle
Common phosphoinositides (PIs) differ in
phosphorylation of the inositol ring at
PI
hydroxyl positions 3, 4 and 5, giving rise to
PA
mono-, bis-, and trisphosphate derivatives,
PIP 4-Pase
ATP
which are abbreviated to PtdIns(4)P,
PtdIns(4,5)P2, and PtdIns(3,4,5)P3.
PI4K
–
PO4H2
Phosphatidylinositol-4,5-bisphosphate
ADP
PtdIns(4)P
(PtdIns(4,5)P2), a lipid found in the inner
leaflet of the plasma membrane, is
Ins(1)P
Rho GTPases
ADP
synthesized from phosphoinositide through
PIP2 5-Pase
ATP
sequential phosphorylations by
DAG
–
PI5K
PO4H2
phosphoinositide 4-kinase (PI4K) and
kinase
PtdIns(4,5)P2
ADP
phosphoinositide 5-kinase (PIP5K, shown
here as PI5K). PtdIns(4,5)P2 can be
ATP
Ins(1,4)P2
metabolized through dephosphorylation by
PI3K
5-phosphatase (PIP2 5-Pase) to PtdIns(4)P.
ATP
ADP PtdIns(3,4,5)P3
–
Type I phosphoinositide 3-kinases (PI3K)
PO4H2
PLC
GPCR
phosphorylate the 3′-OH position of the
inositol ring of PtdIns(4,5)P2, producing the
DAG
DAG
Ins(1,4,5)P3
lipid product phosphatidylinositol-3,4,5lipase
trisphosphate (PtdIns(3,4,5)P3), which is
known to regulate Rho family GTPases121.
When stimulated by G-protein-coupled
IP3R
PKC
receptors (GPCRs), phospholipase C (PLC)
Arachidonic acid
cleaves PtdIns(4,5)P2, producing the second
Phosphate group
Inositol
Fatty acid
Glycerol
messengers inositol-(1,4,5)-trisphosphate
(Ins(1,4,5)P3) and diacylglycerol (DAG).
DAG is the natural activator of the protein kinase C (PKC) family of serine-threonine kinases, whereas Ins(1,4,5)P3
releases Ca2+ from intracellular Ins(1,4,5)P3 receptor (IP3R) stores. DAG can, in turn, be hydrolysed by DAG lipases into
arachidonic acid, another diffusible messenger39, or recycled into phosphoinositide (re)synthesis through DAG kinasemediated phosphorylation to phosphatidic acid (PA). In addition to DAG conversion to PA by DAG kinases, the reverse
reaction can also occur, catalysed by PA phosphohydrolase (not shown). However, this dephosphorylation occurs in a
different signalling pathway, associated with phospholipase D activity. Finally, Ins(1,4,5)P3 is rapidly converted into
inositol through specialized phosphatases that remove specific phosphates. Ins(1)P, inositol phosphate; Ins(1,4)P2,
inositol (1,4)-bisphosphate; PIP 4Pase, phosphatidylinositol-4-phosphate 4-phosphatase.
(a transformed HEK 293 cell line) cells used by the Hille
group29,54, and this has been shown to enhance both
PtdIns(4,5)P2 hydrolysis (measured by fluorescence)
and Kv7.2/Kv7.3 channel inhibition as compared
with situations in which this increase was prevented55.
However, the effects of the Ca2+ rise in these cells can
be attributed solely to the increased PtdIns(4,5)P2
depletion55, and not to activation of channel-associated
calmodulin. Presumably some other factors, such as
the availability of sufficient calmodulin, determine
whether Ca2+/calmodulin activation takes precedence
over PtdIns(4,5)P2 depletion in any given cell as the
prime cause of Kv7 channel closure.
Protein kinase C. Diacylglycerol (DAG, a product of
PtdIns(4,5)P2 hydrolysis, BOX 1) and phorbol esters
(which activate PKC) have been intermittently reported
to inhibit M currents, but previous experiments with
kinase inhibitors have yielded variable results and
have not, in general, supported a major role for PKC
activation in the transmitter-mediated inhibition of
M currents4. This situation has been clarified by the
finding that Kv7 channel proteins bind A-kinase
NATURE REVIEWS | NEUROSCIENCE
anchoring protein (AKAP)25,70, which also binds PKC.
Activation of PKC-induced phosphorylation of the
Kv7.2 channel protein and, importantly, expression
of a mutated AKAP that did not bind PKC, prevented
Kv7.2 phosphorylation and reduced the inhibition of
native ganglionic M channels by muscarinic receptor
stimulation70. This effect was replicated by PKC inhibitors that interact with the DAG binding site, but not by
conventional PKC inhibitors directed against the kinase
site. This suggests that, when PKC is bound to AKAP,
the kinase site is inaccessible or otherwise modified.
Furthermore, the effect of the PKC inhibitors, or of
the mutated AKAP, was not to preclude M-current
inhibition but to reduce sensitivity to inhibition (the
concentration–response curve for muscarinic receptorinduced inhibition was shifted about threefold to
the left). This means that PKC inhibition would be
expected to have no effect when high concentrations
of agonists are used (as is usual for experiments of this
type). A similar (threefold) increase in sensitivity to
muscarinic receptor-mediated inhibition by PKC has
recently been confirmed in striatal neurons9. A possible
mechanism for this enhanced sensitivity to muscarinic
VOLUME 6 | NOVEMBER 2005 | 855
REVIEWS
a
Oxo-M
Kv7/M
M1
also the case among Caenorhabditis elegans KCNQ-like
K+ channel homologues, as KQT1 is more sensitive
than KQT2 or KQT3 REF. 71.
Kv7/M
M1
DAG
PLCβ
PtdIns(4,5)P2
PLCβ
Ins(1,4,5)P3
PLCδ–PH
PLCδ–PH
GFP
GFP
b
Oxo-M
Fluorescence intensity
Control
200
Oxo-M
Control
150
100
50
0
0
50
100
150
200
250
300
Pixel number
c
Oxo-M
700
600
Membrane current (pA)
Oxo-M
Cytosolic fluorescence
M-channel current
500
400
300
200
100
0
–100
0
200
400
600
800
1,000
Time (s)
Figure 5 | Muscarinic inhibition of the M current is accompanied by hydrolysis of
PtdIns(4,5)P2. a | Hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) was
monitored using the green-fluorescent protein (GFP)-tagged pleckstrin homology
(PH)-domain of phospholipase-Cδ (PLCδ–PH). This binds to the inositol phosphate headgroups of PtdIns(4,5)P2 in the inner leaflet of the membrane. When muscarinic receptors (M1)
are stimulated, phospholipase-Cβ (PLCβ) is activated. This hydrolyses PtdIns(4,5)P2, forming
diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (Ins(1,4,5)P3), which enters the cytosol.
The GFP-tagged PLCδ–PH bound to Ins(1,4,5)P3 then leaves the membrane and
translocates to the cytosol. b | The translocation of the probe (left) following stimulation of the
muscarinic receptors with oxotremorine-methiodide (Oxo-M). After washing out the drug, the
probe returns to the membrane as the Ins(1,4,5)P3 is hydrolysed and PtdIns(4,5)P2 is
resynthesized (not illustrated). The right panel shows line scan profiles of the GFP–PLCδ–PH
fluorescence intensity in the presence and absence of Oxo-M. c | Shows simultaneous
recording of the decline in membrane current that occurs as the M channels are inhibited
and the increase in cytosolic fluorescence after two applications of Oxo-M to a rat
sympathetic neuron voltage-clamped with a perforated-patch electrode (J. Winks and
S. J. Marsh, unpublished observations). Right panel in b adapted, with permission, from REF.
48 © (2005) Society for Neuroscience.
receptor stimulation is discussed below. Furthermore,
as with Ca2+/calmodulin regulation, there seems to be
some C-terminal tail-dependent subunit specificity
in sensitivity to PKC-mediated inhibition, in that
oocyte-expressed Kv7.5 subunits have been reported
to be much more sensitive than other Kv7 subunits to
the PKC-activator oleoylacetylglycerol (OAG). This is
856 | NOVEMBER 2005
| VOLUME 6
Cyclic ADP-ribose. Cyclic ADP-ribose is formed from
NAD + by the multifunctional ecto-enzyme ADPribosyl cyclase and releases, or enhances the release
of, Ca2+ through intracellular ryanodine receptors72.
G q-coupled (M1 or M3) muscarinic receptors can
activate ADP-ribosyl cyclase in NG108-15 neuroblastoma cells 73 , and intracellular application of
cyclic ADP-ribose reduces the M current recorded
from these cells74–76. Importantly, depletion of precursor NAD+ levels74 or intracellular instillation of
the cyclic ADP-ribose antagonists, 8-bromo-cyclic
ADP-ribose or 8-amino-cyclic ADP-ribose, reduced
the ability of muscarinic stimulation to inhibit the
M current75,76. However, the effect of cyclic ADP-ribose
did not seem to involve ryanodine receptors or Ca2+
release74,75, so the exact mechanism of action is unclear.
In view of this, and of the fact that no equivalent
effect has been shown in neurons (and bearing in
mind the differences between neuroblastoma cells
and sympathetic, or other, neurons with regard to
muscarinic signalling), the potential role of cyclic
ADP-ribose as a signalling transducer or modulator
for Kv7/M-channel inhibition — although intriguing
— remains uncertain.
Src tyrosine kinase. Like several other K+ channels, the
tyrosine residues of Kv7 channels are phosphorylated
by members of the Src family of non-receptor tyrosine
kinases77. This effect is subunit-specific, being confined
to Kv7.3, Kv7.4 and Kv7.5, and results (in Kv7.3) from
phosphorylation of both amino (N)-terminal Tyr67
and C-terminal Tyr349 REF. 78. Phosphorylation
causes a reduction in the currents carried by these
three subunits, and also those carried by Kv7.2/
Kv7.3 heteromers and by native sympathetic neuron
M channels77. This is not due to reduced channel
expression but to reduced channel open probability,
which results from a shortening of the open state
and lengthening of the closed state 78 . Although
these experiments required expression of Src by
cDNA transfection, the effects of Src were imitated
by acute exposure to a tyrosine-phosphatase inhibitor77, which implies that there was some involvement
of endogenous tyrosine-kinase regulation. However,
Src phosphorylation does not seem to contribute to
muscarinic receptor-mediated inhibition, as the latter
was affected neither by prior Src expression nor by
a Src tyrosine kinase inhibitor, PP2 REF. 79, that
blocked the effect of Src77.
Synthesis of Kv7/M channel messenger systems
We have attempted to generate a model80 of how the
various messenger systems contribute to Kv7/Mchannel closure in sympathetic neurons, as these
are the neurons for which the most information is
available. This synthesis is depicted in FIG. 7 and
summarized below.
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REVIEWS
a
80
70
60
60
PtdIns(4,5)P2
50
40
40
30
KCNQ
20
20
Depletion of PtdIns(4,5)P2 (%)
Inhibition of M current (%)
80
b
1.0
Normalized amount
100
0.8
PtdIns(4)P
Oxo-M
0.6
Gactive
0.4
KCNQ
0.2
10
PtdIns(4,5)P2
0
0
–9
–8
–7
–6
–5
–4
–3
Log [Oxo-M] (M)
0
5
10
15
20
25
Time (s)
Figure 6 | Relationship between PtdIns(4,5)P2 hydrolysis and M-current inhibition produced by the muscarinic
agonist, oxotremorine-methiodide. a | The membrane-to-cytosol translocation of the PLCδ–PH probe (illustrated in
FIG. 5a) was used to calculate the steady-state change in membrane phosphatidylinositol-4,5-bisphosphate
(PtdIns(4,5)P2) after application of increasing concentrations of oxotremorine-methiodide (Oxo-M) (turquoise line).
Starting from an apparent concentration of 261 µM of PtdIns(4,5)P2, as seen by the probe in competition with intracellular
Ins(1,4,5)P3 REF. 48, the purple line shows the expected inhibition of the M current, as predicted from the sensitivity of
Kv7.2/Kv7.3 (KCNQ) channels to 1,2-dioctanoyl-glycerol (diC8)-PtdIns(4,5)P2 reported by Zhang et al.45. The curve
correlates well with the experimentally observed M current inhibition (solid squares). b | Shows the time-course of
G-protein activation (Gactive), PtdIns(4,5)P2 decline and Kv7 (KCNQ) inhibition in a Kv7.2/Kv7.3-transfected tsA-201
(a transformed HEK 293 cell line) cell after a rapid application of Oxo-M calculated by Suh et al.54 using a ‘Virtual Cell’
kinetic model. PtdIns(4)P, phosphatidylinositol-4-phosphate. Panel b modified, with permission, from REF. 54 © (2004)
Rockefeller University Press.
First, we propose that the primary controller for
M-channel activity is PtdIns(4,5)P2. This activates the
channel largely by binding to the membrane-subjacent
C-terminal 45 . We estimate 48 that, in sympathetic
neurons, the current is ~80% of its maximal available strength at resting amounts of PtdIns(4,5)P 2
(although Popen for the individual channels might be
less than this, depending, in part, on intracellular Ca2+
concentrations81, see below). Activation of muscarinic
receptors reduces the amount of PtdIns(4,5)P2 by up
to ~85%, as a result of which most of the channels
will shut.
Then, as one of the Ca2+/calmodulin binding sites60-62
overlaps the putative binding site for PtdIns(4,5)P2
(FIG. 3b), we suggest that occupation of this site can
cause channel closure by reducing the binding of
PtdIns(4,5)P2 to the channel. For example, in the
absence of any change in PtdIns(4,5)P2 concentration,
a fourfold reduction in PtdIns(4,5)P2 binding affinity
would account for the amount of inhibition produced
by bradykinin when it elevates intracellular calcium48
(although this is unlikely to be a truly competitive
interaction because the principal effect of Ca2+ is to
reduce the maximum opening probability81).
Finally, the phosphorylation sites for PKC (on
Ser523 and Ser530 in Kv7.2 REF. 70, see FIG. 3b) overlap
the second binding site for calmodulin. So, perhaps
phosphorylation by PKC (after PtdIns(4,5)P2 hydrolysis
by the G q -coupled muscarinic 64 receptor) might
enhance binding of calmodulin. Because channelbound calmodulin is partially activated at resting Ca2+
concentrations59, it could be proposed that this also
reduces the affinity of the channel for PtdIns(4,5)P2,
NATURE REVIEWS | NEUROSCIENCE
thereby sensitizing the channel to receptor-induced
PtdIns(4,5)P 2 depletion in the manner shown by
Hoshi et al.70. Although still speculative in respect
of Kv7/M channels, there is a precedent for a final
common path for PtdIns(4,5)P2 depletion and PKC
phosphorylation in the regulation of Kir inward
rectifier K + channels. In this case, the effects of
both muscarinic receptor activation and PKC activation show a parallel variation with the apparent
PtdIns(4,5)P2 binding activity among different members of the Kir family82. Sensitization to PtdIns(4,5)P2
reduction by PKC phosphorylation has also been
proposed to drive M3 muscarinic receptor-induced
inhibition of Kir3.1/Kir3.2 channels83. On the other
hand, we cannot yet exclude independent, and
possibly subunit-determined, effects of PKC and
PtdIns(4,5)P2, as seem to occur in the ATP-sensitive
channels Kir6.1 and Kir6.2 REF. 84.
Therefore, in this model, all putative second messengers for the inhibition of M channels by Gq-coupled
receptors are thought to modify the natural gating of
M channels by membrane PtdIns(4,5)P2 as a final endpoint, either by reducing the amount of PtdIns(4,5)P2
through direct hydrolysis, or by modifying the channel’s affinity for PtdIns(4,5)P2 through products of
PtdIns(4,5)P2 hydrolysis (or a combination of both).
The extent to which these mechanisms — reduction
of PtdIns(4,5)P2 or modification of its gating activity
— predominate might then vary both with different
transmitters and in different neurons, depending on
both the microstructural arrangements of the signalling systems64,70 and the Kv7 subunit composition of
the native M channels46,62.
VOLUME 6 | NOVEMBER 2005 | 857
REVIEWS
BK
ACh
Kv7.2–Kv7.5
B2R
Gq/11 PLCβ
M1
PI4K
CaM
NCS1
P
K+
PLCβ
AKAP
PKC
Gq/11
Ca2+
Ins(1,4,5)P3
DAG
PtdIns(4,5)P2
IP3R
IP3R
PtdIns(4)P
F-actin
Endoplasmic reticulum
Figure 7 | Signalling to Kv7/M channels. Kv7.2–Kv7.5 (KCNQ2–5) channels bind phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), calmodulin (CaM) and A-kinase anchoring
protein (AKAP). PtdIns(4,5)P2 is required for KCNQ channel opening45 and AKAP facilitates
phosphorylation of KCNQ serines by protein kinase C (PKC)70. M1 muscarinic and bradykinin
(BK) receptors (B2R) couple to Gq/11 G-proteins and activate phospholipase-Cβ (PLCβ). This
leads to hydrolysis of PtdIns(4,5)P2 to produce inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) and
diacylglycerol (DAG). B2R is closely connected to the Ins(1,4,5)P3 receptor (IP3R) on the
endoplasmic reticulum, assisted by the F-actin cytoskeleton64. Therefore, the local increase
in Ins(1,4,5)P3 produces a vigorous release of Ca2+ that is sufficient to bind to Kv7-attached
calmodulin and close Kv7 channels. B2R also induces PtdIns(4,5)P2 synthesis, concurrent
with its hydrolysis, through the stimulation of phosphoinositide 4-kinase (PI4K) by the
neuronal Ca2+ sensor (NCS1)68. Therefore, bradykinin does not normally inhibit Kv7 currents
through PtdIns(4,5)P2 depletion, but rather through Ca2+ release. M1 receptors are not
directly associated with IP3Rs so Ins(1,4,5)P3 must diffuse further and releases little Ca2+ from
the endoplasmic reticulum. Instead, channel closure results from hydrolysis and depletion of
membrane PtdIns(4,5)P2 REFS 4245. Dissociation of PtdIns(4,5)P2 is facilitated by activation
of AKAP-bound PKC by diacylglycerol and Kv7/M-channel phosphorylation70. ACh,
acetylcholine.
Enhancement (upregulation) of M current?
At normal resting concentrations of PtdIns(4,5)P2,
M channels are not fully open. Therefore, single channel analysis shows that, for both native channels4,81
and recombinant Kv7.2/Kv7.3 channels56,57, maximal
Popen is <<1.0. So, there is considerable scope for
enhancing channel activity. This is important because,
just as reducing the M current enhances neural excitability and predisposes to epilepsy, enhancing the
M current should reduce excitability and counteract
convulsions.
Indeed, this is precisely the effect of the drug retigabine. The main action of this compound is to shift the
current–voltage curve to the left (so that the channels
open at more hyperpolarized membrane potentials),
but it also increases the maximum opening probability85,86. This is due to an interaction of retigabine with
residues in the S5 and S6 domains of the Kv7 subunit, of
which a tryptophan residue in the S5 domain is the key
determinant87,88 (FIG. 3a). This tryptophan is absent from
the cardiac Kv7.1 channel, which is, therefore, resistant
to retigabine action85. From comparisons of the crystal
structures of KcsA K+ channels in the closed state89 and
MthK channels in the open state90, it was proposed that
retigabine binds to a hydrophobic pocket in the cytoplasmic domains of S5 and S6 in Kv7.2–Kv7.5 channels
858 | NOVEMBER 2005
| VOLUME 6
that is created when the channel opens, so stabilizing
the open state87. Because of the consequential increased
outward K+ M current, retigabine strongly suppresses
neuronal firing85 and has potent anticonvulsant activity91. It also enhances M currents in nociceptive sensory
neurons and so exerts anti-nociceptive effects, particularly against neuropathic pain22,92. However, it is not
known whether any endogenous modulators interact
at this site.
Some neurotransmitters have also been reported
to increase M currents in hippocampal neurons.
These include somatostatin93, corticostatin94 and dynorphin95. The effect of somatostatin seems to involve
the phospholipase A2–arachidonic acid pathway96,
probably mediated by a product of 5-lipoxygenase97.
Ca2+-dependent activation of phospholipase A2 REF. 98
is also responsible for the enhancement of M currents
that is seen in frog sympathetic neurons when the
intracellular Ca2+ concentration is increased99–101 and
that generates the over-recovery that is observed after
the removal of a muscarinic receptor agonist99,102 — in
this case, this is mediated by a 12-lipoxygenase product
of arachidonic acid metabolism, not a 5-lipoxygenase
product98. The ubiquitous role of the arachidonic
pathways in the nervous system implies that M-current
activity might be modulated by a great variety of
hormones and drugs that interact with this system.
Currents generated by expressed Kv7.2/Kv7.3 channels are also increased by cysteine-alkylating reagents
such as N-ethylmaleimide (NEM)57. This effect extends
to homomeric Kv7.2, Kv7.4 and Kv7.5 channels, but
not to Kv7.3 channels, and embraces both an increased
maximum Popen and a left-shift of the current–voltage
curve. Although superficially similar to the effect of
retigabine, the molecular mechanism seems to be
quite different, as mutational analysis of Kv7.4 localized the principal site of action of NEM to a cysteine
(Cys519) in the C terminus57. This is also distant from
the assumed PtdIns(4,5)P2-binding site, so presumably
does not involve a change in the affinity of the channels for PtdIns(4,5)P2 — at least, not directly. However,
this cysteine residue is in close proximity to one of the
calmodulin binding sites in the C terminus (FIG 3b);
consequently, alkylation by NEM reduced calmodulin binding to a Kv7.2 C-terminal tail fusion protein
and calmodulin competitively reduced the enhancing
effect of NEM on Kv7 currents62. Furthermore, NEM
prevented the Ca2+/calmodulin-dependent inhibition
of native M channels in sympathetic neurons by bradykinin (see above), but not the effect of a muscarinic
agonist62. Again, it is not known whether any other
endogenous modulators interact with Cys519 (or its
equivalent in other Kv7 subunits). However, it is known
that the kinetic behaviour of other K+ channels can be
dramatically altered by glutathione103, which regulates
the state of cysteine reduction. Therefore, the activity
of M channels as determined by the resting Ca2+ concentrations, or their sensitivity to transmitters that act
by increasing intracellular calcium, might similarly be
modified by changes in cellular glutathione levels or by
other oxidizing agents.
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REVIEWS
HOMER
Homer proteins belong to a
wider family of PDZ domaincontaining proteins and act as
scaffolds, binding clusters of
proteins and glutamate receptors
at postsynaptic sites. PDZ
domains are named after the
proteins in which these sequence
motifs were originally identified
(postsynaptic density 95, discs
large, zona occludens 1).
COINCIDENCE DETECTION
Kv7 channels can be seen as
coincidence detection points of
spatial and temporal signal
integration. As such, an
appropriately timed signal,
otherwise ineffective, can alter
channel gating by sensitizing
the response to a convergent
messenger, thereby playing a
crucial part in adjusting
neuronal output.
Human Kv7.2 subunits can also be phosphorylated
by protein kinase A (PKA) at Ser52 of the N terminus,
and this enhances the activity of expressed Kv7.2/Kv7.3
currents104. Activation of PKA by β-adrenergic receptors also enhances the cardiac Kv7.1 current through
phosphorylation of the N terminus Ser27; this involves
a signalling complex that contains the AKAP protein
Yotiao, which recruits PKA and the protein phosphatase
PP1 to the channel105,106. Stimulation of β-adrenergic
receptors has previously been reported to increase
M currents in frog stomach smooth muscle cells
(presumably by activation of PKA)107, but, curiously,
there have been no reports of similar effects in mammalian neurons, such as sympathetic neurons. This
could be because Yotiao does not seem to contribute
to the signalling complex in these neurons70, and the
AKAP protein that is associated with the M channels in
sympathetic neurons recruits PKC rather than PKA to
the Kv7.2/Kv7.3 channels70. Likewise, dephosphorylation of M channels (or their associated proteins) by
the Ca2+-dependent phosphatase calcineurin induces
channel closure in excised membrane patches from
frog sympathetic neurons108, but, again, no such effect
has so far been reported for mammalian neurons.
EVANESCENT SIGNALS
Signals that are short-lived and
might result in transient, but
not sustained, modification of
channel gating. Such signals
might be due to the fluctuating
activities of kinases and
phosphatases.
HEREDITARY LONGQT
SYNDROME
Familial disorder in which most
affected family members have
delayed ventricular
repolarization manifest as
QT prolongation. Affected
individuals have an increased
propensity to syncope,
polymorphous ventricular
tachycardia and sudden
arrhythmic death.
ANDERSEN’S SYNDROME
A variant of Long-QT
syndrome that is associated
with clinical manifestations,
including periodic paralysis,
prolongation of the QT interval
with ventricular arrhythmias,
and characteristic physical
features, including low-set ears,
micrognathia and clinodactyly.
BARTTER’S SYNDROME
(Also known as K+ wasting).
Involves a group of symptoms
including enlargement of
kidney cells associated with
hypokalemic alkalosis and
increased production of the
hormone aldosterone. The
condition is thought to be
caused by a defect in the
kidney’s ability to reabsorb
potassium.
Integration and discrimination of signals
If all paths following activation of Gq-coupled receptors
lead to changes in [PtdIns(4,5)P2] or PtdIns(4,5)P2–
channel interaction, the question arises: how do neurons separate or integrate signals that are mediated by
different receptors? One mechanism of discrimination
could occur through the segregation of receptors (and
their cognate signalling molecules) into anatomically
distinct microdomains, as exemplified by the bradykinin–IP3R complexes in sympathetic neurons, which
are clearly segregated from the muscarinic acetylcholine receptors64 (for a review, see REF. 80). This is not
unique to bradykinin receptors. In the CNS, group 1
metabotropic glutamate receptors (mGluR1s), which
also inhibit M currents109, form equivalent functional
complexes with IP3Rs through the postsynaptic density
protein HOMER110,111. Furthermore, Homer proteins
regulate the anatomical localization and coupling of
mGluR1s to M channels when these receptors and
proteins are co-expressed in sympathetic neurons112.
Conversely, various interactions at the final common
pathway allow prospective forms of signal integration.
For example, a transmitter-induced Ca2+ signal that, in
itself, is not sufficient to close the channels, might (by
shifting the sensitivity of the channel to PtdIns(4,5)P2)
enhance the response to another transmitter that
depletes PtdIns(4,5)P2 REF. 39. This could provide
a form of COINCIDENCE DETECTION at the channel level.
Alternatively, partial M-channel closure through steady
depletion of PtdIns(4,5)P2 by one transmitter might
sensitize the channels to a Ca2+ transient produced by
another transmitter, or even to the Ca2+ transients that
arise as a result of the Ca2+ influxes that occur during
normal electrical activity. This seems highly plausible,
because M channels are tonically active (and so carry a
steady outward current over many minutes or hours).
NATURE REVIEWS | NEUROSCIENCE
The muscarinic receptors that drive PtdIns(4,5)P2
hydrolysis do not desensitize rapidly and are subject to
continuous bombardment with acetylcholine released
from cholinergic afferents, and the channels will be most
sensitive to changes in their affinity for PtdIns(4,5)P2
when the PtdIns(4,5)P2 concentration is suboptimal for
channel opening. Such interactions could thereby provide a mechanism for long-term ‘setting’ of M-channel
sensitivity to more EVANESCENT SIGNALS.
M channels in health and disease
The linkage of M-channel activity to molecules such as
PtdIns(4,5)P2 and Ca2+, and to products of arachidonic
acid metabolism, implies that M-channel function
might be closely geared to the metabolic state of the
cell. The open probability of the M channels can be
tonically regulated by small changes in external (and,
therefore, presumably internal) calcium58, and the
sensitivity of the channels to PtdIns(4,5)P2 and arachidonic acid metabolites suggests a broad dependence on
lipid metabolism. For example, cardiac Kv7.1 currents
decline after metabolic poisoning, possibly as a result
of diminished PtdIns(4,5)P2 levels47. Both expressed
Kv7.2/Kv7.3 and native M currents are also reduced
by modest reductions in extracellular pH (primarily
through a shift in gating kinetics of Kv7.3 subunits),
and can be increased by modest alkalinization113. Kv7.5
channels are similarly modified by extracellular pH114,
whereas currents through Kv7.1, Kv7.4 and Kv7.5 (but
not Kv7.2/Kv7.3) channels expressed in oocytes are
enhanced by modest osmotic swelling and, conversely,
reduced by osmotic shrinkage114,115. These observations
suggest further ways in which M-channel activity might
be affected by neuronal metabolism and disease states.
Do any of the numerous genetic mutations in
neural Kv7/M channels affect their sensitivity to
PtdIns(4,5)P2, Ca2+/calmodulin or PKC? Although
such an effect has not been shown directly, it seems
likely because the apparent affinity of the homologous
cardiac Kv7.1 channels for PtdIns(4,5)P2 is reduced by
three mutations in the human protein (R243W, R539W
and R555C) that are associated with HEREDITARY LONGQT
116
SYNDROME (LQT1) . Furthermore, mutations in Kir
channels that modify PtdIns(4,5)P2 binding are also
known to generate human diseases such as ANDERSEN’S
117
SYNDROME and BARTTER’S SYNDROME
. Other disease
mutations that are associated with disruption of macromolecular signalling have also been described for Kv7.1
channels. The LQT1 G589D mutation is located in the
leucine zipper binding domain of Kv7.1, and disrupts
the binding of the Yotiao–PKA–PP1 signalling complex
to the C terminus of Kv7.1 REF. 105. This prevents the
regulation of KCNQ1 currents by PKA and has been
linked to LQT1 in a population of Finnish families118.
A similar disease-linked mutation has been described
in LQT5 that disrupts functional PKA regulation of
Kv7.1/KCNE1 REF. 119. Indeed, there are some splice
site mutations in the C terminus of Kv7.2 (at Lys397 and
Cys516) that give rise to juvenile epilepsy18; whether
these (or other) mutations affect the regulation of the
channels remains to be determined.
VOLUME 6 | NOVEMBER 2005 | 859
REVIEWS
Conclusions and future perspectives
The inhibition of M-channel activity by neurotransmitters and hormones is crucially important for
controlling the excitability of neurons. This is exemplified by the fact that a mutation in only one gene for
one of their component subunits (Kv7.2) that results in
a mere 25% reduction in total M-current amplitude is
sufficient to induce juvenile epilepsy104. The receptors
that inhibit these channels are generally coupled to
the G protein Gq, and, historically, several apparently
different biochemical mechanisms have been proposed
to couple Gq activation to M-channel inhibition. We
have used an ‘Occam’s razor’ approach to try to connect these mechanisms within a unifying framework
in which the membrane phospholipid PtdIns(4,5)P2
acts as the primary controller of channel activity; we
envisage that other messengers, such as Ca2+ and PKC,
then serve to modify the interaction of the channels
with PtdIns(4,5)P2. However, although regulation of
Kv7 channels by exogenous PtdIns(4,5)P2 analogues
is now well established42,43,45–48, with the exception
of the results of some tests with homologous Kv7.1
channels120, there is no direct biochemical evidence
for PtdIns(4,5)P2–Kv7 channel interaction; nor is there
yet any direct evidence that Ca2+/calmodulin or PKC
do actually modify PtdIns(4,5)P2–Kv7 channel gating
rather than producing a qualitatively different change
in channel gating. Experiments to establish these
factors need to be done. In the longer term, it will also
be important to have structural information about this
important family of K+ channels — particularly about
their crucially important C terminus: if this is truly
cytoplasmic, as has so far been presumed, this might
not be too daunting.
Evidence is also emerging that the differences
between the amino acid sequences in their C termini
1.
2.
3.
4.
5.
6.
7.
8.
Brown, D. A. & Adams, P. R. Muscarinic suppression of a
novel voltage-sensitive K+ current in a vertebrate neurone.
Nature 283, 673–676 (1980).
Original description of voltage-clamp experiments in
frog sympathetic neurons.
Constanti, A. & Brown, D. A. M-currents in voltageclamped mammalian sympathetic neurones. Neurosci.
Lett. 24, 289–294 (1981).
Brown, D. A. in Ion Channels Vol. 1 (ed. Narahashi, T.)
55–99 (Plenum, New York, 1988).
Marrion, N. V. Control of M-current. Annu. Rev. Physiol. 59,
488–504 (1997).
Peters, H. C., Hu, H., Pongs, O., Storm, J. F. & Isbrandt, D.
Conditional transgenic suppression of M channels in
mouse brain reveals functions in neuronal excitability,
resonance and behavior. Nature Neurosci. 8, 51–60 (2005).
Interesting transgenic approach to Kv7/M-channel
suppression that revealed a number of novel
M-channel functions in relation to hippocampal
electrical activity, spatial memory, motor behaviour
and postnatal brain development.
Aiken, S. P., Zaczek, R. & Brown, B. S. Pharmacology of
the neurotransmitter release enhancer linopirdine (DuP
996) and insights into its mechanism of action. Adv.
Pharmacol. 35, 349–384 (1996).
Yue, C. & Yaari, Y. KCNQ/M channels control spike
afterdepolarization and burst generation in hippocampal
neurons. J. Neurosci. 24, 4614–4624 (2004).
Gu, N., Vervaeke, K., Hu, H. & Storm, J. F. Kv7/KCNQ/M
and HCN/h, but not KCa2/SK channels, contribute to the
somatic medium after-hyperpolarization and excitability
control in CA1 hippocampal pyramidal cells. J. Physiol.
(Lond.) 566, 689–715 (2005).
860 | NOVEMBER 2005
| VOLUME 6
9.
10.
11.
12.
13.
14.
15.
16.
17.
endow individual Kv7 subunits with markedly different sensitivities to Ca2+/calmodulin59 and PtdIns(4,5)P2
REF. 46 . Although the classical M channel in the
sympathetic neuron is primarily composed of Kv7.2/
Kv7.3 heteromers16,26,29, there are neurons or neuronal subcompartments in the brain in which Kv7.2
is expressed without Kv7.3 REF. 23. Furthermore,
the other subunits (Kv7.4 and Kv7.5) also constitute
functional M channels18,27,114 that are, or are likely
to be, of functional importance in some neurons or
neural pathways (Kv7.4 in the auditory system18, and
Kv7.5 (in part) in the hippocampus20 and perhaps
the cerebral cortex21). We would, therefore, expect an
appreciable variation in the sensivity of M channels
in different loci to neurotransmitters and perhaps to
some drugs: this, and its physiological and pharmacological consequences, has yet to be fully explored.
Finally, although our unifying approach provides
scope for integration of channel-controlling signals,
at the same time it is clear that different neurotransmitters can use different intermediary signalling
mechanisms to attain the common end-point of
channel inhibition. In one instance at least — that
of the contrasting effects of activating muscarinic
and bradykinin receptors in sympathetic neurons
— this seems to be achieved (at least in part) through
segregation of the receptors into different signalling
microdomains64. There is also evidence to suggest
that the channels themselves are aggregated with
other proteins, such as AKAP25,70 and calmodulin60–62,
into supramolecular assemblies. Further definition
of these ‘receptorsomes’ and ‘channelosomes’ , their
connections and anatomical localization in living
neurons, is clearly required to understand how the signalling systems work to provide discrete physiological
outputs.
Shen, W., Hamilton, S. E., Nathanson, N. M. &
Surmeier, J. D. Cholinergic suppression of KCNQ channel
currents enhances excitability of striatal medium spiny
neurons. J. Neurosci. 25, 7449–7458 (2005).
Adams, P. R. & Brown, D. A. Synaptic inhibition of the
M-current: slow excitatory post-synaptic potential
mechanism in bullfrog sympathetic neurones. J. Physiol.
(Lond.) 332, 263–272 (1982).
Brown, D. A. & Selyanko, A. A. Membrane currents
underlying the cholinergic slow excitatory post-synaptic
potential in the rat sympathetic ganglion. J. Physiol.
(Lond.) 365, 365–387 (1985).
Gahwiler, B. H. & Brown, D. A. Functional innervation of
cultured hippocampal neurones by cholinergic afferents
from co-cultured septal implants. Nature 313, 577–579
(1985).
Weight, F. F. & Votava, J. Slow synaptic excitation in
sympathetic ganglion cells: evidence for synaptic
inactivation of potassium conductance. Science 170,
755–758 (1970).
Sillito, A. M. The cholinergic neuromodulatory system: an
evaluation of its functional roles. Progr. Brain Res. 98,
371–378 (1993).
Coyle, J. T., Price, D. L. & DeLong, M. R. Alzheimer’s
disease: a disorder of cortical cholinergic innervation.
Science 219, 1184–1190 (1983).
Wang, H.-S. et al. KCNQ2 and KCNQ3 potassium channel
subunits: molecular correlates of the M-channel. Science
282, 1890–1893 (1998).
First identification of M-channel subunits.
Selyanko, A. A. et al. Dominant-negative subunits reveal
potassium channel families that contribute to M-like
potassium currents. J. Neurosci. 22, RC212; 1–5 (2002).
18. Jentsch, T. J. Neuronal KCNQ potassium channels:
physiology and role in disease. Nature Rev. Neurosci. 1,
21–30 (2000).
19. Gutman, G. A. et al. International Union of Pharmacology.
XLI. Compendium of voltage-gated ion channels:
potassium channels. Pharmacol. Rev. 55, 583–586 (2003).
20. Shah, M. M., Mistry, M., Marsh, S. J., Brown, D. A. &
Delmas, P. Molecular correlates of the M-current in cultured
rat hippocampal neurons. J. Physiol. (Lond.) 544, 29–37
(2002)
21. Yus-Najera, E. et al. Localization of KCNQ5 in the normal
and epileptic human temporal neocortex and
hippocampal formation. Neuroscience 120, 353–364
(2003).
22. Passmore, G. M. et al. KCNQ/M currents in sensory
neurons: significance for pain therapy. J. Neurosci. 23,
7227–7236 (2003).
23. Devaux, J. J., Kleopa, K. A., Cooper, E. C. & Scherer, S. S.
KCNQ2 is a nodal K+ channel. J. Neurosci. 24, 1236–1244
(2004).
24. Martire, M. et al. M channels containing KCNQ2 subunits
modulate norepinephrine, aspartate, and GABA release
from hippocampal nerve terminals. J. Neurosci. 24,
592–597 (2004).
25. Cooper, E. C. et al. Colocalization and coassembly of two
human brain M-type potassium channel subunits that are
mutated in epilepsy. Proc. Natl Acad. Sci. USA 97,
4914–4919 (2000).
26. Hadley, J. K. et al. Stoichiometry of expressed KCNQ2/
KCNQ3 potassium channels and subunit composition of
native ganglionic M channels deduced from block by
tetraethylammonium. J. Neurosci. 23, 5012–5019
(2003).
www.nature.com/reviews/neuro
REVIEWS
27. Selyanko, A. A. et al. Inhibition of KCNQ1–4 potassium
channels expressed in mammalian cells via M1 muscarinic
acetylcholine receptors. J. Physiol. (Lond.) 522, 349–355
(2000).
28. Brown, D. A., Selyanko, A. A., Hadley, J. K. & Tatulian, L.
Some pharmacological properties of neural KCNQ
channels. Neurophysiology 34, 111–114 (2002).
29. Shapiro, M. S. et al. Reconstitution of muscarinic
modulation of the KCNQ2/KCNQ3 K+ channels that
underlie the neuronal M current. J. Neurosci. 20,
1710–1721 (2000).
30. Pfaffinger, P. J. Muscarine and t-LHRH suppress M-current
by activating an IAP-insensitive G-protein. J. Neurosci. 8,
3343–3353 (1988).
31. Brown, D. A., Marrion, N. V. & Smart, T. H. On the
transduction mechanisms for muscarine-induced inhibition
of M-current in cultured rat sympathetic neurones.
J. Physiol. (Lond.) 413, 469–488 (1989).
32. Lopez, H. S. & Adams, P. R. A G protein mediates the
inhibition of the voltage-dependent potassium M current
by muscarine, LHRH, substance P and UTP in bullfrog
sympathetic neurons. Eur. J. Neurosci. 5, 529–542 (1989).
33. Caulfield, M. P. et al. Muscarinic M-current inhibition via
G alpha q/11 and alpha-adrenoceptor inhibition of Ca2+
current via G alpha o in rat sympathetic neurones.
J. Physiol. (Lond.) 477, 415–422 (1994).
34. Haley, J. E. et al. The α subunit of Gq contributes to
muscarinic inhibition of the M-type potassium current in
sympathetic neurons. J. Neurosci. 18, 4521–4531 (1998).
35. Haley, J. E. et al. Muscarinic inhibition of calcium current
and M current in Gαq-deficient mice. J. Neurosci. 20,
3973–3979 (2000).
36. Selyanko, A. A., Stansfeld, C. E. & Brown, D. A. Closure of
potassium M-channels by muscarinic acetylcholinereceptor stimulants requires a diffusible messenger. Proc.
R. Soc. Lond. B 250, 119–125 (1992).
37. Marrion, N. V. Selective reduction of one mode of
M-channel gating by muscarine in sympathetic neurons.
Neuron 11, 77–84 (1993).
38. Hille, B. Modulation of ion-channel function by G-proteincoupled receptors. Trends Neurosci. 17, 531–536 (1994).
39. Delmas, P., Coste, B., Gamper, N. & Shapiro, M. S.
Phosphoinositide lipid second messengers: new
paradigms for calcium channel modulation. Neuron 47,
179–182 (2005).
40. Hilgemann, D. W., Feng, S. & Nasuhoglu, C. The complex
and intriguing lives of PIP2 with ion channels and
transporters. Sci. STKE 111, RE19 (2001).
41. Suh, B.-C. & Hille, B. Regulation of ion channels by
phosphatidylinositol 4,5-bisphosphate. Curr. Opin.
Neurobiol. 15, 370–378 (2005).
42. Suh, B.-C. & Hille, B. Recovery from muscarinic
modulation of M-current channels requires
phosphatidylinositol 4,5-bisphosphate synthesis. Neuron
35, 507–520 (2002).
Provides the first published evidence for a role for
PtdIns(4,5)P2 in recovery of M channels from
muscarinic inhibition. The authors report that
recovery of both expressed Kv7.2/Kv7.3 channels
and native M channels in sympathetic neurons
requires hydrolysable ATP and is slowed if
PtdIns(4,5)P2 resynthesis is inhibited with
wortmannin, and introduce the ‘lipid kinase and PI
polyphosphate’ hypothesis.
43. Ford, C. P., Stemkowski, P. L., Light, P. E. & Smith, P. A.
Experiments to test the role of phosphatidylinositol 4,5bisphosphate in neurotransmitter-induced M-channel
closure in bullfrog sympathetic neurons. J. Neurosci. 23,
4931–4941 (2003).
44. Ford, C. P., Stemkowski, P. L. & Smith, P. A. Possible role
of phosphatidylinositol 4,5 bisphosphate in luteinizing
hormone releasing hormone-mediated M-current inhibition
in bullfrog sympathetic neurons. Eur. J. Neurosci. 20,
2990–2998 (2004).
45. Zhang, H. et al. PIP2 activates KCNQ channels, and its
hydrolysis underlies receptor-mediated inhibition of M
currents. Neuron 37, 963–975 (2003).
The authors report that Kv7 channel activity
diminishes in excised patches and can then be
resuscitated by PtdIns(4,5)P2 analogues and
reinhibited by a PtdIns(4,5)P2 antibody or by
polylysine. A mutation that reduced PtdIns(4,5)P2
sensitivity of Kv7 channels also reduced inhibition by
bradykinin.
46. Li, Y., Gamper, N. S., Hilgemann, D. W. & Shapiro, M. S.
Regulation of Kv7 (KCNQ) K+ channel open probability by
phosphatidylinositol (4,5)-bisphosphate. J. Neurosci. (in
the press).
47. Loussouarn, G. et al. Phosphatidylinositol-4,5bisphosphate, PIP2, controls KCNQ1/KCNE1 voltagegated potassium channels: a functional homology
NATURE REVIEWS | NEUROSCIENCE
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
between voltage-gated and inward rectifier K+ channels.
EMBO J. 22, 5412–5421 (2003).
Winks, J. S. et al. Relationship between membrane
phosphatidylinositol-4,5-bisphosphate and receptormediated inhibition of native neuronal M channels.
J. Neurosci. 25, 3400–3413 (2005).
Describes tests on native ganglionic M channels. The
authors used an intracellular Ins(1,4,5)P3 displacement
of a PtdIns(4,5)P2-binding fluorophore to estimate
concentrations of membrane PtdIns(4,5)P2.
Overexpression of PI5K increases PtdIns(4,5)P2 and
reduces muscarinic inhibition. The paper includes a
calculation of the relationship between receptormediated PtdIns(4,5)P2 depletion (measured with the
fluorophore) and M-current inhibition.
McLaughlin, S., Wang, J., Gambhir, A. & Murray, D. PIP2
and proteins: interactions, organization, and information
flow. Annu. Rev. Biophys. Biomol. Struct. 31, 151–175
(2002).
Willars, G. B., Nahorski, S. R. & Challiss, R. A. Differential
regulation of muscarinic acetylcholine receptor-sensitive
polyphosphoinositide pools and consequences for
signaling in human neuroblastoma cells. J. Biol. Chem.
273, 5037–5046 (1998).
Xu, C., Watras, J. & Loew, L. M. Kinetic analysis of
receptor-activated phosphoinositide turnover. J. Cell Biol.
161, 779–791 (2003).
Stauffer, T. P., Ahn, S. & Meyer, T. Receptor-induced
transient reduction in plasma membrane PtdIns(4,5)P2
concentration monitored in living cells. Curr. Biol. 8,
343–346 (1998).
Varnai, P. & Balla, T. Visualization of phosphoinositides that
bind pleckstrin homology domains: calcium and agonistinduced dynamic changes and relationship to myo-[3H]
inositol-labelled phosphoinsitide pools. J. Cell Biol. 143,
501–510 (1998).
Suh, B. C., Horowitz, L. F., Hirdes, W., Mackie, K. & Hille, B.
Regulation of KCNQ2/KCNQ3 current by G protein
cycling: the kinetics of receptor-mediated signaling by Gq.
J. Gen. Physiol. 123, 663–683 (2004).
Provides a comprehensive kinetic description of
the sequence of events that occur between
muscarinic receptor activation and M-channel
closure by PtdIns(4,5)P2 depletion, based on the
University of Connecticut ‘Virtual Cell’ model (see
also reference 51).
Horowitz, L. F. et al. Phospholipase C in living cells:
activation, inhibition, Ca2+ requirement, and regulation of M
current. J. Gen. Physiol. 126, 243–262 (2005).
Selyanko, A. A., Hadley, J. K. & Brown, D. A. Properties of
single M-type KCNQ2/KCNQ3 potassium channels
expressed in mammalian cells. J. Physiol. (Lond.) 534,
15–24 (2001).
Li, Y., Gamper, N. & Shapiro, M. S. Single-channel analysis
of KCNQ K+ channels reveals the mechanism of
augmentation by a cysteine-modifying reagent.
J. Neurosci. 24, 5079–5090 (2004).
Selyanko, A. A. & Brown, D. A. Intracellular calcium directly
inhibits potassium M channels in excised membrane
patches from rat sympathetic neurons. Neuron 16,
151–162 (1996).
M channels in excised patches from sympathetic
neurons are inhibited by Ca2+ with an IC50 of ~100 nM.
However, the authors also noted that another factor
necessary for inhibition is sometimes washed out
after excision. Reference 59 suggests that this factor
is calmodulin.
Gamper, N. & Shapiro, M. S. Calmodulin mediates Ca2+dependent modulation of M-type K+ channels. J. Gen.
Physiol. 122, 17–31 (2003).
The authors show that calmodulin binds to KCNQ2/3
channels through C-terminal tail binding sites (see
also references 60 and 61). Using an ionomycin Ca2+loading method, they confirm the results reported in
reference 58 and further show the calmodulindependence of Ca2+-mediated inhibition. They also
provide evidence that the Ca2+-mediated inhibition of
M channels produced by bradykinin (see reference
63) involves calmodulin.
Yus-Najera, E., Santana-Castro, I. & Villarroel, A. The
identification and characterization of a noncontinuous
calmodulin-binding site in noninactivating voltagedependent KCNQ potassium channels. J. Biol. Chem.
277, 28545–28553 (2002).
Wen, H. & Levitan, I. B. Calmodulin is an auxiliary subunit
of KCNQ2/3 potassium channels. J. Neurosci. 22,
7991–8001 (2002).
Gamper, N., Li, Y. & Shapiro, M. S. Structural requirements
for differential sensitivity of KCNQ K+ channels to
modulation by Ca2+/calmodulin. Mol. Biol. Cell 16,
3538–3551 (2005).
63. Cruzblanca, H., Koh, D. S. & Hille, B. Bradykinin inhibits
M current via phospholipase C and Ca2+ release from
IP3-sensitive Ca2+ stores in rat sympathetic neurons. Proc.
Natl Acad. Sci. USA 95, 7151–7156 (1998).
64. Delmas, P., Wanaverbecq, N., Abogadie, F. C., Mistry, M.
& Brown, D. A. Signalling microdomains define the
specificity of receptor-mediated InsP3 pathways in
neurons. Neuron 34, 209–220 (2002).
The authors used transfected transient receptor
potential channels as biosensors to monitor DAG
and Ins(1,4,5)P3 separately in sympathetic neurons
after stimulation of bradykinin and muscarinic
receptors. They showed that bradykinin receptors
form a tight complex with IP3Rs, whereas muscarinic
receptors do not, which led to the concept of
signalling microdomains. This work helped to
resolve the difference in Ca2+ sensitivity between
muscarinic and bradykinin-induced inhibition
reported in reference 63.
65. Delmas, P. & Brown, D. A. Junctional signaling
microdomains: bridging the gap between the neuronal cell
surface and Ca2+ stores. Neuron 36, 787–790 (2002).
66. Zhao, X. et al. Interaction of neuronal calcium sensor-1
(NCS-1) with phosphatidylinositol 4-kinase β stimulates
lipid kinase activity and affects membrane trafficking in
COS-7 cells. J. Biol. Chem. 278, 40183–40189 (2001).
67. Burgoyne, R. D., O’Callaghan, D. W., Hasdemir, B.,
Haynes, L. P. & Tepikin, A. V. Neuronal Ca2+-sensor
proteins: multitalented regulators of neuronal function.
Trends Neurosci. 27, 203–209 (2004).
68. Gamper, N., Reznikov, V., Yamada, Y., Yang, J. &
Shapiro, M. S. Phosphatidylinositol 4,5-bisphosphate
signals underlie receptor-specific Gq/11-mediated
modulation of N-type Ca2+ channels. J. Neurosci. 24,
10980–10992 (2004).
69. Robbins, J., Marsh, S. J. & Brown, D. A. On the
mechanism of M-current inhibition by muscarinic m1
receptors in DNA-transfected rodent neuroblastoma x
glioma cells. J. Physiol. (Lond.) 469, 153–178 (1993).
70. Hoshi, N. et al. AKAP150 signaling promotes suppression
of the M-current by muscarinic agonists. Nature Neurosci.
6, 564–571 (2003).
Reports that AKAP150 facilitates PKC-mediated
phosphorylation of KCNQ2 channels and that this
contributes to muscarinic inhibition of M currents in
sympathetic neurons. This helps to resolve some of
the previous conflicts regarding PKC, summarized in
reference 4, and also introduces a new function for
AKAP.
71. Wei, A. D., Butler, A. & Salkoff, L. KCNQ-like potassium
channels in Caenorhabditis elegans. J. Biol. Chem. 280,
21337–21345 (2005).
72. Galione, A. & Churchill, G. C. Cyclic ADP ribose as a
calcium-mobilizing messenger. Sci. STKE 41, PE1 (2000).
73. Higashida, H. et al. Muscarinic receptor-mediated dual
regulation of ADP-ribosyl cyclase in NG108-15 neuronal
cell membranes. J. Biol. Chem. 272, 31272–31277
(1997).
74. Higashida, H. et al. Nicotinamide-adenine dinucleotide
regulates muscarinic receptor-coupled K+ (M) channels in
rodent NG108-15 cells. J. Physiol. (Lond.) 482, 317–323
(1995).
75. Bowden, S. E., Selyanko, A. A. & Robbins, J. The role of
ryanodine receptors in the cyclic ADP ribose modulation of
the M-like current in rodent m1 muscarinic receptortransformed NG108-15 cells. J. Physiol. (Lond.) 519,
23–34 (1999).
76. Higashida, H., Brown, D. A. & Robbins, J. Both linopirdineand WAY123,398-sensitive components of IK(M,ng) are
modulated by cyclic ADP ribose in NG108-15 cells.
Pflugers Arch. 441, 228–234 (2000).
77. Gamper, N., Stockand, J. D. & Shapiro, M. S. Subunitspecific modulation of KCNQ potassium channels by Src
tyrosine kinase. J. Neurosci. 23, 84–95 (2003).
78. Li, Y., Langlais, P., Gamper, N., Liu, F. & Shapiro, M. S.
Dual phosphorylations underlie modulation of unitary
KCNQ K+ channels by Src tyrosine kinase. J. Biol. Chem.
279, 45399–45407 (2004).
79. Hanke, J. H. et al. Discovery of a novel, potent, and Src
family-selective tyrosine kinase inhibitor. Study of Lck- and
FynT-dependent T cell activation. J. Biol. Chem. 271,
695–701 (1996).
80. Delmas, P., Crest, M. & Brown, D. A. Functional
organization of PLC signaling microdomains in neurons.
Trends Neurosci. 27, 41–47 (2004).
81. Selyanko, A. A. & Brown, D. A. M-channel gating and
simulation. Biophys. J. 77, 701–713 (1999).
82. Du, X. et al. Characteristic interactions with
phosphatidylinositol 4,5-bisphosphate determine
regulation of Kir channels by diverse modulators. J. Biol.
Chem. 279, 37271–37281 (2004).
VOLUME 6 | NOVEMBER 2005 | 861
REVIEWS
83. Brown, S. G., Thomas, A., Dekker, L. V., Tinker, A. &
Leaney, J. L. Protein Kinase C-δ sensitizes Kir3.1/3.2
channels to changes in membrane phospholipid levels
following M3 receptor activation in HEK293 cells. Am.
J. Physiol. Cell Physiol. C543–C556 (2005).
84. Quinn, K. V., Cui, Y., Giblin, J. P., Clapp, L. H. & Tinker, A.
Do anionic phospholipids serve as cofactors or second
messengers for the regulation of activity of cloned ATPsensitive K+ channels? Circ. Res. 93, 646–655 (2003).
85. Tatulian, L., Delmas, P., Abogadie, F. C. & Brown, D. A.
Activation of expressed KCNQ potassium currents and
native neuronal M-type potassium currents by the anticonvulsant drug retigabine. J. Neurosci. 21, 5535–5545
(2001).
86. Tatulian, L. & Brown, D. A. Effect of the KCNQ potassium
channel opener retigabine on single KCNQ2/3 channels
expressed in CHO cells. J. Physiol. (Lond.) 549, 57–63
(2003).
87. Wuttke, T. V., Seebohm, G., Bail, S., Maljevic, S. &
Lerche, H. The new anticonvulsant retigabine favors
voltage-dependent opening of the Kv7.2 (KCNQ2) channel
by binding to its activation gate. Mol. Pharmacol. 67,
1009–1017 (2005).
88. Schenzer, A. et al. Molecular determinants of KCNQ (Kv7)
K+ channel sensitivity to the anticonvulsant retigabine.
J. Neurosci. 25, 5051–5060 (2005).
89. Doyle, D. A. et al. The structure of the potassium channel:
molecular basis of K+ conduction and selectivity. Science
280, 69–77 (1998).
90. Jiang, Y. et al. The open pore conformation of potassium
channels. Nature 417, 523–526 (2002).
91. Rostock, A. et al. D-23129: a new anticonvulsant with a
broad spectrum activity in animal models of epileptic
seizures. Epilepsy Res. 23, 211–223 (1996).
92. Nielsen, A. N., Mathiesen, C. & Blackburn-Munro, G.
Pharmacological characterisation of acid-induced muscle
allodynia in rats. Eur. J. Pharmacol. 487, 93–103 (2003).
93. Moore, S. D., Madamba, S. G., Joels, M. & Siggins, G. R.
Somatostatin augments the M-current in hippocampal
neurons. Science 239, 278–280 (1988).
94. de Lecea, L. et al. A cortical neuropeptide with neuronal
depressant and sleep-modulating properties. Nature 381,
242–245 (1996).
95. Madamba, S. G., Schweitzer, P. & Siggins, G. R.
Dynorphin selectively augments the M-current in
hippocampal CA1 neurons by an opiate receptor
mechanism. J. Neurophysiol. 82, 1768–1775 (1999).
96. Schweitzer, P., Madamba, S. & Siggins, G. R. Arachidonic
acid metabolites as mediators of somatostatin-induced
increase of neuronal M-current. Nature 346, 464–467
(1990).
97. Lammers, C. H. et al. Arachidonate 5-lipoxygenase and its
activating protein: prominent hippocampal expression and
role in somatostatin signaling. J. Neurochem. 66, 147–152
(1996).
98. Yu, S. P. Roles of arachidonic acid, lipoxygenases and
phosphatases in calcium-dependent modulation of
M-current in bullfrog sympathetic neurons. J. Physiol.
(Lond.) 487, 797–811 (1995).
99. Marrion, N. V., Zucker, R. S., Marsh, S. J. & Adams, P. R.
Modulation of M-current by intracellular Ca2+. Neuron 6,
533–545 (1991).
862 | NOVEMBER 2005
| VOLUME 6
100. Yu, S. P., O’Malley, D. M. & Adams, P. R. Regulation of M
current by intracellular calcium in bullfrog sympathetic
ganglion neurons. J. Neurosci. 14, 3487–3499 (1994).
101. Tokimasa, T., Shirasaki, T. & Kuba, K. Evidence for the
calcium-dependent potentiation of M-current obtained by
the ratiometric measurement of the fura-2 fluorescence in
bullfrog sympathetic neurons. Neurosci. Lett. 236,
123–126 (1997).
102. Villarroel, A. On the role of arachidonic acid in M-current
modulation by muscarine in bullfrog sympathetic neurons.
J. Neurosci. 14, 7053–7066 (1994).
103. Ruppersberg, J. P. Regulation of fast inactivation of cloned
mammalian IK(A) channels by cysteine oxidation. Nature
352, 711–714 (1991).
104. Schroeder, B. C., Kubisch, C., Stein, V. & Jentsch, T. J.
Moderate loss of function of cyclic-AMP-modulated
KCNQ2/KCNQ3 K+ channels causes epilepsy. Nature 396,
687–690 (1998).
105. Marx, S. O. et al. Requirement of a macromolecular
signaling complex for beta adrenergic receptor modulation
of the KCNQ1-KCNE1 potassium channel. Science 295,
496–499 (2002).
106. Kurokawa, J., Motoike, H. K., Rao, J. & Kass, R. S.
Regulatory actions of the A-kinase anchoring protein
Yotiao on a heart potassium channel downstream of PKA
phosphorylation. Proc. Natl Acad. Sci. USA 101,
16374–16378 (2004).
107. Sims, S. M., Clapp, L. H., Walsh, J. V. Jr & Singer, J. J.
Dual regulation of M current in gastric smooth muscle
cells: beta-adrenergic-muscarinic antagonism. Pflugers
Arch. 417, 291–302 (1990).
108. Marrion, N. V. Calcineurin regulates M channel modal
gating in sympathetic neurons. Neuron 16, 163–173
(1996).
109. Charpak, S., Gahwiler, B. H., Do, K. Q. & Knopfel, T.
Potassium conductances in hippocampal neurons
blocked by excitatory amino-acid transmitters. Nature
347, 765–767 (1990).
110. Tu, J. C. et al. Homer binds a novel proline-rich motif and
links group 1 metabotropic glutamate receptors with IP3
receptors. Neuron 21, 717–726 (1998).
111. Thomas, U. Modulation of synaptic signalling complexes
by Homer proteins. J. Neurochem. 81, 407–413 (2002).
112. Kammermeier, P. J., Xiao, B., Tu, J. C., Worley, P. F. &
Ikeda, S. R. Homer proteins regulate coupling of group I
metabotropic glutamate receptors to N-type calcium and
M-type potassium channels. J. Neurosci. 20, 7238–7245
(2000).
113. Prole, D. L., Lima, P. A. & Marrion, N. V. Mechanisms
underlying modulation of neuronal KCNQ2/KCNQ3
potassium channels by extracellular protons. J. Gen.
Physiol. 122, 775–793 (2003).
114. Jensen, H. S., Callo, K., Jespersen, T., Jensen, B. S. &
Olesen, S. P. The KCNQ5 potassium channel from mouse:
a broadly expressed M-current like potassium channel
modulated by zinc, pH, and volume changes. Brain Res.
Mol. Brain Res. 139, 52–62 (2005).
115. Grunnet, M. et al. KCNQ1 channels sense small changes
in cell volume. J. Physiol. (Lond.) 549, 419–427 (2003).
116. Park, K. H. et al. Impaired KCNQ1–KCNE1 and
phosphatidylinositol-4,5-bisphosphate interaction underlies
the long QT syndrome. Circ. Res. 96, 730–739 (2005).
117. Lopes, C. M. et al. Alterations in conserved Kir channelPIP2 interactions underlie channelopathies. Neuron 34,
933–944 (2002).
118. Piippo, K. et al. A founder mutation of the potassium
channel KCNQ1 in long QT syndrome: implications for
estimation of disease prevalence and molecular
diagnostics. J. Am. Coll. Cardiol. 37, 562–568 (2001).
119. Kurokawa, J., Chen, L. & Kass, R. S. Requirement of
subunit expression for cAMP-mediated regulation of a
heart potassium channel. Proc. Natl Acad. Sci. USA 100,
2122–2127 (2003).
120. Thomas, A. M., Giblin, J. P., Wilson, A. & Tinker, A.
A biochemical approach to studying the interaction of
anionic phospholipids with potassium channel domains.
J. Physiol. (Lond.) 557P, PC85 (2004).
121. Cantrell, D. A. Phosphoinositide 3-kinase signalling
pathways. J. Cell Sci. 114, 1439–1445 (2001).
122. Adams, P. R., Brown, D. A. & Constanti, A. M-currents and
other potassium currents in bullfrog sympathetic neurons.
J. Physiol. (Lond.) 330, 537–542 (1982).
123. Brown, D. A. Slow cholinergic excitation — a mechanism
for increasing neuronal excitability. Trends Neurosci. 6,
302–307 (1983).
124. Schwake, M., Pusch, M., Kharkovets, T. & Jentsch, T. J.
Surface expression and single channel properties of
KCNQ2/KCNQ3, M-type K+ channels involved in epilepsy.
J. Biol. Chem. 275, 13343–13348 (2000).
125. Schwake, M., Jentsch, T. J. & Friedrich, T. A carboxyterminal domain determines the subunit specificity of
KCNQ K+ channel assembly. EMBO Rep. 4, 76–81 (2003).
Acknowledgements
The authors’ work has been supported by the UK Medical
Research Council (D.A.B.), the European Union Framework
Programs (D.A.B.), the Wellcome Trust (D.A.B. and P.D.) and the
Centre National de la Recherche Scientifique (CNRS) (P.D.).
Competing interests statement
The authors declare no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
AKAP | LHRH | NCS1 | PLCβ | Src
FURTHER INFORMATION
Geocities web links: http://www.geocities.com/ionchannels/
Potassium channels database: www.receptors.org/KCN/
seq/004/004.SEQ.html
Potassium channel gene nomenclature: www.gene.ucl.
ac.uk/nomenclature/genefamily/KCN.shtml
CNRS Cell Neurophysiology Laboratory: http://www.
univmed.fr/lnpc/Francais/index.htm
Faculty of 1000 author biography: http://www.f1000biology.
com/about/biography/1337430256139691
Brown’s homepage: www.ucl.ac.uk/Pharmacology/Research/
dab.html
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