59

J Physiol 569.1 (2005) pp 59–74

Protein kinase C shifts the voltage dependence of KCNQ/M
channels expressed in Xenopus oocytes
Koichi Nakajo1,2 and Yoshihiro Kubo1,2,3
1Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki,
Aichi, 444-8585, Japan
2
COE Program for Brain Integration and its Disorders, Tokyo Medical and Dental University, Graduate School and Faculty of Medicine,
Bunkyo, Tokyo, 113-8519, Japan
3
Solution Oriented Research for Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan

It is well established that stimulation of Gq -coupled receptors such as the M1 muscarinic
acetylcholine receptor inhibits KCNQ/M currents. While it is generally accepted that thismuscarinic inhibition is mainly caused by the breakdown of PIP2 , the role of the subsequent
activation of protein kinase C (PKC) is not well understood. By reconstituting M currents
in Xenopus oocytes, we observed that stimulation of coexpressed M1 receptors with 10 µM
oxotremorine M (oxo-M) induces a positive shift (4–30 mV, depending on which KCNQ channels
are expressed) in the conductance–voltage relationship (G–V ) of KCNQ channels. When weapplied phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, we observed a large
positive shift (17.8 ± 1.6 mV) in the G–V curve for KCNQ2, while chelerythrine, a PKC inhibitor,
attenuated the shift caused by the stimulation of M1 receptors. By contrast, reducing PIP2
had little effect on the G–V curve for KCNQ2 channels; although pretreating cells with 10 µM
wortmannin for 30 min reduced KCNQ2 current amplitude by 80%, the G–V curve was shifted
only slightly (5 mV). Apparently, the shift induced by muscarinic stimulation in Xenopus oocytes
was mainly caused by PKC activation. When KCNQ2/3 channels were expressed in HEK 293T
cells, the G–V curve seemed already to be shifted in a positive direction, even before activation of
PKC, and PMA failed to shift the curve any further. That alkaline phosphatase in the patch pipetteshifted the G–V curve in a negative direction suggests KCNQ2/3 channels are constitutively
phosphorylated in HEK 293T cells.
(Received 20 July 2005; accepted after revision 21 September 2005; first published online 22 September 2005)
Corresponding author K. Nakajo: Division of Biophysics and Neurobiology, Department of Molecular Physiology,
National Institute for Physiological Sciences, Okazaki, Aichi, 444-8585, Japan. Email: knakajo@nips.ac.jp

KCNQ channels are low-threshold non-inactivating
voltage-gated K+ channels, most of which have been linked
to inherited diseases such as cardiac arrhythmia, benign
familial neonatal convulsions and deafness (Jentsch,2000). Current through KCNQ channels is known as
the M-current and is inhibited by the activation of
Gq/11 -coupled receptors (Brown & Adams, 1980; Caulfield
et al. 1994; Marrion, 1997; Haley et al. 1998). In
sympathetic neurones, for example, M-current amplitude
is reduced by stimulating M1 muscarinic acetylcholine
receptors (Brown & Adams, 1980).
How the M-current is inhibited has long been an
enigma because the complex intracellular signalling
pathway via which it is regulated is comprised ofnumerous components. Recently, however, the M-current
was successfully reconstituted by expressing KCNQ
channels and M1 receptors in Xenopus oocytes andC 2005 The Authors. Journal compilation 
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in mammalian cell lines (Schroeder et al. 1998; Wanget al. 1998; Selyanko et al. 2000; Shapiro et al. 2000).
Using those expression systems, it was shown that
all KCNQ channels can be inhibited by M1 receptor
stimulation, and that receptor-induced hydrolysis ofphosphatidylinositol-4 5 -bisphosphate (PIP2 ) plays a keyrole in the muscarinic inhibition of KCNQ channels (Suh& Hille, 2002; Loussouarn et al. 2003; Zhang et al. 2003).
PIP2 is a negatively charged membrane phospholipidthat interacts with positively charged amino acids in the
C-terminal cytoplasmic region of the KCNQ channel(Zhang et al. 2003; Park et al. 2005). A certain amountof PIP2 is thought to be required to maintain the activityof KCNQ channels, as channels in inside-out patch
membranes quickly run-down without application of PIP2
to the intracellular side (Zhang et al. 2003). A similar
mechanism has also been observed in other ion channels,

DOI: 10.1113/jphysiol.2005.094995

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K. Nakajo and Y. Kubo

including inward rectifier K+ channels and voltage-gated
Ca2+ channels (Huang et al. 1998; Wu et al. 2002).
Protein kinase C (PKC) has also been thought to play arole in M-current inhibition (Higashida & Brown, 1986).
Although its activation by phorbol esters suppresses theM-current, PKC has not been thought to be an essential
factor in the channel’s inhibition. This is because its effectis only partial in sympathetic neurones and because its
activation does not seem to be required for M-current
inhibition (Bosma & Hille, 1989; Marrion, 1994). On theother hand, Hoshi et al. (2003) recently reported that
AKAP150, which is an auxiliary protein that anchors PKC,
directly binds to KCNQ2 channels and that interference
caused by that binding diminishes M-current inhibition.
Although this result suggests that PKC does, indeed, play arole in M-current inhibition, it is still not clear whether it
merely facilitates M-current inhibition by PIP2 or whether
it modulates KCNQ channels independently.
The present work shows that M1 receptor stimulation
shifts the conductance–voltage (G–V ) relationship of all
KCNQ channels expressed in Xenopus oocytes such that thechannels opened at more positive voltages; the magnitudeof the change varied from channel to channel. The phorbol
ester PMA had a similar effect on the KCNQ2 channel, but
induced a smaller shift in KCNQ1 channels. Depletionof PIP2 using wortmannin also induced a shift in voltage
dependence, but one that was much smaller than was
induced by PMA. We propose that PKC participates inmuscarinic receptor-mediated inhibition of the M-current
by modulating the voltage dependence of KCNQ
channels.

J Physiol 569.1

Two electrode voltage clamp

K+ currents were recorded with two-electrode voltage
clamp using an OC725C amplifier (Warner Instruments,
Hamden, CT, USA) and pCLAMP8 software (Axon
Instruments, Union City, CA, USA) 2–5 days after cRNA
injection. Data from the amplifier were digitized at 2 kHz.
The microelectrodes used were drawn from borosilicate
glass capillaries (World Precision Instruments, Sarasota,
FL, USA) to a resistance of 0.2–1 M when filled with
3 m potassium acetate and 10 mm KCl (pH 7.2). The
bath solution (ND96) contained (mm): 96 NaCl, 2 KCl,
1.8 CaCl2 , 1 MgCl2 and 5 Hepes (pH 7.4). Oocytes
were held at −80 mV and were stepped for 2 s todifferent test voltages followed by a 250-ms step pulse
to −30 mV for recording tail currents. Tail current
amplitudes were typically measured as the average value
10–20 ms after the end of the test pulse. All experiments
were carried out at room temperature (25 ± 2◦ C).
For the experiments summarized in Figs 1 and 4, we
pretreated oocytes with 100 nm thapsigargin (an endoplasmic reticulum Ca2+ -ATPase pump inhibitor) for
more than 3 h to deplete the intracellular Ca2+ stores
and, as a result, inhibit endogenous Ca2+ -activatedCl− channels. That pretreatment with thapsigargin did
not affect the muscarinic inhibition of KCNQ channels
indicates a rise in intracellular Ca2+ is not essential formuscarinic inhibition in Xenopus oocytes. If we did notincubate oocytes with thapsigargin, we observed activationof a large Ca2+ -activated Cl− current upon M1 receptor
stimulation. However, the current usually disappeared
within 1 min and did not affect the inhibition of KCNQ
channels.

Methods
Molecular biology

Patch clamp

The accession numbers for the KCNQ channels used (seeAcknowledgements) are AF000571 for human KCNQ1,
NM 133322 for rat KCNQ2, NM 031597 for rat KCNQ3
and AF105202 for human KCNQ4. All KCNQ cDNAs
were subcloned into the expression vector pGEMHE
for Xenopus oocytytes or pCXN2 for HEK 293T
cells.

cDNAs encoding KCNQ2, KCNQ3 and EGFP (5 : 5 : 1)
or KCNQ2 and EGFP (10 : 1) were cotransfected into
HEK 293T cells using Lipofectamine 2000 (Invitrogen,
Carlsbad, CA, USA). K+ currents were then recorded
using an Axopatch 1D amplifier (Axon Instruments) andpCLAMP8 software (Axon Instruments) 24–36 h after
transfection. Data from the amplifier were sampled at
2 kHz. Patch pipettes had resistances of 2–4 M when
filled with a solution of the following composition (mm):140 KCl, 3 MgCl2 , 1 CaCl2 , 3 EGTA, 10 Hepes, 3 Na2 ATP(pH 7.4). For the experiments summarized in Figs 7D and
E and 8B and C, 50 U ml−1 alkaline phosphatase from
calf intestine (Oriental Yeast, Suita, Osaka, Japan) was
also included in the pipette solution. The bath solution
contained (mm): 150 NaCl, 2 KCl, 2 CaCl2 , 2 MgCl2 ,10 Hepes, 10 glucose (pH 7.4). Series resistance wascompensated by 80%. The voltage-clamp experimental
protocols were the same as those used for two-electrodevoltage clamp.

Preparation of Xenopus oocytes

Xenopus oocytes were collected from frogs anaesthetized inwater containing 0.15% tricaine. After the final collection,
the frogs were humanly killed. Isolated oocytes were treatedwith collagenase (2 mg ml−1 , type 1, Sigma) for 5 h, after
which oocytes of similar size at stage V were injected withapproximately 50 nl of cRNA solution and incubated at
17◦ C in frog Ringer solution. All experiments conformed
to the guidelines of the Animal Care Committee of the
National Institute for Physiological Sciences.


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J Physiol 569.1

KCNQ/M channel modulation by PKC

Analysis of channel gating

G–V relationships were plotted using tail current
amplitudes obtained at −30 mV. Normalized tail currents
were fitted using pCLAMP8 software to a two-state
Boltzmann equation (Li-Smerin et al. 2000; Hille, 2001):
−1
G/G max = 1 + e−z F(V −V1/2 )/RT ,where G/Gmax is the normalized tail current amplitude, z is
the effective charge, V1/2 is the half-activation voltage, andT, F and R have their usual meanings.
Mutagenesis

Mutations were introduced into KCNQ2 cDNA usinga QuikChange kit (Stratagene, La Jolla, CA, USA) andconfirmed by sequencing.
Drugs

Oxotremorine-methiodide (oxo-M), phorbol 12myristate 13-acetate (PMA), 4α-PMA, chelerythrine
chloride and wortmannin were all purchased from
Sigma-Aldrich. Stock solutions were made up in DMSO,
stored at −20◦ C, and diluted in the appropriate solution
just before use.
Statistical analyses

The data are expressed as means ± s.e.m., with n indicating
the number of samples. Differences between means were
evaluated using Student’s paired or unpaired t tests. Valuesof P ≤ 0.05 were considered statistically significant.
Results
Muscarinic activity induces a positive shift in the
conductance–voltage relationship of KCNQ channels
in Xenopus oocytes

In order to reconstitute the M-current in Xenopus oocytes,
cRNAs encoding KCNQ channels (human KCNQ1, rat
KCNQ2, rat KCNQ2 + rat KCNQ3, and human KCNQ4)
were injected together with cRNA encoding the porcine
M1 muscarinic acetylcholine receptor. Each channel
showed a slowly activating, non-inactivating outward
current during 2-s depolarization (Fig. 1A, left column).
When we added 10 µm oxo-M, a muscarinic acetylcholine
receptor agonist, to the external bath solution in
the absence of thapsigargin, an endoplasmic reticulum
Ca2+ -ATPase pump inhibitor, large Ca2+ -activatedCl− currents were activated, but they usually disappeared
within 1 min (see Methods). Regardless of whether or notcells were pretreated with thapsigargin, KCNQ current
amplitudes gradually declined in the presence of oxo-M
until reaching a steady level after about 5 min. The effect
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61of the M1 receptor stimulation seemed relatively mildin Xenopus oocytes; even application of the agonist for
10 min failed to fully inhibit KCNQ currents (Fig. 1A, rightcolumn).
That KCNQ currents retained substantial amplitudes
after M1 receptor-mediated inhibition, provided us withthe opportunity to analyse the properties of these channels
under conditions of muscarinic inhibition. To do so, werecorded tail current amplitudes at −30 mV after 2-s
depolarizations and plotted the G–V curves obtained
before and after M1 receptor stimulation (Fig. 1B). The
curves were then fitted with a two-state Boltzmann
equation (see the equation in Methods). We also
compared the maximum tail current amplitude obtained
before and after M1 receptor stimulation (Fig. 1C).
After receptor stimulation, the maximum tail current
amplitudes of KCNQ1, KCNQ2 + KCNQ3, and KCNQ4
were, respectively, reduced to 58.5 ± 10.5%, 71.5 ± 8.4%
and 39.8 ± 12.8% of the amplitudes seen before M1
stimulation (n = 5 for each). By contrast, the maximum
tail current amplitude of homomeric KCNQ2 channels
was unchanged or even slightly increased after receptor
stimulation (108.5 ± 11.6%; n = 12).
We also noticed that the G–V curves for some KCNQ
channels were shifted; that is, they started to activate ata more depolarized potential after M1 stimulation. As
shown in Fig. 2A, the G–V curves for KCNQ2, KCNQ2 +KCNQ3 and KCNQ4 were all substantially shifted tomore positive potentials after stimulation. The G–V curvefor KCNQ1 was also shifted, but only slightly. The
corresponding shifts in V1/2 (midpoint of the G–V curve)
were 4.5 ± 1.7 mV (KCNQ1), 10.3 ± 1.5 mV (KCNQ2),
10.4 ± 1.9 mV (KCNQ2 + KCNQ3) and 29.6 ± 5.6 mV(KCNQ4), respectively (Fig. 2B and Table 1, n = 5 foreach). The z-value (effective charge, see the equation in
Methods) was slightly decreased for all KCNQ channels(Fig. 2C and Table 1).
Although all KCNQ channels were inhibited by
M1 receptor stimulation in Xenopus oocytes, thecharacteristics of the inhibition varied from channel tochannel. In KCNQ2 channels, for example, maximum
current amplitude was unaffected by M1 receptor
stimulation, though the G–V curve was substantiallyshifted. Conversely, in KCNQ1 channels the G–V curve
was shifted only slightly, while the maximum current
amplitude was substantially diminished. We therefore
decided to focus on KCNQ2 and KCNQ1 channels withthe aim of identifying the mechanisms underling the shifts
in the G–V curve.
PMA induces a positive shift in the conductancevoltage relationship in KCNQ2 channels

PKC is known to be involved in Gq -coupled receptor
signalling and may play a role in muscarinic inhibition

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K. Nakajo and Y. Kubo

(Higashida & Brown, 1986; Hoshi et al. 2003). We therefore
tested whether PMA, a potent PKC activator, would also
induce a positive shift in the G–V curves for KCNQ
channels. In this experiment, the exogenous M1 receptor

J Physiol 569.1

was not expressed. After addition of 1 µm PMA to thebath solution, homomeric KCNQ2 current amplitudes
at −30 mV gradually declined until reaching a new
steady level, usually within 20 min (Fig. 3A, inset). After

Figure 1. KCNQ/M channels are inhibited by M1 receptor stimulation in Xenopus oocytes
KCNQ channels were coexpressed with M1 muscarinic acetylcholine receptors in Xenopus oocytes. A, representative
recordings of currents through KCNQ1 (homotetramer), KCNQ2 (homo), KCNQ2 + KCNQ3 (hetero), and KCNQ4(homo) channels before and 10 min after the application of 10 µM oxo-M. K+ currents were elicited by voltage steps
between −80 and +40 mV followed by a step to −30 mV for tail current analysis. B, tail currents at −30 mV before
and after muscarinic receptor stimulation are plotted against test potentials. Current amplitudes are normalized
to the maximum tail current amplitude before receptor stimulation. Smooth curves were fitted using a two-state
Boltzmann function (see Methods). C, remaining maximum current amplitudes after muscarinic stimulation are
represented as percentages.

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KCNQ/M channel modulation by PKC

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63

Table 1. Changes in V1/2 and z for the KCNQ1, KCNQ2, KCNQ2/KCNQ3 and KCNQ4 channel induced by stimulating M1
muscarinic acetylcholine receptors
V1/2 (mV)

KCNQ1
KCNQ2
Q2/Q3
KCNQ4

z

n

Before oxo-M

After oxo-M

V1/2 (mV)

Before oxo-M

After oxo-M

z

5
5
5
5

−23.3 ± 1.2−39.2 ± 1.1−34.3 ± 1.1−15.8 ± 2.3−18.8 ± 1.2−28.9 ± 1.0−23.9 ± 1.6
13.8 ± 5.1

4.5 ± 1.7
10.3 ± 1.5
10.4 ± 1.9
29.6 ± 5.6

2.8 ± 0.1
4.1 ± 0.1
3.2 ± 0.2
1.8 ± 0.2

2.4 ± 0.1
3.7 ± 0.1
2.8 ± 0.1
1.3 ± 0.1

−0.4 ± 0.2−0.4 ± 0.2−0.4 ± 0.2−0.5 ± 0.2

20 min, the current showed slower activation and faster
deactivation (Fig. 3A). More importantly, the G–V curve
was positively shifted by 17.8 ± 1.6 mV (Fig. 3C and D;
n = 5), an even larger shift than was induced by oxo-M.
By comparison, 4α-PMA, an inactive isomer of PMA,
induced a G–V shift of only 4.4 ± 0.6 mV (Fig. 3C and
D; n = 6; P < 0.001 versus PMA). These results suggestthat activation of PKC induces a shift in the G–V curvefor KCNQ2 in Xenopus oocytes. On the other hand, PMA
induced a smaller shift in the G–V curve for KCNQ1(5.8 ± 0.6 mV; n = 6; Fig. 3B, C and D). As was seen
with M1 receptor stimulation (Fig. 2), the G–V curve for
KCNQ1 channels was little affected by the presence ofactivated PKC.
To determine whether the G–V shift induced bymuscarinic stimulation was mediated exclusively via
activation of PKC, we applied oxo-M and PMA
sequentially to oocytes expressing both M1 receptors

Figure 2. G–V curves for KCNQ/M channels are
shifted to more depolarized potentials by M1
receptor stimulation
A, G–V curves for KCNQ channels before (open symbols)
and after (filled symbols) muscarinic stimulation. Smooth
curves were fitted using a two-state Boltzmann function.
B and C, V1/2 (B) and z (C) obtained from the two-state
Boltzmann function are plotted.

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and KCNQ channels. In the first experiment, we first
applied oxo-M and then PMA. When we applied
10 µm oxo-M to stimulate coexpressed M1 receptors,
the G–V curve for KCNQ2 shifted 10.3 ± 1.5 mV in
10 min (Fig. 4A left, grey triangles; n = 5). When wethen added 1 µm PMA to the external solution andwaited another 20 min, the G–V curve was further shiftedby another 10.4 ± 1.4 mV (Fig. 4A left, filled triangles;n = 5). In the second experiment, we first pretreatedoocytes with 30 nm PMA for 30 min, after which theaveraged V1/2 for the G–V curves was −18.3 ± 0.7 mV(Fig. 4A right, open diamonds; n = 5). When we thenapplied 10 µm oxo-M to the PMA-treated oocytes, weobserved no shift (0.0 ± 0.9 mV; Fig. 4A right, filled
diamonds; n = 5). Because PMA had a stronger effectthan M1 receptor stimulation on the G–V relationship(Figs 2 and 3), PMA might have obscured the effect of
oxo-M.

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K. Nakajo and Y. Kubo

We next tested whether the shift in the G–V relationshipcould be prevented by a PKC inhibitor. After oocytes werepretreated with 20 µm chelerythrine for at least 3 h, theoxo-M-induced shift in the G–V curve was significantly

J Physiol 569.1

attenuated from 10.3 ± 1.5 mV (control) to 5.4 ± 0.8 mV(Fig. 4B; n = 5). This suggests that the shift induced by M1
receptor stimulation is due at least in part to activation ofa chelerythrine-sensitive PKC subtype.

Figure 3. The G–V curve for KCNQ2 is shifted by PMA
A, representative current recordings from KCNQ2 channels before and 20 min after application of 1 µM PMA.
Currents were elicited by depolarization from −100 to +40 mV with a subsequent step to −30 mV for the tailcurrent. The time course of PMA-induced inhibition is shown in the inset. Current amplitudes at −30 mV wereplotted against time; 0 min indicates the time at which PMA was applied to the bath solution. B, representative
current recordings from KCNQ1 channels before and 20 min after application of 1 µM PMA. C, G–V curves for the
KCNQ channels before (open symbols) and 20 min after (filled symbols) application of 1 µM PMA or 1 µM 4α-PMA(inactive isomer of PMA). Smooth curves were drawn by fitting the data with a two-state Boltzmann function.
D, V1/2 induced by 1 µM PMA or 1 µM 4α-PMA are plotted. The asterisk denotes statistical significance determinedusing an unpaired Student’s t test.

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J Physiol 569.1

KCNQ/M channel modulation by PKC

Reducing PIP2 reduces KCNQ2 current amplitude andslightly shifts the conductance–voltage relationshipof the channel

When Gq -coupled receptors are activated, PIP2 ishydrolysed by phospholipase C. Because KCNQ channels
are known to require PIP2 to maintain their activity,hydrolysis of PIP2 is thought to be a principle mechanism
by which the M-current is inhibited. For instance, with
10 µm wortmannin (known to inhibit both PI4 and PI3
kinases) in the external bath solution, KCNQ channels
fail to recover after M1 receptor-mediated inhibition
because PIP2 is unable to be re-synthesized (Suh &Hille, 2002; Zhang et al. 2003). In addition, incubating
oocytes with 30 µm wortmannin reduces KCNQ2/3
current amplitudes by ∼95% (Zhang et al. 2003). We
found that KCNQ2 current amplitudes gradually declined

after addition of 10 µm wortmannin to the externalbath solution (Fig. 5A), so that they were reduced byapproximately 40% after 15 min and by 80% after 30 min(Fig. 5B). This is consistent with the idea that KCNQ2
channels in Xenopus oocytes require PIP2 for activity,
even though the maximum current amplitude was notdiminished by M1 receptor stimulation (see Fig. 1). We
next pretreated oocytes with 30 nm PMA for 30 min
in order to induce a shift in the G–V curve before
making recordings, after which the cells were placed inexternal solution containing both 10 µm wortmannin and
30 nm PMA. The PMA-conditioned KCNQ2 currents werealso inhibited by wortmannin, declining in amplitude
by about 60% within 15 min and 80% within 30 min(Fig. 5C). Thus, PMA-treated KCNQ2 channels showing
a maximally shifted G–V curve still require PIP2 for theactivity.

Figure 4. PKC is a main pathway via which receptor stimulation induces G–V shifts
A, left, G–V curves for KCNQ2 channels obtained before and after sequential application of 10 µM oxo-M and 1 µM
PMA. Right, G–V curves for KCNQ2 channels before and after application of 10 µM oxo-M to PMA-pretreatedcells. Oocytes expressing KCNQ2 channels were incubated with 30 nM PMA for 30 min before the recording.
Smooth curves were drawn by fitting the data with a two-state Boltzmann function. B, left, G–V curves for
the KCNQ2 channel before and 10 min after application of 10 µM oxo-M (control). Right, G–V curves for thechelerythrine-pretreated KCNQ2 channel before and 10 min after application of 10 µM oxo-M. Oocytes expressing
KCNQ2 channels were incubated with 20 µM chelerythrine for 3 h before the recording. Smooth curves weredrawn by fitting the data with a two-state Boltzmann function.
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Figure 5. Reduction of PIP2 reduces KCNQ current amplitude and slightly affects G–V curves
A, representative current recordings before and 15 min and 30 min after application of wortmannin. B, G–V
curves for KCNQ2 channels before and 15 min and 30 min after application of wortmannin. Forty per cent of
the KCNQ2 current was inhibited after 15 min and 80% after 30 min. C, G–V curves for PMA-treated KCNQ2
channels before and 15 min and 30 min after application of wortmannin. Sixty per cent of the KCNQ2 current
was inhibited after 15 min and 80% after 30 min. D, G–V curves for KCNQ2 channels with ( e) or without() 30 min pretreatment with 10 µM wortmannin. The dashed curve represents the G–V curve for PMA-treated
KCNQ2 channels (from Fig. 3C). V1/2 values were −41.0 ± 1.0 mV (control; n = 6), −36.0 ± 0.9 mV (wortmannin;
n = 6; P < 0.005) and −23.1 ± 0.5 mV (PMA; n = 6; P < 0.001). E, G–V curve for KCNQ1 channels, which were
not affected by PMA (see Fig. 2), were not shifted by wortmannin; V1/2 was −25.8 ± 0.7 mV (control; n = 5) or−25.3 ± 1.9 mV (wortmannin; n = 5; P > 0.05), though the KCNQ1 current was inhibited by 78% after incubationfor 30 min with 10 µM wortmannin (data not shown).

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J Physiol 569.1

KCNQ/M channel modulation by PKC

To determine whether wortmannin would also inducea shift in the G–V relationship, we pretreated oocyteswith 10 µm wortmannin for 30 min, which should havecaused about 20% of the channels to be inhibited due
to a lack of PIP2 (Fig. 5B). Under those conditions,
the V1/2 of the G–V curve was −36.0 ± 0.9 mV (n = 6),
whereas it was −41.0 ± 1.0 mV without wortmannin(n = 6; P < 0.005) (Fig. 5D). The 5.0-mV shift wassignificant, but was far smaller than the shift induced by
PMA.
KCNQ1 currents also required PIP2 and were inhibitedby 80% after incubating 30 min with 10 µm wortmannin(data not shown). The G–V curve for KCNQ1 wascompletely unaffected by wortmannin; the V1/2 of the G–V
curve was −25.8 ± 0.7 mV in the absence of wortmannin(n = 5) and −25.3 ± 1.9 mV in its presence (n = 5;
P > 0.05) (Fig. 5E).

Some of KCNQ2 serine-to-aspartate mutants
within the A-domain show a positive shift
in their conductance–voltage relationship

When M1 receptors are stimulated, some of the serine
residues in KCNQ channels are phosphorylated by PKC(Hoshi et al. 2003). The KCNQ2 channel protein has
a long intracellular C-terminal domain, which contains
more than 500 amino acids, including 64 serine and27 threonine residues. Hoshi et al. (2003) identified
two serine residues, S534 and S541, that are perhaps
responsible for the muscarinic inhibition of KCNQ2
channels. These residues are both located in the middleof the intracellular C-terminal region and are very close toor within the ‘A-domain’, a region that is highly conserved
among KCNQ family proteins and forms the coiled-coilstructure needed for tetramerization of KCNQ channels(Fig. 6A) (Jenke et al. 2003; Maljevic et al. 2003; Schwake
et al. 2003).
If S534 and/or S541 are the only residues
phosphorylated by PKC during muscarinic inhibition, theG–V relationships of single S534A and S541A mutants
or a double S534A + S541A mutant should be unaffectedby muscarinic inhibition or PKC activation. We foundthat the G–V curve for the S541A mutant was negatively
shifted (∼10 mV) under control conditions (Table 2),
but that PMA induced a large positive G–V shift inall three S→A mutants tested, as it did with wild-type
KCNQ2 (Fig. 6B and Table 2). Apparently, these two
serine residues are not the only sites phosphorylated by
PKC, which is not surprising given the large numberof serine and threonine residues in the intracellulardomain of KCNQ2 channels. In addition, these two
serine residues are not fully conserved throughout
KCNQ family (Fig. 6A). For example in KCNQ4,
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A491 corresponds to S534 and phosphorylatable T498
corresponds to S541 while the G–V curve for KCNQ4
was also shifted by muscarinic stimulation (see Figs 1
and 2).
Bearing that in mind, we decided to change our strategy
and replaced five serine residues (S534, S541, S563,
S567 and S570) in the A-domain with aspartic acid. S563,
S567 and S570 are somewhat conserved throughout the
KCNQ family except KCNQ1 (Fig. 6A). Because aspartic
acid has a negative charge, we expected it to mimic
phosphorylated serine and, even before PMA treatment,
the G–V curves for the S541D, S563D and S570D mutants
were already shifted in a positive direction (Fig. 6C). For
example, the V1/2 for S541D was −17.7 ± 0.8 mV (n = 7),
which was close to that for PMA-treated wild-type KCNQ2
channels (the right dotted curve in Fig. 6C; data are thesame as in Fig. 3C). S534D and S567D also showed amild positive shift in their G–V curves (Fig. 6B and C
and Table 2), and their averaged current amplitudes werecomparable to those seen with the wild-type channel(Table 2).
When PMA was applied, the G–V curve for each mutant
showed a further positive shift, although it was not aslarge as that seen with the wild-type KCNQ2 channel.
S541D, S563D and S570D, which all showed a large positive
shift before the application of PMA, showed a shift of
9.9 ± 1.0, 6.1 ± 1.4 and 7.6 ± 0.8 mV, respectively, while
S534D and S567D, which showed a small shift before
treatment, showed a PMA-induced shift of 13.3 ± 1.1 and17.6 ± 1.4 mV, respectively (Fig. 6B and C and Table 2).
It thus appears that phosphorylation of a few residues
is sufficient to induce a large positive shift in the G–V
curve, even though the protein contains numerous otherphosphorylatable sites. S541D and S570D also showedsignificantly smaller current amplitudes than that ofwild-type KCNQ2 after PMA treatment (Table 2). We do
not yet have a good explanation for this.

Conductance–voltage relationship of KCNQ2/3
channels is constitutively shifted in HEK 293T cells

Shapiro et al. (2000) have already shown that muscarinic
stimulation inhibits KCNQ2/3 channels without affecting
the voltage dependence in human tsA-201 cells(a derivative of the HEK 293 cell line). In their experiments,
however, the PKC inhibitor staurosporine was included in
the patch pipette to block the effect of PKC activation.
We therefore decided to test whether the G–V curves for
KCNQ channels expressed in mammalian cells were also
modifiable by PKC activation. In this experiment, both
KCNQ2 and KCNQ3 were transfected into HEK 293T cells,
after which heteromultimeric KCNQ2/3 channels carrieda slowly activating, non-inactivating outward current

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K. Nakajo and Y. Kubo

J Physiol 569.1

Figure 6. Single S→D mutations within the A-domain of KCNQ2 affects the voltage dependenceA, amino acid sequences of A-domain of rat KCNQ2, rat KCNQ3, human KCNQ4, human KCNQ5 and human
KCNQ1. Serine and threonine residues are shaded in grey. Asterisks indicate mutated serine residues in this study.
B, changes in V1/2 (V1/2 ) after 30-min incubation with 30 nM PMA. Mutants that caused a significant decrease ofV1/2 relative to wild-type (WT, top) are indicated with asterisks. The number of measurements (n) for each data
point is indicated in parentheses; the left number indicating n for recordings without PMA incubation and the rightnumber indicating n for recordings after 30-min PMA incubation. C, representative current traces for S→D KCNQ2
mutants with or without 30-min incubation with 30 nM PMA. Currents were elicited by depolarization from −100
to +40 mV. The G–V curve for each mutant with (filled symbols) or without (open symbols) PMA incubation is
also shown in the right column. Smooth curves correspond to a two-state Boltzmann function. Dotted curves
correspond to fittings obtained with the two-state Boltzmann function for wild-type KCNQ2 channels before andafter application of PMA (from Fig. 3C).

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KCNQ/M channel modulation by PKC

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69

Table 2. Changes of V1/2 induced by PMA for KCNQ2 mutantsnbefore PMA

I−30mV (µA)
before PMA

V1/2 (mV)before PMAnafter PMA

I−30mV (µA)
after PMA

V1/2 ( mV)
after PMA

V1/2 (mV)WT

18

1.66 ± 0.20

−38.2 ± 0.6

231.85 ± 0.23

−20.8 ± 0.8

17.5 ± 1.0

S534A
S541A
S563A
S567A

10
5
5
5

2.06 ± 0.20
1.06 ± 0.50
1.65 ± 0.45
0.69 ± 0.18

−39.9 ± 0.5−46.3 ± 0.7−39.7 ± 0.6−39.4 ± 0.2

10
5
5
5

2.15 ± 0.21
3.33 ± 0.59
3.65 ± 0.38
0.84 ± 0.18

−17.6 ± 0.5−23.9 ± 1.7−16.5 ± 1.1−18.2 ± 2.0

22.4 ± 0.7
22.4 ± 1.8
23.2 ± 1.3
21.2 ± 2.0

S570A
S534A/S541A
S534A/S541A/S563A/
S567A/S570A
S534D

5
5
5

1.88 ± 0.20
1.89 ± 0.20
0.83 ± 0.12

−39.2 ± 0.3−47.6 ± 0.6−44.9 ± 0.74342.21 ± 0.53
1.91 ± 0.36
1.10 ± 0.42

−13.8 ± 0.6−20.6 ± 1.3−26.4 ± 0.8

25.4 ± 0.6
27.0 ± 1.4
18.4 ± 1.1

10

2.13 ± 0.31

−35.5 ± 0.7

101.73 ± 0.28

−22.2 ± 0.8

13.3 ± 1.1

S541D
S563D
S567D
S570D

7
8
5
5

0.80 ± 0.17
1.31 ± 0.17
4.04 ± 0.50
1.93 ± 0.35

−17.7 ± 0.8−27.0 ± 0.8−33.2 ± 0.9−23.5 ± 0.5

7
2
5
4

0.17 ± 0.03
0.60 ± 0.33
1.67 ± 0.21
0.19 ± 0.01

−7.7 ± 0.5−21.0 ± 1.2−15.6 ± 1.1−15.9 ± 0.6

9.9 ± 1.0
6.1 ± 1.4
17.6 ± 1.4
7.6 ± 0.8

similar to that seen in Xenopus oocytes (Fig. 7A left).
However, the G–V relationship in HEK 293T cells wasquite different from that in Xenopus oocytes. The V1/2of the G–V curve for KCNQ2/3 channels in HEK 293T
cells was −23.6 ± 1.4 mV (Fig. 7B and C; n = 18), whichis comparable to the V1/2 of −21 mV seen previously by
Shapiro et al. (2000) tsA-201 cells and to the V1/2 of−20.8 ± 0.8 mV seen with PMA-treated KCNQ2 channels
in Xenopus oocytes (right dotted curve in Fig. 7C; also
see Fig. 3 and Table 2). When we then recorded KCNQ2/3
currents from PMA-treated HEK 293T cells (Fig. 7A right),
we found that PMA did not elicit a significant reduction
in current amplitude or a voltage shift (Fig. 7B and C); V1/2of the G–V relationship in PMA-treated HEK 293T cells
was −20.2 ± 1.3 mV (Fig. 7B and C; n = 16; P = 0.08).
Because KCNQ2/3 currents in HEK 293T cells werelargely insensitive to PMA and the G–V curve was similar
to that obtained with PMA-treated KCNQ2 channels
in oocytes, we hypothesized that KCNQ2/3 channels in
HEK 293T cells might already be phosphorylated andtherefore the G–V curve might already be shifted. If so,
dephosphorylation of KCNQ2/3 channels could bring theG–V curve back to a more hyperpolarized potential. To
test that idea, we added alkaline phosphatase (50 U ml−1 )to the patch pipette in order to dephosphorylate KCNQ2/3
channels. After rupture of the cell membrane, the V1/2 of the
G–V relationship gradually shifted in a positive direction(Fig. 7D and E): V1/2 was −23.8 ± 2.1 mV at 0 min (justafter the rupture) and −28.0 ± 2.5 mV at 10 min (n = 9
for each; P = 0.01 by paired t test). This implies thatKCNQ2/3 channels (or their auxiliary proteins) areconstitutively phosphorylated in HEK 293T cells, which
could account for the insensitivity of the channels to
PMA.
Finally, we examined the behaviour of the S→D mutantsin HEK 293T cells. If KCNQ2 channels are constitutively
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phosphorylated in HEK 293T cells, and that is the reason
why the G–V curve for KCNQ2/3 channels was shifted
in Fig. 7, the G–V curves for the S→D mutants should
be similar to that of wild-type KCNQ2 channels. To test
that idea, we separately transfected the S541D, S563D or
S570D mutant or wild-type KCNQ2 channels into HEK
293T cells. KCNQ3 channels were not cotransfected inthis experiment. The S563D mutant was not expressed
well in HEK 293T cells and so was not analysed further.
The other homomultimeric channels were successfully
expressed, and their averaged current amplitudes at−30 mV were 889 ± 188, 147 ± 22 and 502 ± 148 pA for
the wild-type (n = 7), S541D (n = 6) and S570D (n = 6)channels, respectively, while the averaged V1/2 -values were
−10.7 ± 1.9, −4.0 ± 1.7 and −5.7 ± 2.7 mV (Fig. 8A).
The differences in V1/2 between the S→D mutants andwild-type KCNQ2 in HEK 293T cells were 6.7 and 5.0 mV
for S541D and S570D, respectively, values much smaller
than those in Xenopus oocytes (20.7 and 14.7 mV for
S541D and S570D, respectively; see Fig. 6 and Table 2).
This suggests that because of the possible phosphorylationof KCNQ2 channels, the effect of S→D mutationon the G–V curve might be obscured in HEK 293T
cells.
We also tested whether alkaline phosphatase in the patch
pipette would elicit negative shifts in the G–V curve for
KCNQ2 channels in HEK 293T cells. Along with run-downof the current, the V1/2 for wild-type KCNQ2 was shifted
toward more negative potentials, from −11.3 ± 2.2 to−17.5 ± 3.0 mV (n = 4; Fig. 8B and C). On the other
hand, the current amplitude and V1/2 for the S541D
mutant were relatively stable with alkaline phosphatasein the patch pipette (from −9.0 ± 0.4 to −6.8 ± 4.5 mV;
n = 3; Fig. 8B and C), which supports our hypothesisthat S541D mutants mimic phosphorylated KCNQ2
channels.

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Discussion
KCNQ channels are inhibited by the activation of
Gq -coupled receptors such as the M1 muscarinic acetylcholine receptor, and depletion of PIP2 and/or theactivation of PKC may play a significant role during thatprocess. In the present study, we showed that activationof PKC modulates the gating properties of all KCNQ
channels except KCNQ1 in Xenopus oocytes. Moreover,whereas the depletion of PIP2 reduces maximum current
amplitude, activation of PKC shifts the G–V relationshipof the channel. Although KCNQ channels expressed in

J Physiol 569.1

HEK 293T cells are insensitive to PMA, our experimentusing alkaline phosphatase suggests KCNQ channels areconstitutively modulated by phosphorylation in thosecells.
Voltage dependence of KCNQ channels

In our present work, we primarily used Xenopus oocytes
as an expression system. Using this preparation, we foundthat muscarinic stimulation or PKC activation induced
substantial G–V shifts for all KCNQ channels except
KCNQ1. For example, PMA caused the V1/2 of the G–V

Figure 7. Voltage dependence of KCNQ2/3 channels in HEK 293T cells is insensitive to PMA but sensitive
to alkaline phosphataseA, representative KCNQ2/3 current traces recorded from HEK 293T cells before (left) and after 30-min incubation
with 30 nM PMA (right). B, mean tail current amplitudes at −30 mV plotted against prepulse voltages. C, G–V
curve for KCNQ2/3 channels with or without PMA treatment. PMA did not shift G–V curves in HEK 293T cells. For
comparison, dotted curves represent the G–V curves for KCNQ2 channels in Xenopus oocytes with and without
PMA treatment (from Fig. 3C). D, representative KCNQ2/3 current traces recorded from HEK 293T cells 0 min(left) and 10 min (right) after membrane rupture. The patch pipette contained 50 U ml−1 alkaline phosphatase.
E, change in V1/2 induced by alkaline phosphatase is plotted (n = 9; P = 0.01).
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KCNQ/M channel modulation by PKC

curve for KCNQ2 channels to go from −38.2 ± 0.6 mV
to −20.8 ± 0.8 mV (Fig. 3). Muscarinic inhibition also
induced a positive shift in the G–V curves for KCNQ
channels (Figs 1 and 2). By contrast, Shapiro et al. (2000)
reported that muscarinic inhibition of KCNQ2/3 channels
is voltage independent; that is, oxo-M elicited no apparent
G–V shift in tsA-201 cells. This looks to be a major
discrepancy between the two expression systems, but wenoticed a few things which may explain this discrepancy.
First, with staurosporine in their patch pipette to exclude
the effects of PKC activation, those investigators foundthat PIP2 does not modulate G–V curves. This result is
consistent with our finding that depletion of PIP2 does notsubstantially modulate the voltage dependence of KCNQ2
channels (Fig. 5). Second, G–V curves for KCNQ2/3 in
tsA-201 and HEK 293T cells look positively shifted even
before muscarinic modulation; the V1/2 was −21 mV in
tsA-201 cells (Shapiro et al. 2000) and −23.6 ± 1.4 mV
in HEK 293T (Fig. 7). As these values are very close to
the V1/2 for KCNQ2 channels in PMA-treated Xenopus
oocytes (Fig. 7C), we hypothesize that KCNQ2/3 channels
are constitutively phosphorylated in both tsA-201 andFigure 8. Voltage dependence of wild-type KCNQ2 channels and S→D mutants in HEK 293T cells
A, homomeric wild-type KCNQ2 channels and S541D and S570D mutants were expressed in HEK 293T cells. Shown
are representative traces and G–V curves acquired from tail currents at −30 mV; V1/2 is −10.7 ± 1.9, −4.0 ± 1.7
and −5.7 ± 2.7 mV for wild-type, S541D and S570D, respectively. B, tail current amplitudes at −30 mV fromKCNQ2 current traces recorded at 0 min (open symbols) and 5 min (filled symbols) after membrane rupture. The
patch pipette contained 50 U ml−1 alkaline phosphatase. C, changes in V1/2 induced by alkaline phosphatase areplotted (n = 4 for wild-type and n = 3 for S541D).
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K. Nakajo and Y. Kubo

HEK 293T cells. We have no direct evidence showing
KCNQ2/3 channels to be constitutively phosphorylated
in HEK 293T cells. But consistent with that idea is our
finding that addition of alkaline phosphatase to the patch
pipette elicited a gradual shift in the G–V curve back tomore negative potentials (Fig. 7D and E), presumably due
to dephosphorylation of the channels.
Our data, as well as those of Shapiro et al. (2000),
indicate that the PIP2 concentration does not have alarge effect on the G–V curve. Still, our data show thata reduction in PIP2 can shift the G–V curve up to 5 mV
(Fig. 5), which may explain why inhibition of the shift bychelerythrine is only partial (Fig. 4).

Mechanisms underlying the shift in the G–V curves
for KCNQ channels

Hoshi et al. (2003) showed that serine residues in KCNQ2
channels are phosphorylated when the M1 receptor is
stimulated and that muscarinic inhibition of KCNQ2
channels is attenuated in S534A, S541A and R543A
mutants. We failed to reproduce a PKC-insensitive mutant
even with multiple S→A substitutions (Table 2), whichsuggests that other phosphorylatable serine residues are
responsible for the shift in the G–V curve. It would be
difficult to identify all putative phosphorylatable sites in
KCNQ2 given that the cytoplasmic C-terminal region of
the protein in rat, for example, contains 64 serine and27 threonine residues. On the other hand, some S→D
substitution mutants (e.g. S541D) showed a positively
shifted G–V curve in the absence of PMA (Fig. 6),
which implies that phosphorylation of a few sites
is sufficient to induce a positive shift in the G–V
curve.
Other binding proteins could also be targets ofphosphorylation by PKC. For instance, in addition
to the PKC-anchoring protein AKAP150, a ubiquitous
calmodulin is known to bind to KCNQ2 (Wen & Levitan,
2002; Yus-Najera et al. 2002), where it senses intracellular Ca2+ and regulates channel activity (Gamper& Shapiro, 2003). Calmodulin and other auxiliary
proteins might be phosphorylated by PKC and inturn modulate the voltage dependence of KCNQ
channels.
How might phosphorylation by PKC induce the shift
in the G–V curves for KCNQ channels? S534, S541, S563,
S567 and S570 all reside in the A-domain, which probably
mediates tetramerization of the channel (Jenke et al. 2003;
Maljevic et al. 2003; Schwake et al. 2003). In Shaker (Kv1)
K+ channels, the N-terminal T1 domain responsible fortetramerization is also involved in channel gating, and amutation that stabilizes the T1 tetramer also stabilizes theclosed state (Cushman et al. 2000; Minor et al. 2000). If
the aforementioned serine residues within the A-domain

J Physiol 569.1

of KCNQ channels are truly phosphorylated, a resultant
increase in the stability of the domain might affect channel
gating as it does in Shaker K+ channels.

Physiological function of PKC during muscarinic
inhibition of KCNQ channels

The function of PKC in muscarinic inhibition has long
been controversial. In frog sympathetic neurones, smoothmuscle cells and NG108-15 neuroblastoma–glioma cells,
phorbol esters or diacylglycerols suppress the M-current(Higashida & Brown, 1986; Brown & Higashida, 1988;
Pfaffinger et al. 1988). In NG108-15 cells, moreover, PKC
19–31 peptide, an effective inhibitor of PKC, elicits a shift inthe voltage dependence of the M-current such that the V1/2
goes from −34 mV to −44 mV (Schmitt & Meves, 1993).
However, because the inhibition induced by phorbol esters
or diacylglycerols is partial at most, and because PKC
inhibitors fail to prevent receptor-mediated M-current
inhibition, PKC has not been considered necessary formuscarinic inhibition (Bosma & Hille, 1989; Chen et al.
1994). In the present study, we found that PKC positively
shifts the G–V relationship of KCNQ channels; in otherwords, PKC made KCNQ channels reluctant to open. If
the main role of PKC is changing the gating properties of
KCNQ channels but not the number of available channels,
it is reasonable that PKC only partially suppresses theM-current. By contrast, PIP2 dramatically affected current
amplitude with little change in the gating properties(see Fig. 5). Because PIP2 is required for the activityof KCNQ channels, there would be no channel activity
if PIP2 were completely depleted. This can be seen in
Fig. 5C, where even though PKC was fully activated,some KCNQ current remained and was abolished bywortmannin.
The availability of PKC may differ depending on thecellular or molecular environment, which includes not
only PKC itself, but also adaptor and/or auxiliary proteins.
Recently, AKAP150 was shown to bind to KCNQ2 channels(Hoshi et al. 2003), where it can recruit cytoplasmic
PKC to the cytoplasmic domain of KCNQ channels,
which should facilitate PKC signalling. It is thus obviousthat AKAP150 changes the molecular environment of
KCNQ2 channels. On the other hand, the density orconcentration of PIP2 is controlled by the activities ofnumerous kinases and phosphatases (Andersen, 2005).
In the presence of excess PIP2 , activation of the M1
receptor may not be sufficient to suppress KCNQ channel
activity. Even in this case, however, PKC could induce
a positive shift of the G–V curve. In addition, in
frog sympathetic ganglion cells, the response to PMA
obscured that to substance P but not that to luteinizing
hormone releasing hormone (Bosma & Hille, 1989).
This may be indicative of how the cellular environmentC 2005 The Authors. Journal compilation 
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KCNQ/M channel modulation by PKC

around each kind of receptor differs, probably becauseof its subcellular localization and associated binding
proteins.
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Acknowledgements
We are grateful to Dr D. McKinnon for providing us with
cDNA encoding rat KCNQ2 and rat KCNQ3, Dr T. J. Jentsch
for cDNA encoding human KCNQ4, and Dr T. Kubo forproviding cDNA encoding porcine M1 receptor. Expression
vector pCXN2 was kindly provided by Dr J. Miyazaki. We
thank Drs M. Tateyama, T. Misaka and Y. Fujiwara for helpful
discussions and advice. We also thank Ms R. Watanabe, T.
Yamamoto and Y. Asai for technical assistance. This work
was supported partly by the research grants from the Ministry of Education, Culture, Sports, Science and Technology ofJapan and from Japan Society for Promotion of Science toY.K. K.N. was supported by the Inoue Foundation for Science.


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