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

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 You might find this additional info useful This article cites 25 articles, 17 of which you can access for free at: http://jn.physiology.org/content/99/5/2126.full#ref-list-1 This article has been cited by other HighWire-hosted articles: http://jn.physiology.org/content/99/5/2126#cited-by Updated information and services including high resolution figures, can be found at: http://jn.physiology.org/content/99/5/2126.full This information is current as of June 8, 2013 Journal of Neurophysiology publishes original articles on the function of the nervous system It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991 Copyright © 2008 by the American Physiological Society ISSN: 0022-3077, ESSN: 1522-1598 Visit our website at http://www.the-aps.org/ Downloaded from http://jn.physiology.org/ by guest on June 8, 2013 Additional material and information about Journal of Neurophysiology can be found at: http://www.the-aps.org/publications/jn 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 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) 2126 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, KCl, 20 NaCl, 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, 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 0022-3077/08 $8.00 Copyright © 2008 The American Physiological Society www.jn.org Downloaded from http://jn.physiology.org/ by guest on June 8, 2013 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 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 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 J Neurophysiol • VOL 99 • MAY 2008 • www.jn.org Downloaded from http://jn.physiology.org/ by guest on June 8, 2013 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 and 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 and and defined as the scaled conductance (S-Gx) as follows: S-Gx ⫽ 2127 2128 TOYODA ET AL 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 J Neurophysiol • VOL 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 in Kang et al 2007 and see also Fig in this paper), effects of pH changes on the 8-Br-cGMP–induced current can be safely examined at least for 20 –30 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 99 • MAY 2008 • www.jn.org Downloaded from http://jn.physiology.org/ by guest on June 8, 2013 2⫹ FIG 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 2129 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 99 • MAY 2008 • www.jn.org Downloaded from http://jn.physiology.org/ by guest on June 8, 2013 FIG 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 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 current traces were obtained at the respective times indicated with *1 (Control, black trace) and *2 (8-Br-cGMP, gray trace) in Da 2130 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 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– under the voltage-clamp condition (Fig 6, A and B) There were no significant differences in 99 • MAY 2008 • www.jn.org Downloaded from http://jn.physiology.org/ by guest on June 8, 2013 FIG 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 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 2131 B FIG 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 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 J Neurophysiol • VOL 99 • MAY 2008 • www.jn.org Downloaded from http://jn.physiology.org/ by guest on June 8, 2013 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– 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- 2132 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 (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 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 and 6) 99 • MAY 2008 • www.jn.org Downloaded from http://jn.physiology.org/ by guest on June 8, 2013 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 REFERENCES 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 heteromeric 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 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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) 2126 that... 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... 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,