cGMP Activates a pH-Sensitive Leak K+ Current in the Presumed Cholinergic Neuron of Basal Forebrain

cGMP Activates a pH-Sensitive Leak K+ Current in the
Presumed Cholinergic Neuron of Basal Forebrain
Hiroki Toyoda, Mitsuru Saito, Hajime Sato, Yoshie Dempo, Atsuko Ohashi, Toshihiro
Hirai, Yoshinobu Maeda, Takeshi Kaneko and Youngnam Kang
J Neurophysiol 99:2126-2133, 2008. First published 20 February 2008;
doi: 10.1152/jn.01051.2007
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J Neurophysiol 99: 2126 –2133, 2008.
First published February 20, 2008; doi:10.1152/jn.01051.2007.

cGMP Activates a pH-Sensitive Leak K⫹ Current in the Presumed
Cholinergic Neuron of Basal Forebrain
Hiroki Toyoda,1,* Mitsuru Saito,1,* Hajime Sato,1 Yoshie Dempo,3 Atsuko Ohashi,4 Toshihiro Hirai,3
Yoshinobu Maeda,2 Takeshi Kaneko,5 and Youngnam Kang1,3
1

Department of Neuroscience and Oral Physiology, Osaka University Graduate School of Dentistry; 2Division for Interdisciplinary
Dentistry, Osaka University Dental Hospital, Osaka; 3The Research Institute of Personalized Health Science and 4Department of Clinical
Pharmacology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Hokkaido; and 5Department
of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan
Submitted 23 September 2007; accepted in final form 18 February 2008

INTRODUCTION

As demonstrated in an earlier study (Kang et al. 2007),
S-nitroso-N-acetylpenicillamine (SNAP) or 8-bromoguanosine-3⬘,5⬘-cyclomonophosphate (8-Br-cGMP) induced a
membrane hyperpolarization in the presumed basal forebrain
cholinergic (BFC) neurons by activating K⫹ currents that usually displayed Goldman–Hodgkin–Katz (GHK) rectification,
most likely the leak K⫹ current. However, it has been reported
that nitric oxide (NO) increased membrane excitability in striatal medium spiny neurons, presumably by inhibition of leak
K⫹ channels (West and Grace 2004). It has also been reported
* These authors contributed equally to this work.
Address for reprint requests and other correspondence: Y. Kang, Department of Neuroscience and Oral Physiology, Osaka University Graduate School
of Dentistry, 1-8, Yamadaoka, Suita, Osaka 565-0871, Japan (E-mail: kang
@dent.osaka-u.ac.jp).
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that long-term activation of the NO– cGMP–protein kinase G
(PKG) pathway in injured motoneurons resulted in an inhibition of a pH-sensitive leak K⫹ current, suggesting an involvement of NO in inhibiting TWIK-related acid-sensitive K⫹
(TASK) current (Gonzalez-Forero et al. 2007). Thus activation
of the NO– cGMP pathway may have differential effects on
neuronal excitability among different brain regions.
In the present study, we examined whether the presumed
BFC neurons express any pH-sensitive K⫹ current and whether
8-Br-cGMP can modulate the activity of such pH-sensitive K⫹
current. We found that the presumed BFC neurons displayed a

pH-sensitive K⫹ current similar to TASK1 current in response
to changes in the external pH and that 8-Br-cGMP dramatically
enhanced the K⫹ current only at pH 7.3, leaving it almost
unchanged at pH 6.3 and 8.3.
METHODS

The procedure for slice preparation is the same as that in an earlier
study (Kang et al. 2007).

Electrophysiological recording
Details of the whole cell patch-clamp recording method were also
described in an earlier study (Kang et al. 2007). The composition of
extracellular solution was the same as previously reported (in mM):
124 NaCl, 1.8 KCl, 2.5 CaCl2, 1.3 MgCl2, 26 NaHCO3, 1.2 KH2PO4,
and 10 glucose. When changing the external pH, 26 mM NaHCO3 in
the extracellular solution was substituted with 10 mM HEPES and 12
mM NaCl, and pH was adjusted using NaOH (Talley et al. 2000). The
composition of the internal solution was the same as the modified
internal solution previously reported (in mM): 123 K-gluconate, 8
KCl, 20 NaCl, 2 MgCl2, 0.5 ATP-Na2, 0.3 GTP-Na3, 10 HEPES, and
0.1 EGTA; the pH was adjusted to 7.3 with KOH. All recordings were
obtained in the presence of tetrodotoxin (1 ␮M). Under the voltageclamp condition, the baseline current at the holding potential of ⫺70
mV was continuously measured except during the depolarizing ramp
(⫺130 to ⫺40 mV, 1-s duration) and step (to ⫺90 mV, 0.1-s duration)
pulses applied alternately every 10 s. The conductance was measured
using linear regression across the linear part of the current–voltage
(I–V) plot (⫺70 to ⫺95 mV) in response to the ramp pulses.

Drug application
8-Br-cGMP, a membrane-permeable cGMP analog (Sigma–Aldrich, St. Louis, MO), and BaCl2 (Wako Pure Chemicals, Osaka,

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Toyoda H, Saito M, Sato H, Dempo Y, Ohashi A, Hirai T, Maeda
Y, Kaneko T, Kang Y. cGMP activates a pH-sensitive leak K⫹
current in the presumed cholinergic neuron of basal forebrain. J
Neurophysiol 99: 2126 –2133, 2008. First published February 20,
2008; doi:10.1152/jn.01051.2007. In an earlier study, we demonstrated that nitric oxide (NO) causes the long-lasting membrane
hyperpolarization in the presumed basal forebrain cholinergic (BFC)
neurons by cGMP–PKG-dependent activation of leak K⫹ currents in
slice preparations. In the present study, we investigated the ionic
mechanisms underlying the long-lasting membrane hyperpolarization
with special interest in the pH sensitivity because 8-Br-cGMP–
induced K⫹ current displayed Goldman–Hodgkin–Katz rectification
characteristic of TWIK-related acid-sensitive K⫹ (TASK) channels.
When examined with the ramp command pulse depolarizing from
⫺130 to ⫺40 mV, the presumed BFC neurons displayed a pHsensitive leak K⫹ current that was larger in response to pH decrease
from 8.3 to 7.3 than in response to pH decrease from 7.3 to 6.3. This
K⫹ current was similar to TASK1 current in its pH sensitivity,
whereas it was highly sensitive to Ba2⫹, unlike TASK1 current. The
8-Br-cGMP–induced K⫹ currents in the presumed BFC neurons were
almost completely inhibited by lowering external pH to 6.3 as well as
by bath application of 100 ␮M Ba2⫹, consistent with the nature of the

leak K⫹ current expressed in the presumed BFC neurons. After
8-Br-cGMP application, the K⫹ current obtained by pH decrease from
7.3 to 6.3 was larger than that obtained by pH decrease from pH 8.3
to 7.3, contrary to the case seen in the control condition. These
observations strongly suggest that 8-Br-cGMP activates a pH- and
Ba2⫹-sensitive leak K⫹ current expressed in the presumed BFC
neurons by modulating its pH sensitivity.


cGMP ACTIVATES A pH-SENSITIVE LEAK K⫹ CURRENT IN BFC NEURONS

Japan) were dissolved in distilled water for preparing respective stock
solutions. They were bath-applied at a dilution ⬎1:1,000 to give a
final concentration of 0.2 mM (8-Br-cGMP) and 0.1 mM (BaCl2).

Data analysis
Numerical data were expressed as means ⫾ SD. The statistical
significance was assessed using paired or unpaired Student’s t-test, or
using ANOVA followed by Fisher’s PLSD (protected least-significant
difference) post hoc test.
RESULTS

The presumed BFC neurons display a pH-sensitive leak
K⫹ current

A

B

(Gx ⫺ GpH6.3)/(GpH8.3 ⫺ GpH6.3), where x is the pH of the

external solution. The S-G values at pH 6.3, 7.3, and 8.3 were
0, 0.34 ⫾ 0.07, and 1, respectively (Fig. 1D, n ⫽ 5). Thus the
presumed BFC neurons displayed a pH-sensitive leak K⫹
current, similar to TASK1 current expressed in the recombinant systems (Duprat et al. 1997; Kim et al. 1998; Leonoudakis
et al. 1998). In the next experiments, we examined whether this
pH-sensitive current is sensitive to Ba2⫹.
Ba2⫹ sensitivity of pH-sensitive currents
in the presumed BFC neurons
After the current responses to the ramp pulse were obtained
at pH 7.3 and 8.3 (Fig. 2Aa, black and gray traces, respectively), 100 ␮M Ba2⫹ was added in the extracellular solution
maintained at pH 8.3. Ba2⫹ substantially reduced the current
response at pH 8.3 (Fig. 2Ab, gray trace). Thereafter, when pH
was decreased from 8.3 to 7.3 in the presence of Ba2⫹, the
current response remained almost unchanged (Fig. 2Ab, compare gray and black traces). Ba2⫹-sensitive currents at pH 8.3
and 7.3 (Fig. 2Ba) were obtained by subtracting currents
obtained after application of Ba2⫹ (Fig. 2Ab) from those
obtained before application of Ba2⫹ (Fig. 2Aa) and their I–V
relationships were revealed to be inwardly rectified (Fig. 2Bb).
The pH-sensitive currents were also obtained by subtracting
the current responses obtained at pH 7.3 from those at pH 8.3,
before and after application of Ba2⫹ (Fig. 2Ca, black and gray
traces). As revealed in the I–V relationship, the pH-sensitive
current in the absence of Ba2⫹ was slightly outwardly rectified
(Fig. 2Cb, black trace), whereas in the presence of Ba2⫹ there
was little pH-sensitive current over the voltage range from
⫺130 to ⫺40 mV (Fig. 2Cb, gray trace). In six presumed BFC
neurons, when the possible conductance decrease following
decreasing pH from 8.3 to 7.3 was measured in the presence of
Ba2⫹, the conductance changed from 6.4 ⫾ 1.6 to 6.1 ⫾ 1.8 nS
by ⫺0.2 ⫾ 0.6 nS. There was no significant (P ⬎ 0.4) decrease

in the conductance in the presence of Ba2⫹, contrasting to large
conductance decreases observed in the absence of Ba2⫹ following the same decrease in the external pH (⫺7.0 ⫾ 4.4 nS,
n ⫽ 5, P ⬍ 0.04).

C
FIG. 1. External-pH sensitivity in the presumed basal forebrain cholinergic (BFC) neurons. A: plotting of baseline currents against time following changes in the external pH from
8.3 to 6.3 in a presumed BFC neuron. Note that lowering
external pH from 8.3 to 7.3 caused a much larger inward shift
of baseline current than did that from 7.3 to 6.3. B: pooled data
showing the scaled baseline currents obtained at pH 6.3, 7.3,
and 8.3, respectively (n ⫽ 5). The baseline currents (Ix) were
scaled by using an equation: S-Ix ⫽ (Ix ⫺ I pH6.3)/(IpH8.3 ⫺
IpH6.3), where x is the pH of the external solution. C: sample
current traces evoked by applying a ramp command pulse
recorded in a presumed BFC neuron at external pH 8.3, 7.3, and
6.3. Note that these 3 current traces crossed each other around
the theoretical EK (⫺95 mV), indicated with a vertical dotted
line. D: pooled data showing the scaled conductances at pH 6.3,
7.3, and 8.3, respectively (n ⫽ 5). The conductances were
scaled by using an equation: S-Gx ⫽ (Gx ⫺ GpH6.3)/(GpH8.3 ⫺
GpH6.3), where x is the pH of the external solution.

D

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Given that the leak K⫹ current was mediated by the activity
of TASK channels, the leak K⫹ current in the presumed BFC
neurons would be sensitive to changes in the external pH. This
possibility was investigated under the voltage-clamp condition
at a holding potential of ⫺70 mV. The external pH was
changed after the baseline current reached the respective steady
levels that remained constant for ⱖ30 s at respective pH values
(Fig. 1A). Following changes of external pH from 8.3 to 6.3,
the baseline current decreased from a positive value to a
minimum level (Fig. 1, A and Cb). To isolate pH-sensitive
components, the amplitude of the baseline current (Ix) was
scaled between 0 and 1 and defined as the scaled baseline
current (S-Ix) as follows: S-Ix ⫽ (Ix ⫺ IpH6.3)/(IpH8.3 ⫺ IpH6.3),
where x is the pH of the external solution. The amplitudes of
S-I at pH 6.3, 7.3, and 8.3 were 0, 0.34 ⫾ 0.05, and 1,
respectively (Fig. 1B, n ⫽ 5).
The I–V relationship examined with the depolarizing ramp
pulse from ⫺130 to ⫺40 mV was almost linear at pH 8.3 (Fig.
1Cb), but became more outwardly rectified with decreasing pH
to 6.3 (Fig. 1Cb). Respective current responses obtained at pH
8.3, 7.3, and 6.3 crossed each other around the theoretical K⫹
equilibrium potential (EK ⫽ ⫺95 mV), indicating the presence
of pH-sensitive K⫹ currents (Fig. 1Cb). To isolate pH-sensitive
components, the conductance was scaled between 0 and 1 and
defined as the scaled conductance (S-Gx) as follows: S-Gx ⫽

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On the other hand, when the possible conductance increase
following raising pH from 7.3 to 8.3 was measured in the
absence and presence of 100 ␮M Ba2⫹ in the same presumed
BFC neurons, the conductance increases were 3.4 ⫾ 2.6 and
⫺0.1 ⫾ 0.2 nS, respectively (n ⫽ 5). Thus the conductance did
not increase but decreased very slightly following raising
external pH in the presence of Ba2⫹ in the presumed BFC
neurons that displayed a prominent conductance increase following the same increase in the external pH in the absence of
Ba2⫹. Taken together, no pH-sensitive current remained in the
presence of Ba2⫹ following the pH decrease from 8.3 to 7.3,
whereas the pH increase from 7.3 to 8.3 often resulted in a very
slight increase in the blockade by Ba2⫹ seen at pH 7.3 in three
of five presumed BFC neurons examined, in spite of the relief
from the proton blockade. However, this latter effect was not
statistically significant (P ⬎ 0.2). At any rate, the pH-sensitive
leak K⫹ current expressed in the presumed BFC neurons
appeared to be highly sensitive to Ba2⫹. In the next series of
experiments, we examined whether 8-Br-cGMP activates the
pH- and Ba2⫹-sensitive leak K⫹ current.
Differential effects of 8-Br-cGMP on the leak K⫹ current
between pH 6.3 and pH 7.3
8-Br-cGMP (0.2 mM) was applied at pH 7.3 after examining
the control current responses to the ramp pulse at pH 8.3, 7.3,
and 6.3 (Fig. 3, A and B). Following application of 8-Br-cGMP
at pH 7.3, both the baseline current and the conductance

increased considerably, exceeding their original values at pH
7.3, as revealed in the continuous recording (Fig. 3, A and B, a
and b; compare *1 and *3) and by the superimposed traces of
current responses (Fig. 3Ca). The 8-Br-cGMP–induced current
can be obtained by subtraction of the current response (Fig. 3B,
*1) at pH 7.3 before application of 8-Br-cGMP from that (Fig.
3B, *3) at pH 7.3 during application of 8-Br-cGMP (Fig. 3Cb,
*3 ⫺ *1, gray trace). By contrast, there was nearly no difference in the current responses at pH 6.3 obtained before and
after 8-Br-cGMP application (Fig. 3Ba; compare *2 and *4), as
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revealed by the current obtained by subtraction of *2 from *4
(Fig. 3Cb, *4 ⫺ *2, black trace). In agreement with this
observation, neither the baseline current nor the ramp response
was affected significantly (Fig. 3D, a and b) when 8-Br-cGMP
was applied at pH 6.3. Thus 8-Br-cGMP increased the pHsensitive leak K⫹ current at pH 7.3, but failed to increase at
pH 6.3.
8-Br-cGMP–induced current is greater at pH 7.3 than at pH
8.3
To further examine the sensitivity of 8-Br-cGMP–induced
current to external pH changes, current responses were recorded at various external pH values before, during, and after
application of 8-Br-cGMP. Since even the brief application of
8-Br-cGMP caused a long-lasting hyperpolarization (half-duration, 29 ⫾ 12 min, n ⫽ 5) in the presumed BFC neurons (see
Figs. 2B, 4B, and 5 in Kang et al. 2007 and see also Fig. 6 in
this paper), effects of pH changes on the 8-Br-cGMP–induced
current can be safely examined at least for 20 –30 min after the
removal of 8-Br-cGMP. Therefore 8-Br-cGMP was applied
only once in this experiment. The external pH was changed
only after the baseline current reached a steady level that
remained constant for ⱖ30 s.

8-Br-cGMP (0.2 mM) was applied at pH 7.3 after examining
the control current responses to the ramp pulse at pH 8.3, 7.3,
and 6.3 (Fig. 4, A and B). An application of 8-Br-cGMP at pH
7.3 dramatically enhanced the current response to the ramp
pulse (Fig. 4Ba, compare *2 and *4), as revealed by the
superimposed traces (Fig. 4Ca) and by the 8-Br-cGMP–induced current obtained by subtraction of the current response
denoted by *2 from that denoted by *4 (Fig. 4Cb, *4 ⫺ *2,
gray trace). However, when the external pH was decreased to
6.3 during washout of 8-Br-cGMP, there was no apparent
difference in the current responses at pH 6.3 obtained before
and after 8-Br-cGMP application (Fig. 4Ba, compare *3 and
*5), as revealed by the current obtained by subtraction of *3
from *5 (Fig. 4Cb, *5 ⫺ *3, black trace). Nevertheless, when

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2⫹
FIG. 2. Ba
sensitivity of pH-sensitive
currents. A–C, top: voltage command pulses.
A: sample current traces obtained at pH 7.3
and 8.3 (black and gray traces, respectively)
before (a) and during 100 ␮M Ba2⫹ application (b). Note that the current responses
obtained at pH 7.3 and 8.3 in the presence of
Ba2⫹ were almost the same. B: Ba2⫹-sensitive currents obtained by subtracting the
currents obtained after Ba2⫹ application

from the control currents, at pH 7.3 and
8.3 (black and gray traces, respectively,
a). Inwardly rectified current–voltage (I–V) relationships of Ba2⫹-sensitive currents at pH
7.3 and 8.3 (black and gray traces, respectively, b). Ca: pH-sensitive currents obtained
by subtracting the currents evoked at pH 7.3
from those evoked at pH 8.3, before and
during Ba2⫹ application (black and gray
traces, respectively). Cb: a slightly outwardly rectified I–V relationship of pH-sensitive current in the absence of Ba2⫹ (black
trace). In the presence of Ba2⫹, no apparent
pH-sensitive current remained over the voltage range from ⫺130 to ⫺40 mV (gray
trace).


cGMP ACTIVATES A pH-SENSITIVE LEAK K⫹ CURRENT IN BFC NEURONS

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A

B

C

D

the external pH was increased from 6.3 to 8.3 or 7.3 even after
washout of 8-Br-cGMP, the current responses and conductances were still larger than their controls (Fig. 4B, a and b). As
shown in the I–V relationship (Fig. 4Cb), however, 8-BrcGMP–induced current at pH 8.3 obtained by subtraction of *1
from *6 (*6 ⫺ *1, black trace) was much smaller than that at
pH 7.3 (*4 ⫺ *2, gray trace). These observations clearly

indicate the long-lasting nature of 8-Br-cGMP–induced responses and its sensitivity to acidification. This long-lasting
nature of 8-Br-cGMP–induced responses seen under the voltage-clamp condition was consistent with that seen under the
current-clamp condition as described in our previous study
(Kang et al. 2007).
Thus 8-Br-cGMP–induced current was completely and reversibly inhibited by lowering the external pH to 6.3. These
observations clearly indicate that 8-Br-cGMP–induced current
is sensitive to acidification, although its I–V relationship did
J Neurophysiol • VOL

not always display a clear GHK rectification, especially at
depolarized or hyperpolarized membrane potentials (Figs. 3C
and 4C). Since native BFC neurons would display multiple
components of K⫹ currents flowing through not only leak K⫹
channels but also other K⫹ channels including voltage-activated K⫹ (Kv) channels (Markram and Segal 1990) and inwardly rectifying K⫹ (Kir) channels (Farkas et al. 1994) in
response to the ramp command pulse, the I–V relationship
would neither be linear nor display GHK rectification (Fig.
4Ca, *2). When the leak K⫹ conductance was increased by
8-Br-cGMP or by raising pH, the space clamp would become
less stringent, resulting in less activation of voltage-dependent
currents (Fig. 4Ca, *4). Since 8-Br-cGMP–induced K⫹ currents can be isolated only by the subtraction method following
application of 8-Br-cGMP in native BFC neurons (Fig. 4C, a
and b), the I–V relationship (Fig. 4Cb, gray trace) may be less
accurate, especially at very depolarized or hyperpolarized

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FIG. 3. Differential effects of 8-bromoguanosine-3⬘,5⬘-cyclomonophosphate (8-Br-cGMP) on the leak K⫹ current between pH 6.3 and pH 7.3. A: a continuous recording of current
responses to repetitively applied step-and-ramp voltage pulses
under the voltage-clamp condition. External pH was serially
changed as indicated with gray horizontal bars, which represent
the duration and timing of perfusion of external solution at
respective pH values. 8-Br-cGMP was applied at pH 7.3 and 6.3
as indicated with a black horizontal bar. B: plotting of baseline
currents (a) and conductances (b) against time. The current
responses to the ramp pulses were considerably enhanced after
the application of 8-Br-cGMP at pH 7.3 (compare *1 and *3).
Note that the 8-Br-cGMP–induced enhancement of current
responses at pH 7.3 was completely blocked by lowering external pH to 6.3 even in the presence of 8-Br-cGMP (compare *2
and *4). Ca, top: voltage command pulse. Bottom: sample
current traces obtained at pH 7.3 before and during 8-Br-cGMP
application (black and gray traces, respectively). The superimposed 2 current responses were obtained at the respective times
indicated with *1 (Control, black trace) and *3 (8-Br-cGMP,
gray trace) in Ba. Cb: the I–V relationships of 8-Br-cGMP–
induced currents at pH 7.3 and 6.3 (gray and black traces,
respectively). 8-Br-cGMP–induced currents at pH 7.3 and 6.3
were obtained by the subtraction of currents recorded before
application of 8-Br-cGMP (*1 and *2, respectively) from those
recorded after application of 8-Br-cGMP (*3 and *4, respectively). 8-Br-cGMP–induced current at pH 7.3 displayed a slight
sigmoidal I–V relationship. Note no apparent 8-Br-cGMP–induced current at pH 6.3 examined at any potential from ⫺120 to
⫺50 mV. Da: the baseline currents were indistinguishable
before and after application of 8-Br-cGMP when applied at pH
6.3. Db, top: voltage command pulse. Bottom: sample current
responses obtained at pH 6.3 before and during 8-Br-cGMP
application (black and gray traces, respectively). The superimposed 2 current traces were obtained at the respective times
indicated with *1 (Control, black trace) and *2 (8-Br-cGMP,
gray trace) in Da.

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TOYODA ET AL.

A

B

C

membrane potentials due to the larger contamination by Kv
and Kir currents, respectively, in the control condition (Fig.
4Ca, *2).
External pH-dependent effects of 8-Br-cGMP on leak
K⫹ currents
Summary data of the external pH-dependent effects of 8-BrcGMP are shown in Fig. 5. Bath application of 8-Br-cGMP
increased the conductance of the leak K⫹ current measured
between ⫺70 and ⫺95 mV in a manner dependent on the
external pH. The conductance obtained after application of
8-Br-cGMP at pH 7.3 was 2.24 ⫾ 0.43-fold larger than the
control (Fig. 5A, P ⬍ 0.02, n ⫽ 6). However, those at pH 8.3
and 6.3 were only 1.10 ⫾ 0.09-fold (P ⬎ 0.05, n ⫽ 6) and
1.03 ⫾ 0.03-fold (P ⬎ 0.1, n ⫽ 6) larger than their controls,
respectively (Fig. 5A). Using these values of normalized conductances and the scaled conductances in the control condition
(Fig. 1D), the possible scaled conductances of 8-Br-cGMP–
induced leak K⫹ currents at the respective pH levels were
calculated. The scaled conductances at pH 6.3, 7.3, and 8.3
J Neurophysiol • VOL

following application of 8-Br-cGMP were 0, 0.90, and 1,
respectively (Fig. 5B, hollow columns). As represented by
solid (control) and hollow (8-Br-cGMP) columns (Fig. 5B), the
pH profile of scaled conductances was dramatically changed by
8-Br-cGMP. Although the modified pH profile was not necessarily obtained following pH changes in the same neurons, it is
likely that 8-Br-cGMP changed the pH sensitivity of the leak
K⫹ current, from the one similar to that of TASK1 to the other
rather similar to that of TASK3 current (Berg et al. 2004; Kang
et al. 2004). Indeed, after 8-Br-cGMP application, the K⫹
current obtained by pH decrease from 7.3 to 6.3 was larger than
that obtained by pH decrease from pH 8.3 to 7.3 (n ⫽ 3, Fig.
4), contrary to the case seen in the control condition (Fig. 1). In
the next experiment, Ba2⫹ sensitivity of 8-Br-cGMP–induced
current was examined.
Ba2⫹ sensitivity of 8-Br-cGMP–induced current
In the presence of Ba2⫹, 0.2 mM 8-Br-cGMP was bath
applied for 5– 6 min under the voltage-clamp condition
(Fig. 6, A and B). There were no significant differences in

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FIG. 4. 8-Br-cGMP–induced current is
greater at pH 7.3 than at pH 8.3. A: a continuous recording of current responses to
repetitively applied step-and-ramp voltage
pulses at ⫺70 mV under the voltage-clamp

condition at various external pH obtained
before, during, and after application of 8-BrcGMP. External pH was serially changed as
indicated with gray horizontal bars, which
represent the duration and timing of perfusion of external solution at respective pH
values. 8-Br-cGMP was applied at pH 7.3
as indicated with a black horizontal bar.
B: plotting of baseline currents (a) and conductances (b) against time. The current responses to the ramp pulses were dramatically
enhanced after the application of 8-Br-cGMP
at pH 7.3 (compare *2 and *4). Note that the
8-Br-cGMP–induced enhancement of current responses was completely blocked by
lowering external pH to 6.3 (compare *3 and
*5). Ca, top: voltage command pulse. Bottom: sample current traces obtained at pH 7.3
before and during 8-Br-cGMP application
(black and gray traces, respectively). The
superimposed 2 current responses were obtained at the respective times indicated with
*2 (Control, black trace) and *4 (8-BrcGMP, gray trace) in Ba. Cb: the I–V relationships of 8-Br-cGMP–induced currents at
pH 8.3, 7.3, and 6.3. 8-Br-cGMP–induced
currents at pH 8.3, 7.3, and 6.3 were obtained
by the subtraction of currents recorded before application of 8-Br-cGMP (*1, *2, and
*3, respectively) from those recorded after
application of 8-Br-cGMP (*6, *4, and *5,
respectively). 8-Br-cGMP–induced current
at pH 7.3 displayed a sigmoidal I–V relationship. Note that the 8-Br-cGMP–induced current was greater at pH 7.3 than at pH 8.3.
Also note that no apparent 8-Br-cGMP–induced current was observed at pH 6.3 at any
potential from ⫺120 to ⫺50 mV.


cGMP ACTIVATES A pH-SENSITIVE LEAK K⫹ CURRENT IN BFC NEURONS

A

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B

FIG. 5. External-pH– dependent effects of 8-Br-cGMP. A: pooled data showing the conductances normalized to their controls at pH 6.3, 7.3, and 8.3 following
application of 8-Br-cGMP. Note the most prominent change at pH 7.3 and no or less apparent changes at pH 6.3 and 8.3. *P ⬍ 0.02 compared with its control. B: the
solid (control) and hollow (8-Br-cGMP) columns represent the scaled conductances obtained before and after application of 8-Br-cGMP, respectively. The scaled
៮
៮
៮
៮
conductance at pH 7.3 after 8-Br-cGMP application was calculated by using an equation: S(8-Br-cGMP)-G
pH7.3 ⫽ [(GpH7.3 ⫻ 2.24) ⫺ (GpH6.3 ⫻ 1.03)]/[(GpH8.3 ⫻ 1.10) ⫺
៮
៮
៮
៮
(G
pH6.3 ⫻ 1.03)]. GpH6.3, GpH7.3, and GpH8.3 represent the mean conductances at respective pH levels shown in Fig. 1D.

A

rent response at the time point of *1 from that at *3 in Fig.
6B displayed slight inward rectification (Fig. 6C, *3 ⫺ *1).
By contrast, 8-Br-cGMP induced no marked current at
potentials examined by the ramp pulse in the presence of
Ba2⫹, as revealed by subtraction of the current response at
the time point of *1 from that at *2 in Fig. 6B (Fig. 6C, *2 ⫺
*1). The long-lasting nature and Ba2⫹ sensitivity to 8-BrcGMP–induced conductance increase were confirmed by the

second brief application of Ba2⫹ (Fig. 6, A and B). These
observations clearly indicate that 100 ␮M Ba2⫹ completely
antagonized the action of 8-Br-cGMP. Thus 8-Br-cGMP–
induced K⫹ current was almost completely blocked at any
potential examined, by lowering external pH to 6.3 as well
as by bath application of 100 ␮M Ba2⫹, as was the case with
the pH-sensitive current expressed in the presumed BFC
neurons. Therefore the 8-Br-cGMP–induced K⫹ current is

C

2⫹
FIG. 6. Ba
sensitivity of 8-Br-cGMP–induced currents. A: a continuous recording of current responses to the ramp and hyperpolarizing
pulses in a presumed BFC neuron. Gray and
black horizontal bars represent the duration and
timing of bath application of Ba2⫹ and 8-BrcGMP, respectively. B: 8-Br-cGMP showed no
significant effects on either the baseline current
(a) or the conductance (b) in the presence of Ba2⫹
(compare *1 and *2), whereas these values were
markedly increased following the simultaneous
washout of 8-Br-cGMP and Ba2⫹ (*3). The second brief application of Ba2⫹ transiently suppressed these responses, suggesting that 8-BrcGMP had long-lasting effects on the current
responses. C: the I–V relationship of 8-BrcGMP–induced current in the presence of Ba2⫹
obtained by *2 ⫺ *1, showing complete inhibition of 8-Br-cGMP response by Ba2⫹ at potentials over the range between ⫺120 and ⫺50 mV
(black trace). An inwardly rectified I–V relationship of Ba2⫹-sensitive component of the 8-BrcGMP–induced current obtained by *3 ⫺ *1
(gray trace). D: pooled data showing that 8-BrcGMP had no significant effect on either the
baseline current (a) or the conductance (b) in the
presence of Ba2⫹, whereas these values were
significantly increased following the simultaneous washout of 8-Br-cGMP and Ba2⫹. *P ⬍
0.002, **P ⬍ 0.001 (ANOVA followed by

PLSD).

B
D

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either the baseline current level (P ⬎ 0.9) or the conductance (P ⬎ 0.8) between the current responses obtained
before (9 ⫾ 33 pA and 3.9 ⫾ 1.2 nS, respectively) and 5– 6
min after application of 8-Br-cGMP (10 ⫾ 23 pA and 4.0 ⫾
1.2 nS, respectively) in five presumed BFC neurons examined (Fig. 6B, compare *1 and *2; see also Fig. 6D, a and b).
Nevertheless, following the simultaneous washout of Ba2⫹
and 8-Br-cGMP, the baseline current level was significantly
(P ⬍ 0.001) shifted outwardly from 10 ⫾ 23 to 88 ⫾ 24 pA
by 78 ⫾ 27 pA (n ⫽ 5) when measured from the original
baseline current level, and the conductance was also significantly (P ⬍ 0.002) increased from 4.0 ⫾ 1.2 to 7.2 ⫾ 2.5
nS by 3.2 ⫾ 1.5 nS (n ⫽ 5) (Fig. 6B, compare *2 and *3; see
also Fig. 6D, a and b). Consistent with the I–V relationship
shown in Fig. 2Bb, the Ba2⫹-sensitive component of 8-BrcGMP–induced current obtained by subtraction of the cur-


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TOYODA ET AL.

likely to be mediated by a pH- and Ba2⫹-sensitive leak K⫹
current expressed in the presumed BFC neurons.
DISCUSSION

Expression of pH-sensitive leak K⫹ channels similar
to TASK1 in the presumed BFC neurons

Contamination of GHK rectification with voltage-dependent
Kir and Kv currents
The 8-Br-cGMP–induced K⫹ current was invariably and
completely inhibited by the external acidification to pH 6.3,
regardless of whether it displayed a clear GHK rectification
(Figs. 3–5). This clearly indicates the acid sensitivity of 8-BrcGMP–induced K⫹ currents in the presumed BFC neurons, which
displayed pH-sensitive leak K⫹ current similar to TASK1 currents in its pH sensitivity. However, the 8-Br-cGMP–induced
K⫹ currents did not necessarily display GHK rectification,
unlike TASK1 current. This is because the 8-Br-cGMP–induced K⫹ current was often contaminated with Kv and Kir
currents at very depolarized or hyperpolarized membrane potentials, respectively. When the leak K⫹ conductance was
increased by 8-Br-cGMP or by raising pH, the space clamp
would become less stringent, resulting in less activation of
voltage-dependent currents (Figs. 2Aa, 3Ca, and 4Ca, gray
traces). Then, the I–V relationship of the 8-Br-cGMP–induced
or pH-sensitive current isolated by the subtraction method in
native neurons (Fig. 2Cb, black trace; Figs. 3Cb and 4Cb, gray
traces) may be less accurate, especially at very depolarized or
hyperpolarized membrane potentials due to the contamination
with Kv and Kir currents, respectively (Figs. 2Aa, 3Ca, and
4Ca, black traces). Thus the apparent inconsistency with GHK
rectification does not necessarily exclude the possibility of
involvement of leak K⫹ or TASK current in 8-Br-cGMP–

induced pH-sensitive K⫹ current.
J Neurophysiol • VOL

In the absence of 8-Br-cGMP, the conductance increase was
significantly larger following raising pH from 7.3 to 8.3 than
raising pH from 6.3 to 7.3 (Fig. 1). On the contrary, after the
application of 8-Br-cGMP, the conductance increase was significantly larger following raising pH from 6.3 to 7.3 than
raising pH from 7.3 to 8.3, as was confirmed in three neurons
tested (Fig. 4). This suggests that 8-Br-cGMP may have
changed the pH sensitivity of the leak K⫹ current, from the one
similar to that of TASK1 to the other rather similar to that of
TASK3 current, as seen in the pH profiles of the scaled
conductances obtained in the control condition and after 8-BrcGMP application (Fig. 5B, solid and hollow columns, respectively).
Similar upregulations of TWIK-related K⫹ channel 1 (TREK1)
and TWIK-related alkaline pH-activated K⫹ channel (TALK)
channels by cGMP have been reported in nonneuronal cells;
the NO– cGMP pathway acts to open TREK1 in smooth muscles (Koh et al. 2001) and TALK in the acinar cell of the
exocrine pancreas (Duprat et al. 2005). However, since TREK1
and TALK channels are much less sensitive to the acidification
to pH 6.3 (Duprat et al. 2005; Patel and Honore 2001), it is
unlikely that these channels are responsible for the acidsensitive 8-Br-cGMP–induced K⫹ current in the presumed
BFC neurons.
Many neuromodulators closing leak K⫹ channels including
TASK1 channels have been reported in a variety of neurons in
the thalamus and cortex (McCormick 1992), cerebellum (Abudara et al. 2002; Millar et al. 2000), and brain stem (Talley
et al. 2000). By contrast, the endogenous neuromodulators
opening leak K⫹ channels in neurons remained unknown,
although the volatile general anesthetics have been found to
open TASK1 channels in neurons of the locus coeruleus (Sirois
et al. 2000) and TASK1/3 channels in neurons of the raphe

nucleus (Washburn et al. 2002). The present study demonstrates for the first time in neurons that cGMP activates leak
K⫹ channels in the presumed BFC neurons, although we did
not identify the detailed subtype of the acid-sensitive leak K⫹
channel. This identification would be a very important issue in
a future study.
Ba2⫹ sensitivity of the pH-sensitive K⫹ current
Ba2⫹ sensitivities of cloned rTASK (Leonoudakis et al.
1998) or TASK1 (Millar et al. 2000) channels appeared to be
lower (IC50 ⫽ 0.35 mM) than those of the pH-sensitive current
or 8-Br-cGMP–induced responses seen in the present study
(Figs. 2 and 6). However, Ba2⫹ sensitivity was increased by
replacing some amino acids of the channel proteins with
histidine in TASK1 channels, although its acid sensitivity was
reduced (O’Connell et al. 2005). Then, it may be possible that
native wild-type TASK1 channels are more sensitive to Ba2⫹
than recombinant TASK1 channels in expression systems,
given the unknown posttranslational modification of TASK1
channels, partly similar to replacement of the amino acids.
Indeed, a similar high Ba2⫹ sensitivity of TASK1/3 channels
has been reported in thalamocortical neurons, in which no
pH-sensitive K⫹ current remained in the presence of 150 ␮M
Ba2⫹ (Meuth et al. 2003), as seen in the present study (Figs. 2
and 6).

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Among the 2P-domain K⫹ channels, TASK channels (Duprat
et al. 1997; Talley et al. 2000) are the most likely candidates for
the leak K⫹ channels. Indeed, the presumed BFC neurons displayed pH-sensitive currents in the present study (Figs. 1–5), and
the external pH decrease from 8.3 to 7.3 caused significantly
larger changes in the conductance than did the pH decrease from
7.3 to 6.3 (Fig. 1). Therefore the presumed BFC neurons express
K⫹ channels similar to TASK1 channels in the recombinant
systems (Duprat et al. 1997; Kim et al. 1998; Leonoudakis et al.
1998).
As reported in the previous studies using in situ hybridization, many neurons in nuclei of medial septum/diagonal band
(MS/DB) expressed a moderate to abundant amount of mRNA
of TASK1 channels (Karschin et al. 2001; Talley et al. 2001),
whereas there were only few cells in MS/DB that abundantly
express mRNA of TASK3 channels (Karschin et al. 2001). Our
electrophysiological findings are in good agreement with these
histological observations. Given the expression of TASK1
channels in the BFC neurons as reported histologically, TASK1
currents should be reflected, at least partly, in our electrophysiological observations.

Modulation of pH-sensitive leak K⫹ current by cGMP in the
presumed BFC neurons


cGMP ACTIVATES A pH-SENSITIVE LEAK K⫹ CURRENT IN BFC NEURONS

GRANTS

This work was partly supported by the Academic Frontier Project from
Japan Ministry of Education, Culture, Sports, Science and Technology
(MEXT) to Health Sciences University of Hokkaido and also partly supported

by Grant-in-Aid 17021027 for Scientific Research on Priority Areas (A) from
Japan MEXT to Y. Kang.
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Ba2⫹-sensitive currents or Ba2⫹-sensitive components of
8-Br-cGMP–induced currents obtained by the subtraction

method did not display GHK rectification. Instead, these usually displayed an inward rectification (Figs. 2B and 6C).
However, this is completely consistent with the previous report, in which the voltage-dependent blockade of TASK1
channels by Ba2⫹ became apparent as [Ba2⫹]o is increased
(O’Connell et al. 2005). As the membrane potential was
hyperpolarized, the attraction of positively charged blocking
ions to the channel pore would increase, resulting in an increase in the degree of channel block (Hille 2001). Then, the
“inward rectification” of Ba2⫹-sensitive K⫹ current is not due
to the rectification of the channel itself, and has nothing to do
with the inwardly rectifying nature of Kir channels mediated
by intracellular Mg2⫹ (Matsuda et al. 1987) and polyamine
(Ficker et al. 1994; Lopatin et al. 1994). Therefore the apparent
inwardly rectifying nature of Ba2⫹-sensitive current does not
necessarily mean the involvement of Kir channels in generating the inward rectification, as were the cases with recombinant
TASK1 channels (O’Connell et al. 2005) and TASK1/3 channels in thalamocortical neurons (Meuth et al. 2003).

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