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

doi: 10.1152/jn.01051.2007

99:2126-2133, 2008 First published 20 February 2008;

J Neurophysiol

Hirai, Yoshinobu Maeda, Takeshi Kaneko and Youngnam Kang

Hiroki Toyoda, Mitsuru Saito, Hajime Sato, Yoshie Dempo, Atsuko Ohashi, Toshihiro

Presumed Cholinergic Neuron of Basal Forebrain

cGMP Activates a pH-Sensitive Leak K+ Current in the

You might find this additional info useful

25 articles, 17 of which you can access for free at:

This article cites

http://jn.physiology.org/content/99/5/2126.full#ref-list-1

1 other HighWire-hosted articles:

This article has been cited by

http://jn.physiology.org/content/99/5/2126#cited-by

including high resolution figures, can be found at:

Updated information and services

http://jn.physiology.org/content/99/5/2126.full

can be found at:

Journal of Neurophysiology

about

Additional material and information

http://www.the-aps.org/publications/jn

This information is current as of June 8, 2013

at http://www.the-aps.org/

Copyright © 2008 by the American Physiological Society ISSN: 0022-3077, ESSN: 1522-1598 Visit our website

times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991

publishes original articles on the function of the nervous system It is published 12

Journal of Neurophysiology

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 Kang 1,3

1Department 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 and4Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Hokkaido; and5Department

of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan

Submitted 23 September 2007; accepted in final form 18 February 2008

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

demon-strated 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

pH-sensitive 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 Ba 2⫹ , 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.

I N T R O D U C T I O N

As demonstrated in an earlier study (Kang et al 2007),

S-nitroso-N-acetylpenicillamine (SNAP) or

8-bromo-guanosine-3⬘,5⬘-cyclomonophosphate (8-Br-cGMP) induced a

membrane hyperpolarization in the presumed basal forebrain

cholinergic (BFC) neurons by activating K⫹currents that

usu-ally 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

stri-atal medium spiny neurons, presumably by inhibition of leak

K⫹channels (West and Grace 2004) It has also been reported

that long-term activation of the NO– cGMP–protein kinase G (PKG) pathway in injured motoneurons resulted in an inhibi-tion of a pH-sensitive leak K⫹current, suggesting an involve-ment 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

M E T H O D S

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 NaHCO3in 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 voltage-clamp 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–Al-drich, St Louis, MO), and BaCl2 (Wako Pure Chemicals, Osaka,

* These authors contributed equally to this work.

Address for reprint requests and other correspondence: Y Kang,

Depart-ment 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).

The costs of publication of this article were defrayed in part by the payment

of page charges The article must therefore be hereby marked “advertisement”

in accordance with 18 U.S.C Section 1734 solely to indicate this fact.

First published February 20, 2008; doi:10.1152/jn.01051.2007.

2126 0022-3077/08 $8.00 Copyright © 2008 The American Physiological Society www.jn.org

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.

R E S U L T S

The presumed BFC neurons display a pH-sensitive leak

K⫹current

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 forⱖ30 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-I x ) as follows: S-I x ⫽ (I x ⫺ 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-G x ) as follows: S-G x⫽

(G x ⫺ 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 recombi-nant 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⫹

Ba 2⫹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,

respec-tively), 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, com-pare 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⫹ fol-lowing the same decrease in the external pH (⫺7.0 ⫾ 4.4 nS,

n ⫽ 5, P ⬍ 0.04).

FIG 1 External-pH sensitivity in the presumed basal

fore-brain cholinergic (BFC) neurons A: plotting of baseline

cur-rents 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 (I x) were

scaled by using an equation: S-I x ⫽ (I x ⫺ 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-G x ⫽ (G x ⫺ GpH6.3)/(GpH8.3⫺

GpH6.3), where x is the pH of the external solution.

2127 cGMP ACTIVATES A pH-SENSITIVE LEAK K CURRENT IN BFC NEURONS

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

fol-lowing 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

differ-ence in the current responses at pH 6.3 obtained before and

after 8-Br-cGMP application (Fig 3Ba; compare *2 and *4), as

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 pH-sensitive 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 re-corded 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-du-ration, 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 forⱖ30 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–in-duced 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

FIG 2 Ba2⫹ 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 Ba 2⫹

appli-cation (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⫹ -sensi-tive 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)

re-lationships of Ba2⫹-sensitive currents at pH

7.3 and 8.3 (black and gray traces, respec-tively, 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

out-wardly rectified I–V relationship of

pH-sen-sitive current in the absence of Ba2⫹(black

trace) In the presence of Ba2⫹, no apparent pH-sensitive current remained over the volt-age range from ⫺130 to ⫺40 mV (gray

trace).

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

conduc-tances were still larger than their controls (Fig 4B, a and b) As

shown in the I–V relationship (Fig 4Cb), however,

8-Br-cGMP–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

re-sponses and its sensitivity to acidification This long-lasting

nature of 8-Br-cGMP–induced responses seen under the

volt-age-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

re-versibly 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

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-acti-vated K⫹ (Kv) channels (Markram and Segal 1990) and in-wardly 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⫹ cur-rents 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

A

B

C

D

FIG 3 Differential effects of 8-bromoguanosine-3 ⬘,5⬘-cy-clomonophosphate (8-Br-cGMP) on the leak K⫹ current

be-tween 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

exter-nal 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

superim-posed 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,

respec-tively) 8-Br-cGMP–induced current at pH 7.3 displayed a slight

sigmoidal I–V relationship Note no apparent

8-Br-cGMP–in-duced 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

superim-posed 2 current traces were obtained at the respective times

indicated with *1 (Control, black trace) and *2 (8-Br-cGMP,

gray trace) in Da.

2129 cGMP ACTIVATES A pH-SENSITIVE LEAK K CURRENT IN BFC NEURONS

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-Br-cGMP are shown in Fig 5 Bath application of 8-Br-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

con-ductances and the scaled concon-ductances 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

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 neces-sarily 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

Ba 2⫹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

A

B

C

FIG 4 8-Br-cGMP–induced current is

greater at pH 7.3 than at pH 8.3 A: a

con-tinuous 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-Br-cGMP External pH was serially changed as indicated with gray horizontal bars, which represent the duration and timing of perfu-sion 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

con-ductances (b) against time The current

re-sponses 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 cur-rent responses was completely blocked by

lowering external pH to 6.3 (compare *3 and

*5) Ca, top: voltage command pulse Bot-tom: 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 ob-tained at the respective times indicated with

*2 (Control, black trace) and *4

(8-Br-cGMP, gray trace) in Ba Cb: the I–V

rela-tionships 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

be-fore 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

relation-ship Note that the 8-Br-cGMP–induced cur-rent was greater at pH 7.3 than at pH 8.3 Also note that no apparent 8-Br-cGMP–in-duced current was observed at pH 6.3 at any potential from ⫺120 to ⫺50 mV.

either the baseline current level (P ⬎ 0.9) or the

conduc-tance (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

exam-ined (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

signif-icantly (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-Br-cGMP–induced current obtained by subtraction of the

cur-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-Br-cGMP–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

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⫽ [(G៮pH7.3⫻ 2.24) ⫺ (G៮pH6.3⫻ 1.03)]/[(G៮pH8.3⫻ 1.10) ⫺

(G៮pH6.3⫻ 1.03)] G៮pH6.3, G៮pH7.3, and G៮pH8.3represent the mean conductances at respective pH levels shown in Fig 1D.

A

B

C

D

FIG 6 Ba 2 ⫹ sensitivity of

8-Br-cGMP–in-duced currents A: a continuous recording of

cur-rent 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-Br-cGMP, 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

sec-ond brief application of Ba 2 ⫹ transiently sup-pressed these responses, suggesting that 8-Br-cGMP had long-lasting effects on the current

responses C: the I–V relationship of

8-Br-cGMP–induced current in the presence of Ba 2 ⫹

obtained by *2 ⫺ *1, showing complete

inhibi-tion of 8-Br-cGMP response by Ba 2 ⫹ at poten-tials over the range between ⫺120 and ⫺50 mV

(black trace) An inwardly rectified I–V

relation-ship of Ba2⫹-sensitive component of the

8-Br-cGMP–induced current obtained by *3 ⫺ *1 (gray trace) D: pooled data showing that

8-Br-cGMP had no significant effect on either the

baseline current (a) or the conductance (b) in the

presence of Ba 2 ⫹ , whereas these values were significantly increased following the simulta-neous washout of 8-Br-cGMP and Ba 2 ⫹ *P⬍

0.002, **P ⬍ 0.001 (ANOVA followed by PLSD).

2131 cGMP ACTIVATES A pH-SENSITIVE LEAK K CURRENT IN BFC NEURONS

likely to be mediated by a pH- and Ba2⫹-sensitive leak K⫹

current expressed in the presumed BFC neurons

D I S C U S S I O N

Expression of pH-sensitive leak K⫹channels similar

to TASK1 in the presumed BFC neurons

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

dis-played 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

hybridiza-tion, 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

electrophysio-logical observations

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-Br-cGMP–induced K⫹currents in the presumed BFC neurons, which

displayed pH-sensitive leak K⫹ current similar to TASK1

cur-rents 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–in-duced K⫹ current was often contaminated with Kv and Kir

currents at very depolarized or hyperpolarized membrane

po-tentials, 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

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

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 sig-nificantly 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-Br-cGMP application (Fig 5B, solid and hollow columns,

respec-tively)

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 mus-cles (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 acid-sensitive 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 (Abu-dara 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 demon-strates 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

Ba 2⫹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)

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

usu-ally displayed an inward rectification (Figs 2B and 6C).

However, this is completely consistent with the previous

re-port, 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

in-crease 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

generat-ing the inward rectification, as were the cases with recombinant

TASK1 channels (O’Connell et al 2005) and TASK1/3

chan-nels in thalamocortical neurons (Meuth et al 2003)

G R A N T S

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.

R E F E R E N C E S

Abudara V, Alvarez AF, Chase MH, Morales FR Nitric oxide as an

anterograde neurotransmitter in the trigeminal motor pool J Neurophysiol

88: 497–506, 2002.

Berg AP, Talley EM, Manger JP, Bayliss DA Motoneurons express

hetero-meric TWIK-related acid-sensitive K⫹(TASK) channels containing TASK-1

(KCNK3) and TASK-3 (KCNK9) subunits J Neurosci 24: 6693– 6702, 2004.

Duprat F, Girard C, Jarretou G, Lazdunski M Pancreatic two P domain

K⫹channels TALK-1 and TALK-2 are activated by nitric oxide and reactive

oxygen species J Physiol 562: 235–244, 2005.

Duprat F, Lesage F, Fink M, Reyes R, Heurteaux C, Lazdunski M TASK,

a human background K⫹ channel to sense external pH variations near

physiological pH EMBO J 16: 5464 –5471, 1997.

Farkas RH, Nakajima S, Nakajima Y Neurotensin excites basal forebrain

cholinergic neurons: ionic and signal-transduction mechanisms Proc Natl

Acad Sci USA 91: 2853–2857, 1994.

Ficker E, Taglialatela M, Wible BA, Henley CM, Brown AM Spermine

and spermidine as gating molecules for inward rectifier K⫹ channels.

Science 266: 1068 –1072, 1994.

Gonzalez-Forero D, Portillo F, Gomez L, Montero F, Kasparov S,

Moreno-Lopez B Inhibition of resting potassium conductances by

long-term activation of the NO/cGMP/protein kinase G pathway: a new

mecha-nism regulating neuronal excitability J Neurosci 27: 6302– 6312, 2007.

Hille B Ion Channels of Excitable Membranes (3rd ed.) Sunderland, MA:

Sinauer, 2001, p 814.

Kang D, Han J, Talley EM, Bayliss DA, Kim D Functional expression of

TASK-1/TASK-3 heteromers in cerebellar granule cells J Physiol 554:

64 –77, 2004.

Kang Y, Dempo Y, Ohashi A, Saito M, Toyoda H, Sato H, Koshino H, Maeda Y, Hirai T Nitric oxide activates leak K⫹currents in the presumed

cholinergic neuron of basal forebrain J Neurophysiol 98: 3397–3410, 2007.

Karschin C, Wischmeyer E, Preisig-Muller R, Rajan S, Derst C, Grz-eschik KH, Daut J, Karschin A Expression pattern in brain of TASK-1,

TASK-3, and a tandem pore domain K⫹channel subunit, TASK-5,

associ-ated with the central auditory nervous system Mol Cell Neurosci 18:

632– 648, 2001.

Kim D, Fujita A, Horio Y, Kurachi Y Cloning and functional expression of

a novel cardiac two-pore background K⫹channel (cTBAK-1) Circ Res 82:

513–518, 1998.

Leonoudakis D, Gray AT, Winegar BD, Kindler CH, Harada M, Taylor

DM, Chavez RA, Forsayeth JR, Yost CS An open rectifier potassium

channel with two pore domains in tandem cloned from rat cerebellum.

J Neurosci 18: 868 – 877, 1998.

Lopatin AN, Makhina EN, Nichols CG Potassium channel block by

cyto-plasmic polyamines as the mechanism of intrinsic rectification Nature 372:

366 –369, 1994.

Markram H, Segal M Electrophysiological characteristics of cholinergic and

non-cholinergic neurons in the rat medial septum-diagonal band complex.

Brain Res 513: 171–174, 1990.

Matsuda H, Saigusa A, Irisawa H Ohmic conductance through the inwardly

rectifying K channel and blocking by internal Mg2⫹ Nature 325: 156 –159,

1987.

McCormick DA Neurotransmitter actions in the thalamus and cerebral cortex

and their role in neuromodulation of thalamocortical activity Prog

Neuro-biol 39: 337–388, 1992.

Meuth SG, Budde T, Kanyshkova T, Broicher T, Munsch T, Pape HC.

Contribution of TWIK-related acid-sensitive K⫹channel 1 (TASK1) and TASK3 channels to the control of activity modes in thalamocortical neurons.

J Neurosci 23: 6460 – 6469, 2003.

Millar JA, Barratt L, Southan AP, Page KM, Fyffe RE, Robertson B, Mathie A A functional role for the two-pore domain potassium channel

TASK-1 in cerebellar granule neurons Proc Natl Acad Sci USA 97:

3614 –3618, 2000.

O’Connell AD, Morton MJ, Sivaprasadarao A, Hunter M Selectivity and

interactions of Ba2⫹ and Cs⫹ with wild-type and mutant TASK1 K⫹

channels expressed in Xenopus oocytes J Physiol 562: 687– 696, 2005.

Patel AJ, Honore E Properties and modulation of mammalian 2P domain K⫹

channels Trends Neurosci 24: 339 –346, 2001.

Sirois JE, Lei Q, Talley EM, Lynch C 3rd, Bayliss DA The TASK-1

two-pore domain K⫹channel is a molecular substrate for neuronal effects of

inhalation anesthetics J Neurosci 20: 6347– 6354, 2000.

Talley EM, Lei Q, Sirois JE, Bayliss DA TASK-1, a two-pore domain K⫹

channel, is modulated by multiple neurotransmitters in motoneurons

Neu-ron 25: 399 – 410, 2000.

Talley EM, Solorzano G, Lei Q, Kim D, Bayliss DA CNS distribution of

members of the two-pore-domain (KCNK) potassium channel family.

J Neurosci 21: 7491–7505, 2001.

Washburn CP, Sirois JE, Talley EM, Guyenet PG, Bayliss DA

Serotoner-gic raphe neurons express TASK channel transcripts and a TASK-like pH-and halothane-sensitive K⫹conductance J Neurosci 22: 1256 –1265, 2002.

West AR, Grace AA The nitric oxide-guanylyl cyclase signaling pathway

modulates membrane activity states and electrophysiological properties of

striatal medium spiny neurons recorded in vivo J Neurosci 24: 1924 –1935,

2004.

2133 cGMP ACTIVATES A pH-SENSITIVE LEAK K CURRENT IN BFC NEURONS

J Neurophysiol• VOL 99 • MAY 2008 • www.jn.org