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• W1975620408 abstract "Synaptic cleft acidification occurs following vesicle release. Such a pH change may affect synaptic transmissions in which G-protein-coupled inward rectifier K+ (GIRK) channels play a role. To elucidate the effect of extracellular pH (pHo) on GIRK channels, we performed experiments on heteromeric GIRK1/GIRK4 channels expressed in Xenopus oocytes. A decrease in pHo to 6.2 augmented GIRK1/GIRK4 currents by ∼30%. The channel activation was reversible and dependent on pHo levels. This effect was produced by selective augmentation of single channel conductance without change in the open-state probability. To determine which subunit was involved, we took advantage of homomeric expression of GIRK1 and GIRK4 by introducing a single mutation. We found that homomeric GIRK1-F137S and GIRK4-S143T channels were activated at pHo 6.2 by ∼20 and ∼70%, respectively. Such activation was eliminated when a histidine residue in the M1-H5 linker was mutated to a non-titratable glutamine, i.e.H116Q in GIRK1 and H120Q in GIRK4. Both of these histidines were required for pH sensing of the heteromeric channels, because the mutation of one of them diminished but not abolished the pHosensitivity. The pHo sensitivity of the heteromeric channels was completely lost when both were mutated. Thus, these results suggest that the GIRK-mediated synaptic transmission is determined by both neurotransmitter and protons with the transmitter accounting for only 70% of the effect on postsynaptic cell and protons released together with the transmitter contributing to the other 30%. Synaptic cleft acidification occurs following vesicle release. Such a pH change may affect synaptic transmissions in which G-protein-coupled inward rectifier K+ (GIRK) channels play a role. To elucidate the effect of extracellular pH (pHo) on GIRK channels, we performed experiments on heteromeric GIRK1/GIRK4 channels expressed in Xenopus oocytes. A decrease in pHo to 6.2 augmented GIRK1/GIRK4 currents by ∼30%. The channel activation was reversible and dependent on pHo levels. This effect was produced by selective augmentation of single channel conductance without change in the open-state probability. To determine which subunit was involved, we took advantage of homomeric expression of GIRK1 and GIRK4 by introducing a single mutation. We found that homomeric GIRK1-F137S and GIRK4-S143T channels were activated at pHo 6.2 by ∼20 and ∼70%, respectively. Such activation was eliminated when a histidine residue in the M1-H5 linker was mutated to a non-titratable glutamine, i.e.H116Q in GIRK1 and H120Q in GIRK4. Both of these histidines were required for pH sensing of the heteromeric channels, because the mutation of one of them diminished but not abolished the pHosensitivity. The pHo sensitivity of the heteromeric channels was completely lost when both were mutated. Thus, these results suggest that the GIRK-mediated synaptic transmission is determined by both neurotransmitter and protons with the transmitter accounting for only 70% of the effect on postsynaptic cell and protons released together with the transmitter contributing to the other 30%. The G-protein-coupled inward rectifier K+(GIRK) 1The abbreviations used for: GIRK, G-protein-coupled inward rectifier K+; pHo, extracellular pH; pHi, intracellular pH; PIPES, 1,4-piperazinediethanesulfonic acid; ANOVA, analysis of variance; P, pore. 1The abbreviations used for: GIRK, G-protein-coupled inward rectifier K+; pHo, extracellular pH; pHi, intracellular pH; PIPES, 1,4-piperazinediethanesulfonic acid; ANOVA, analysis of variance; P, pore. channels are important players in cellular communications in several excitable tissues (1Hille B. Ionic Channels of Excitable Membranes. 3rd Ed. Sinauer Associates, Inc., Sunderland, MA2001: 201-236Google Scholar, 2Luscher C. Jan L.Y. Stoffel M. Malenka R.C. Nicoll R.A. Neuron. 1997; 19: 687-695Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 3Yamada M. Inanobe A. Kurachi Y. Pharmacol. Rev. 1998; 50: 723-760PubMed Google Scholar). The GIRK channels are activated by βγ-subunits of G-proteins, which are dissociated from the αβγ-trimer as a result of receptor binding to neurotransmitters or hormones (3Yamada M. Inanobe A. Kurachi Y. Pharmacol. Rev. 1998; 50: 723-760PubMed Google Scholar). Four members of GIRK channels have been identified in mammals with GIRK1/GIRK4 expressed abundantly in the heart and brain (4Dascal N. Cell. Signalling. 1997; 9: 551-573Crossref PubMed Scopus (265) Google Scholar). GIRK channels are modulated by several intracellular signal molecules such as Na+, ATP, and phospholipids (5Sui J.L. Chan K.W. Logothetis D.E. J. Gen. Physiol. 1996; 108: 381-391Crossref PubMed Scopus (102) Google Scholar, 6Ho I.H. Murrell-Lagnado R.D. J. Physiol. (London). 1999; 520: 645-651Crossref Scopus (85) Google Scholar, 7Ho I.H. Murrell-Lagnado R.D. J. Biol. Chem. 1999; 274: 8639-8648Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 8Otero A.S. Breitwieser G.E. Szabo G. Science. 1988; 242: 443-445Crossref PubMed Scopus (55) Google Scholar, 9Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (755) Google Scholar, 10Kim D. Bang H. J. Physiol. (London). 1999; 517: 59-74Crossref Scopus (24) Google Scholar, 11Zhang J. Kong C. Xie H. McPherson P.S. Grinstein S. Trimble W.S. Curr. Biol. 1999; 9: 1458-1467Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 12Kobrinsky E. Mirshahi T. Zhang H. Jin T. Logothetis D.E. Nat. Cell Biol. 2000; 2: 507-514Crossref PubMed Scopus (203) Google Scholar). Extracellular molecules including hormones, neurotransmitters, and integrins directly or indirectly modulate GIRK channel activity through signaling transduction pathways (13Kreienkamp H.J. Honck H.H. Richter D. FEBS Lett. 1997; 419: 92-94Crossref PubMed Scopus (70) Google Scholar, 14Stevens E.B. Shah B.S. Pinnock R.D. Lee K. Mol. Pharmacol. 1999; 55: 1020-1027Crossref PubMed Scopus (43) Google Scholar, 15Liao Y.J. Jan Y.N. Jan L.Y. J. Neurosci. 1996; 16: 7137-7150Crossref PubMed Google Scholar, 16Dutar P. Petrozzino J.J. Vu H.M. Schmidt M.F. Perkel D.J. J. Neurophysiol. 2000; 84: 2284-2290Crossref PubMed Scopus (39) Google Scholar, 17Lei Q. Talley E.M. Bayliss D.A. J. Biol. Chem. 2001; 276: 16720-16730Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 18McPhee J.C. Dang Y.L. Davidson N. Lester H.A. J. Biol. Chem. 1998; 273: 34696-34702Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). GIRK channels are also the major targets of ethanol, anesthetics, and opioids (19Kobayashi T. Ikeda K. Kojima H. Niki H. Yano R. Yoshioka T. Kumanishi T. Nat. Neurosci. 1999; 2: 1091-1097Crossref PubMed Scopus (210) Google Scholar, 20Lewohl J.M. Wilson W.R. Mayfield R.D. Brozowski S.J. Morrisett R.A. Harris R.A. Nat. Neurosci. 1999; 2: 1084-1090Crossref PubMed Scopus (206) Google Scholar, 21Weigl L.G. Schreibmayer W. Mol. Pharmacol. 2001; 60: 282-289Crossref PubMed Scopus (39) Google Scholar, 22Zhou W. Arrabit C. Choe S. Slesinger P.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6482-6487Crossref PubMed Scopus (74) Google Scholar, 23Kobayashi T. Ikeda K. Ichikawa T. Togashi S. Kumanishi T. Br. J. Pharmacol. 1996; 119: 73-80Crossref PubMed Scopus (22) Google Scholar). Another potentially important modulator of the GIRK channels is hydrogen ion. Increasing evidence indicates that H+ can act as a messenger modulating multiple cellular functions (24Yuli I. Oplatka A. Science. 1987; 235: 340-342Crossref PubMed Scopus (72) Google Scholar). In the central nervous system, protons have been shown to modulate synaptic transmission, neuronal plasticity, and membrane excitability (25Kaila K. Ransom B.R. pH and Brain Function. John Wiley & Sons, Inc., New York1998: 3-10Google Scholar). It is known that the pH level in synaptic vesicles is ∼1.5 pH units lower than in cytosol (26Liu Y. Edwards R.H. Annu. Rev. Neurosci. 1997; 20: 125-156Crossref PubMed Scopus (236) Google Scholar). These protons are released from synaptic vesicles together with neurotransmitters during synaptic transmission, leading to extracellular acidification in the synaptic cleft (27Miesenbock G. De Angelis D.A. Rothman J.E. Nature. 1998; 394: 192-195Crossref PubMed Scopus (1901) Google Scholar). If the GIRK channels are sensitive to extracellular pH (pHo), such extracellular acidification can have a major impact on synaptic transmission. Indeed, certain unidentified GIRK channels in the brainstem that play a part in the generation and control of central respiratory activity have been suggested being sensitive to hypercapnic acidosis (28Smith J.C. Funk G.D. Johnson S.M. Feldman J.L. Trouth C.O. Millis R.M. Kiwull-Schöne H. Schläfke M.E. Ventral Brainstem Mechanisms and Control of Respiration and Blood Pressure. Marcel Dekker, Inc., New York1995: 463-496Google Scholar, 29Johnson S.M. Smith J.C. Feldman J.L. J. Appl. Physiol. 1996; 80: 2120-2133Crossref PubMed Scopus (109) Google Scholar, 30Ballanyi K. Onimaru H. Homma I. Prog. Neurobiol. 1999; 59: 583-634Crossref PubMed Scopus (262) Google Scholar, 31Nattie E. Prog. Neurobiol. 1999; 59: 299-331Crossref PubMed Scopus (315) Google Scholar). It is possible that the GIRK channels are pH-sensitive, because several inward rectifier K+ channels are directly gated by intracellular and/or extracellular protons. To test the hypothesis that the GIRK channels are modulated by pHo, we performed experiments on the heteromeric GIRK1/GIRK4 channels. Our results indicate that these channels are augmented by extracellular acidification through an increase in single channel conductance, and such activation relies on a histidine residue in the extracellular loop. Experiments were performed as we described previously (32Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (London). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 33Qu Z. Zhu G. Yang Z. Cui N. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 34Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Oocytes from Xenopus laevis were used in this study. Frogs were anesthetized by bathing them in 0.3% 3-aminobenzoic acid ethyl ester. A few lobes of ovaries were removed after a small abdominal incision (∼5 mm). The surgical incision then was closed, and the frogs were allowed to recover from the anesthesia.Xenopus oocytes were treated with 2 mg/ml collagenase (Type IA, Sigma) in the OR2 solution (in mm) as follows: 82 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES, pH 7.4, for 60 min at room temperature. After three washes (10 min each) of the oocytes with the OR2 solution, cDNAs (25–50 ng in 50 nl water) were injected into the oocytes. The oocytes were then incubated at 18 °C in the ND-96 solution containing (in mm) 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES, and 2.5 sodium pyruvate with 100 mg/liter Geneticin added, pH 7.4. Rat GIRK1 (Kir3.1) cDNA (GenBankTM accession numberU01071) and rat GIRK4 (Kir3.4) cDNA (GenBankTMaccession number X83584) are gifts from Dr. Norman Davidson (California Institute of Technology). These cDNAs were subcloned into a eukaryotic expression vector pcDNA3.1 (Invitrogen) and used forXenopus oocyte expression without in vitro cRNA synthesis. PCR was used to generate GIRK1/GIRK4 dimer. Two PCR fragments encoding the entire length of GIRK1 and GIRK4 were joined with an XbaI restriction site created using primers with the GIRK1 at the 5′ end and then cloned to the pcDNA3.1. Five extra amino acids (RCQQQ) were created between the C terminus of GIRK1 and the N terminus of GIRK4. We did not find any detectable effect of these additional residues on channel expression, current profile, and pH sensitivity. Site-specific mutations were made using a site-directed mutagenesis kit (Stratagene, La Jolla, CA). Correct constructions and mutations were confirmed with DNA sequencing. Whole-cell currents were studied on the oocytes 2–4 days after injection. Two-electrode voltage clamp was performed using an amplifier (Geneclamp 500, Axon Instruments Inc., Foster City, CA) at room temperature (∼24 °C). The extracellular solution contained (in mm): 90 KCl, 3 MgCl2, and 5 HEPES, pH 7.4. Extracellular acidification was done by perfusing the oocytes with the extracellular solution containing PIPES buffer, pH 5.2 and 6.2. HEPES buffer was used for extracellular alkalization, pH 8.4. These buffers were chosen because their buffering ranges are suitable for these pH levels and none of them is membrane-permeable as shown in our previous studies (33Qu Z. Zhu G. Yang Z. Cui N. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 34Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Several control experiments were done in which inward rectifying currents of oocytes receiving an injection of the expression vector or Kir2.1 channel were not affected by these PIPES and HEPES buffers. Currents were also measured at various pH points in oocytes receiving an injection of the expression vector or the pH-insensitive Kir2.1 as controls. Intracellular acidification was produced using 90 mm KHCO3 to replace all KCl (90 mm) in the extracellular solution, which acidified the cytosol to pH 6.6 (see “Results”). This solution was titrated to pH 7.4 immediately before use (33Qu Z. Zhu G. Yang Z. Cui N. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 34Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 50Zhu G.Y. Chanchevalap S. Liu C. Xu H. Jiang C. J. Cell. Physiol. 2000; 183: 53-64Crossref PubMed Scopus (38) Google Scholar). pHo and intracellular pH (pHi) were measured using ion-selective microelectrodes as we detailed previously (34Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 50Zhu G.Y. Chanchevalap S. Liu C. Xu H. Jiang C. J. Cell. Physiol. 2000; 183: 53-64Crossref PubMed Scopus (38) Google Scholar). Two single-barreled microelectrodes were employed. One of them (ion-selective) was exposed to hexamethyldisilazan vapor (Fluka Chemie AG, Buchs, Switzerland) for 30 min and then baked at 125 °C for 8 h. The tip of the ion-selective microelectrode was filled with H+ liquid exchanger (Hydrogen Ion Ionophore l-Mixture A, Fluka Chemie AG), and the remainder of the microelectrode was backfilled with phosphate buffer, pH 7.0, for both pHi and pHo measurements. This ionophore is greatly selective for H+ (e.g. H+:K+, Na+ or Ca2+ > 1,000,000:1). The other microelectrode was filled with 3 m KCl. Electrodes were used only if they had high frequency response (90% response time ≤ 5 s) and showed an excellent sensitivity (a voltage change > 55 mV when pH changed from 6.0 to 7.0). A high input-resistance amplifier (Duo773, World Precision Instruments, Inc., Sarasota, FL) was used for pH measurements. The ion-selective electrode was connected to the high input-resistance channel (1015ohms), and the KCl electrode was connected to the other (1012 ohm). Voltage was removed by subtracting records between these two channels. Serial calibrations of ion-selective microelectrodes were made with potassium phosphate buffer at pH 6.0, 7.0, and 8.0 (Fisher Scientific). The time profile of current response to pHo was studied using a perfusion chamber with a total volume of 1 ml (RC-3Z, Warner Instruments, New Haven, CT). The current amplitude was plotted against time together with pHo measured using a H+-selective electrode (34Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 45Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar). The pH electrode was positioned in the extracellular solution near the cell. When the extracellular solution was switched to a low pH perfusate, the pHo started to change in ∼1 min and reached a plateau level in ∼3 min. Single channel currents were studied in outside-out patch configuration using solutions containing equal concentrations of K+applied to the bath and recording pipettes. The bath solution contained (in mm): 140 KCl, 10 Na2H2P2O7, 5 NaF, 0.1 Na3VO3, 0.2 ATP, 0.2 GTP, 10 EGTA, and 10 HEPES, pH 7.4. The pipette was filled with the same solution (32Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (London). 1999; 516: 699-710Crossref Scopus (69) Google Scholar). The open-state probability (P o) was calculated as we described previously (32Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (London). 1999; 516: 699-710Crossref Scopus (69) Google Scholar). The single channel conductance was measured in negative membrane potential using a ramp command potential (from −100 to 100 mV). To change pHo, the patch membrane was perfused with solutions in different pH levels with no dead space. Data are presented as the means ± S.E. ANOVA or Student'st test was used. The differences of pH effects beforeversus during exposures were considered to be statistically significant if p ≤ 0.05. Whole-cell currents were studied in the two-electrode voltage-clamp mode using an extracellular solution containing 90 mm K+ (see “Materials and Methods”). Depolarizing and hyperpolarizing command pulses were given to the cell in a range from −140 mV (or −160 mV) to 100 mV with a 20-mV increment at a holding potential of 0 mV. Under this condition, inward rectifying currents were observed 2–4 days after coinjection of GIRK1 and GIRK4 cDNAs. The GIRK1/GIRK4 currents showed a clear inward rectification (Fig.1 A) and were sensitive to micromolar concentrations of Ba2+ (data not shown). These currents should be recorded from the heteromeric GIRK1/GIRK4 rather than the homomeric channels, because similar inward rectifying currents were seen following an injection of a tandem dimer of GIRK1/GIRK4 cDNA (data not shown). Exposure of the oocytes to a perfusate of pH 6.2 augmented the heteromeric GIRK/GIRK4 currents (Fig.1 A). Evident increase in the GIRK/GIRK4 currents was seen within 1 min into the exposure and reached the maximal level in 3–4 min (Fig. 1 B). Washout with the pH 7.4 perfusate led to a clear recovery (Fig. 1 A). When the current amplitude was plotted together with pHo against time, the current response curve was almost identical to the pHo profile with only an ∼0.5-min delay (Fig. 1 B). This effect was not produced by a change in pHi, because the perfusate containing membrane-impermeable PIPES buffer does not change pHi as shown in our previous studies (33Qu Z. Zhu G. Yang Z. Cui N. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 34Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). To strengthen this argument, we selectively reduced pHi to 6.6 without changing pHousing 90 mm bicarbonate as demonstrated previously (34Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar,50Zhu G.Y. Chanchevalap S. Liu C. Xu H. Jiang C. J. Cell. Physiol. 2000; 183: 53-64Crossref PubMed Scopus (38) Google Scholar). Such intracellular acidification for 10 min did not produce any significant increase in the GIRK1/GIRK4 currents (n = 6, data not shown). Thus, these results indicate that extracellular acidification augments the GIRK1/GIRK4 currents. Fig. 1 C shows concentration dependence of the GIRK/GIRK4 currents on the pHo levels. The currents were moderately stimulated at pHo 6.8 and inhibited at pHo 8.4. The maximal activation occurred at pHo 6.2, whereas a further drop in pHo to 5.2 did not have any additional effect, suggesting that the most sensitive pH range of the GIRK/GIRK4 channels is between pH 7.0 and 7.4. At the maximal activation with pHo 6.2, the amplitude of heteromeric GIRK/GIRK4 currents was enhanced by 33.4 ± 3.6% (n = 11). Similar channel activation was also observed in the tandem-dimeric GIRK1/GIRK4 channel whose amplitude increased by 33.9 ± 3.6% (n = 6). To understand how single channel properties and voltage dependence underlie the change in whole-cell currents, we performed single channel recordings in the outside-out patch configuration. The heteromeric GIRK/GIRK4 channels showed low base-line activity at pH 7.4 in the absence of exogenous G-protein. These currents had single channel conductance of 28.0 ± 0.4 picoSiemens (n = 6) (Fig.2 A). Exposure of external patch membranes to a perfusate of pH 6.2 enhanced the single channel conductance by 29.8 ± 2.9% (n = 6) (Fig.2 B). Unlike the single channel conductance, theP o did not show significant change with pHo 6.2 (p > 0.05, n = 9) (Fig. 2, C–F). In whole-cell recordings, the increase in GIRK1/GIRK4 currents at acidic pHo did not show any voltage dependence in a voltage range from −160 to 0 mV (Fig. 2,G–I). Thus, the increase in whole-cell currents at acidic pHo is likely to be produced by augmentation of the single channel conductance. To elucidate which subunit is responsible for proton sensing, homomeric expression of these GIRK channels was carried out. It is known that the homomeric GIRK1 and GIRK4 produce only small currents with or without Gβγ-subunits, whereas the small currents are probably derived from heteromeric channels formed by Xenopus endogenous GIRK5 with the exogenous GIRK subunits (35Vivaudou M. Chan K.W. Sui J.L. Jan L.Y. Reuveny E. Logothetis D.E. J. Biol. Chem. 1997; 272: 31553-31560Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 36Hedin K.E. Lim N.F. Clapham D.E. Neuron. 1996; 16: 423-429Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). However, previous studies have demonstrated that the mutation of a single amino acid residue in the pore-forming sequence (P) allows both GIRK1 and GIRK4 to be expressed homomerically (35Vivaudou M. Chan K.W. Sui J.L. Jan L.Y. Reuveny E. Logothetis D.E. J. Biol. Chem. 1997; 272: 31553-31560Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). This homomeric expression technique has been conducive to elucidating the modulation of GIRK channels by several modulators (11Zhang J. Kong C. Xie H. McPherson P.S. Grinstein S. Trimble W.S. Curr. Biol. 1999; 9: 1458-1467Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 35Vivaudou M. Chan K.W. Sui J.L. Jan L.Y. Reuveny E. Logothetis D.E. J. Biol. Chem. 1997; 272: 31553-31560Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 37Hill J.J. Peralta E.G. J. Biol. Chem. 2001; 276: 5505-5510Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 38Rogalski S.L. Chavkin C. J. Biol. Chem. 2001; 276: 14855-14860Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Therefore, we constructed the GIRK1 with Phe-137 mutated to serine (GIRK1-F137S) and GIRK4 with Ser-143 mutated to threonine (GIRK4-S143T) as shown previously by Vivaudou et al. (35Vivaudou M. Chan K.W. Sui J.L. Jan L.Y. Reuveny E. Logothetis D.E. J. Biol. Chem. 1997; 272: 31553-31560Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Consistent with their results, functional expressions of both these homomeric GIRK1 and GIRK4 channels were seen with the mutations (Fig. 3, A andB). When the channels were challenged with extracellular acidosis (pHo 6.2), the amplitude of the homomeric GIRK1 and GIRK4 currents increased by 21.0 ± 2.9% (n = 9) and 66.7 ± 6.2% (n = 6), respectively (Fig.3 C). These data indicated that both GIRK1 and GIRK4 possess proton-sensing mechanism with the one in GIRK4 more prominent. If these GIRK channels are inherently sensitive to protons, there should be specific protein domain or amino acid residue that is accessible to extracellular protons and responsible for the pHo-induced channel activation. To test this hypothesis, we performed site-directed mutagenesis on potentially titratable histidine residues, an amino acid with its side-chain pK of 6.04 most close to pHolevels for the channel activation. Therefore, we examined amino acid sequences from the first membrane-spanning helix (M1), the P-loop, to the second membrane-spanning sequence (M2) helix and found a histidine in the M1-P linker (Fig. 4 A). The sequence alignment using the BLAST 2 sequences shows that this histidine is conserved in all GIRK channels but not seen in any other Kir channels (Fig. 4 A). Thereafter, we site-specifically mutated this histidine to a neutral polar glutamine. We found that the histidine mutation totally abolished the pHo sensitivity in both homomeric GIRK1-F137S and GIRK4-S143T channels (Figs. 4,B and C, and5 A), indicating that the histidine residue is the proton sensor in these homomeric channels.Figure 5Effects of histidine mutations on homomeric and heteromeric GIRK channels. A, mutation of His-116 and His-120 abolished the pHo sensitivity of homomeric GIRK1 and GIRK4 channels, respectively. These mutant channels responded to pHo 6.2 similarly to Kir2.1 known as a pH-insensitive channel.I, current. B, in the heteromeric GIRK1/GIRK4 channels, mutations of any of these histidine residues alone reduced but not eliminated the pHo sensitivity. The heteromeric channels became pHo-insensitive only when both of these histidines were replaced with a non-titratable residue. Data are presented as the means ± S.E. (n = 4–9).View Large Image Figure ViewerDownload (PPT) Because the homomeric GIRK4-S143T is more sensitive to pHo than the GIRK1-F137S, it is possible that the His-120 in GIRK4 plays a more important role in the pHo sensitivity of the heteromeric GIRK1/GIRK4 channels. To test this hypothesis, we studied the heteromeric channels with the mutation of the histidine residue in GIRK1 and/or GIRK4. We found that heteromeric channels carrying any one of the histidines (GIRK1-H116Q/GIRK4, GIRK1/GIRK4-H120Q) remained pHo-sensitive, although their pHo sensitivity was significantly lower than the wild-type channels. Simultaneous mutations of the histidine in both GIRK1 and GIRK4 (GIRK1H116Q-GIRK4H120Q) completely eliminated the pHo sensitivity of the heteromeric channels (Figs. 5 B and 6). Thus, these results suggest that all four histidine residues in both GIRK1 and GIRK4 subunits are involved in proton sensing in their heteromeric channels. This is the first demonstration of the pHo sensitivity in the GIRK channels. We have found that the heteromeric GIRK1/GIRK4 are stimulated by extracellular acidification and inhibited by alkalization. A decrease in pHo to 6.2 enhances the GIRK1/GIRK4 currents by ∼30%, which has been observed in both GIRK1/GIRK4 coexpression and the tandem-dimeric channel. The increase in whole-cell currents is attributed to augmentation of the single-channel conductance without changes in P o and voltage dependence. A histidine residue in the extracellular domain is crucial, a mutation of which eliminates the pH sensitivity of the homomeric channels, whereas the pH sensitivity in the heteromeric GIRK1/GIRK4 channels requires this histidine in both subunits. The GIRK1/GIRK4 channels are activated by extracellular but not intracellular acidification for the following reasons. 1) In our previous studies, we have previously shown that a decrease in extracellular pH does not cause intracellular acidification using the same PIPES buffer (32Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (London). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 33Qu Z. Zhu G. Yang Z. Cui N. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). 2) Our data indicate that augmentation of these currents is associated with a change in pHo. 3) A decrease in pHi does not enhance the currents. 4) Similar current augmentation was seen in excised patched. Thus, the increase in current amplitude seen in this study should be produced by pHoindeed. Biophysical mechanisms underlying the change of whole-cell currents were examined in this study. Our data indicate that the increase in whole-cell currents is produced by selective augmentation of single-channel conductance without affecting the open-state probability and the voltage dependence. The channel activation is reversible and dependent on pH levels. The linear working range of the GIRK1/GIRK4 heteromeric channels is between pH 7.0 and 7.4, suggesting that the heteromeric GIRK1/GIRK4 channels can detect pH changes in most physiologic and pathophysiologic conditions. Several members of K+ channels in the Kir family are known to be pH-sensitive, such as Kir1.1, Kir1.2, Kir2.3, Kir2.4, Kir4.1, Kir6.1, Kir6.2, and heteromeric Kir4.1-Kir5.1 (34Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 39Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar, 40Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 41Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 273: F516-F529PubMed Google Scholar, 42Hughes B.A. Kumar G. Yuan Y. Swaminathan A. Yan D. Sharma A. Plumley L. Yang-Feng T.L. Swaroop A. Am. J. Physiol. 2000; 279: C771-C784Crossref PubMed Google Scholar, 43Pearson W.L. Dourado M. Schreiber M. Salkoff L. Nichols C.G. J. Physiol. (London). 1999; 514: 639-653Crossref Scopus (68) Google Scholar, 44Yang Z. Jiang C. J. Physiol. (London). 1999; 520: 921-927Crossref Scopus (47) Google Scholar, 45Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar). In comparison with other Kir channels, the GIRK1/GIRK4 heteromeric channels are characterized by 1) sensitivity to pHo, which make them similar only to the Kir2.3 (32Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (London). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 40Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar), and 2) stimulation by acidic pH, which renders them more like Kir6 channels (34Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In contrast, most of the Kir channels are inhibited by acidic pH with the proton sensors mostly located on the cytosolic side of the plasma membranes (39Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar, 40Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 41Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 273: F516-F529PubMed Google Scholar, 42Hughes B.A. Kumar G. Yuan Y. Swaminathan A. Yan D. Sharma A. Plumley L. Yang-Feng T.L. Swaroop A. Am. J. Physiol. 2000; 279: C771-C784Crossref PubMed Google Scholar, 43Pearson W.L. Dourado M. Schreiber M. Salkoff L. Nichols C.G. J. Physiol. (London). 1999; 514: 639-653Crossref Scopus (68) Google Scholar, 44Yang Z. Jiang C. J. Physiol. (London). 1999; 520: 921-927Crossref Scopus (47) Google Scholar, 45Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar). The proton detection in the GIRK1/GIRK4 channels depends on a histidine residue in the extracellular protein domain. The pHo sensitivity of GIRK1 and GIRK4 homomeric channels is lost when this histidine residue is mutated to a non-titratable amino acid. Thus, the pH sensing in these GIRK channels is similar to several other Kir channels as shown previously (46Chanchevalap S. Yang Z. Cui N. Qu Z. Zhu G. Liu C. Giwa L.R. Abdulkadir L. Jiang C. J. Biol. Chem. 2000; 275: 7811-7817Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 47Qu Z. Yang Z. Cui N. Zhu G. Liu C. Xu H. Chanchevalap S. Shen W. Wu J. Li Y. Jiang C. J. Biol. Chem. 2000; 275: 31573-31580Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 48Xu H. Wu J. Cui N. Abdulkadir L. Wang R. Mao J. Giwa L.R. Chanchevalap S. Jiang C. J. Biol. Chem. 2001; 276: 38690-38696Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). This histidine residue is conserved among all GIRK channels, suggesting that other GIRK subunits (i.e. GIRK2, GIRK3) are probably pH-sensitive as well. Because this histidine does not exist in other Kir channels, these results may explain why most of them, with the exception of Kir2.3 that is inhibited by both intracellular and extracellular acidifications and is also regulated by G-proteins (see Refs. 32Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (London). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 40Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar, and 49Cohen N.A. Sha Q. Makhina E.N. Lopatin A.N. Linder M.E. Snyder S.H. Nichols C.G. J. Biol. Chem. 1996; 271: 32301-32305Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), are insensitive to pHo. One interesting finding from our current studies is the graded pHo sensitivity of the heteromeric GIRK1/GIRK4 channels with the histidine mutation. We have found that mutation of the histidine in either GIRK1 or GIRK4 leads to a reduction but not elimination of the pHo sensitivity, whereas the heteromeric channel loses the pHo sensitivity only when both histidines in GIRK1 and GIRK4 are simultaneously mutated. Based on this observation, it is possible that the GIRK channel modulation by extracellular protons requires protonation of this histidine residue in all four subunits and perhaps movements of these extracellular protein domains as well. The pHo sensitivity of these GIRK channels has a profound impact on synaptic transmission. In the presynaptic terminals, neurotransmitter uptake relies on ATP-dependent proton pumps whose activity results in acidification inside synaptic vesicles up to 1.5 pH units lower than cytosolic pH (26Liu Y. Edwards R.H. Annu. Rev. Neurosci. 1997; 20: 125-156Crossref PubMed Scopus (236) Google Scholar). Following each action potential at presynaptic terminals, protons are released with neurotransmitters into the synaptic cleft. This has been shown to lead to significant extracellular acidification in the synaptic cleft (27Miesenbock G. De Angelis D.A. Rothman J.E. Nature. 1998; 394: 192-195Crossref PubMed Scopus (1901) Google Scholar). As the GIRK channels are major targets of the neurotransmitters on the postsynaptic membranes, the pHo sensitivity provides these channels with an important modulatory mechanism by which they are first activated by neurotransmitters, and subsequently, channel activity is further enhanced by protons released from presynaptic terminals. Such a modulatory mechanism appears to enhance greatly the synaptic efficiency, because according to our data, the neurotransmitter accounts for only 70% of the synaptic transmission and protons released together with the transmitter contribute to the other 30%. How does the pHo sensitivity affect specificity of synaptic transmission? Our results show that extracellular protons do not seem to affect GIRK channel gating, because they act on single channel conductance and augment the GIRK channels by 33% at most. Therefore, the synaptic cleft acidification should have very little effect on the GIRK channels before they are opened by specific neurotransmitters. Such a modulation instead of channel gating may prevent these channels from being activated by protons released with irrelevant neurotransmitters. This is remarkable, because protons released from presynaptic terminals may contribute significantly to the GIRK-mediated synaptic transmission without compromising synaptic specificity. Thus, the demonstration of the modulation of GIRK1/GIRK4 channels by extracellular acidification contributes valuable information to the understanding of these GIRK channels and their function in synaptic transmission. We thank Dr. Norman Davidson (California Institute of Technology, Pasadena, CA) for the GIRK1 and GIRK4 cDNA." @default.
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