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J. Biol. Chem., Vol. 277, Issue 48, 46166-46171, November 29, 2002

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Molecular Determinants for Activation of G-protein-coupled Inward Rectifier K⁺ (GIRK) Channels by Extracellular Acidosis^*

Jinzhe Mao, Lilly Li, Maurine McManus, Jianping Wu, Ningren Cui, and Chun Jiang

From the Department of Biology, Georgia State University, Atlanta, Georgia 30302-4010

Received for publication, June 1, 2002, and in revised form, October 1, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 (pH_o) on GIRK channels, we performed experiments on heteromeric GIRK1/GIRK4 channels expressed in Xenopus oocytes. A decrease in pH_o to 6.2 augmented GIRK1/GIRK4 currents by ~30%. The channel activation was reversible and dependent on pH_o 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 pH_o 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 pH_o sensitivity. The pH_o 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%.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The G-protein-coupled inward rectifier K⁺ (GIRK)¹ channels are important players in cellular communications in several excitable tissues (1-3). The GIRK channels are activated by -subunits of G-proteins, which are dissociated from the -trimer as a result of receptor binding to neurotransmitters or hormones (3). Four members of GIRK channels have been identified in mammals with GIRK1/GIRK4 expressed abundantly in the heart and brain (4).

GIRK channels are modulated by several intracellular signal molecules such as Na⁺, ATP, and phospholipids (5-12). Extracellular molecules including hormones, neurotransmitters, and integrins directly or indirectly modulate GIRK channel activity through signaling transduction pathways (13-18). GIRK channels are also the major targets of ethanol, anesthetics, and opioids (19-23). 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 (24). In the central nervous system, protons have been shown to modulate synaptic transmission, neuronal plasticity, and membrane excitability (25). It is known that the pH level in synaptic vesicles is ~1.5 pH units lower than in cytosol (26). These protons are released from synaptic vesicles together with neurotransmitters during synaptic transmission, leading to extracellular acidification in the synaptic cleft (27). If the GIRK channels are sensitive to extracellular pH (pH_o), 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 (28-31).

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 pH_o, 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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Experiments were performed as we described previously (32-34). 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 (GenBank^TM accession number U01071) and rat GIRK4 (Kir3.4) cDNA (GenBank^TM accession 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 for Xenopus 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 (33, 34). 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 (33, 34, 50).

pH_o and intracellular pH (pH_i) were measured using ion-selective microelectrodes as we detailed previously (34, 50). 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 pH_i and pH_o 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 (10¹⁵ ohms), 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 pH_o 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 pH_o measured using a H⁺-selective electrode (34, 45). The pH electrode was positioned in the extracellular solution near the cell. When the extracellular solution was switched to a low pH perfusate, the pH_o 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 (32). The open-state probability (P_o) was calculated as we described previously (32). The single channel conductance was measured in negative membrane potential using a ramp command potential (from 100 to 100 mV). To change pH_o, 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's t test was used. The differences of pH effects before versus during exposures were considered to be statistically significant if p  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Base-line Activity of Heteromeric GIRK1/GIRK4-- 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. 1A) 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).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Activation of heteromeric GIRK1/GIRK4 channels by extracellular acidification. The GIRK1 and GIRK4 cDNAs were cloned in a eukaryotic expression vector pcDNA3.1 and expressed in Xenopus oocytes. Whole-cell currents were studied in two-electrode voltage clamp using an extracellular solution containing 90 mM K+. A, inward rectifying currents were recorded from an oocyte 3 days after a coinjection of the GIRK1 and GIRK4 cDNAs. Exposure to a perfusate of pH 6.2 for 10 min increased the currents by 38%. The current activation was reversible as washout at pH 7.4 led to almost complete recovery. B, the current amplitude together with pHo was plotted against time. The pHo (gray line) started to change in ~0.5 min with acid exposure and reached a plateau in ~3 min. The current response curve (black circle) was almost the same as the pHo profile, although it showed ~0.5-min delay. Note that the data are obtained from A, and the currents are measured at 120 mV. C, concentration dependence of the heteromeric GIRK1/GIRK4 channels on pHo levels. The heteromeric GIRK1/GIRK4 channels were studied at different pHo levels as indicated in the x axis. The currents were inhibited at alkaline pH and stimulated at acidic pH levels with the maximal activation at pHo 6.2. I, current. Data are presented as the means ± S.E. (n = 4-11).

Response to Extracellular pH-- Exposure of the oocytes to a perfusate of pH 6.2 augmented the heteromeric GIRK/GIRK4 currents (Fig. 1A). 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. 1B). Washout with the pH 7.4 perfusate led to a clear recovery (Fig. 1A). When the current amplitude was plotted together with pH_o against time, the current response curve was almost identical to the pH_o profile with only an ~0.5-min delay (Fig. 1B). This effect was not produced by a change in pH_i, because the perfusate containing membrane-impermeable PIPES buffer does not change pH_i as shown in our previous studies (33, 34). To strengthen this argument, we selectively reduced pH_i to 6.6 without changing pH_o using 90 mM bicarbonate as demonstrated previously (34, 50). 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. 1C shows concentration dependence of the GIRK/GIRK4 currents on the pH_o levels. The currents were moderately stimulated at pH_o 6.8 and inhibited at pH_o 8.4. The maximal activation occurred at pH_o 6.2, whereas a further drop in pH_o 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 pH_o 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. 2A). 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. 2B). Unlike the single channel conductance, the P_o did not show significant change with pH_o 6.2 (p > 0.05, n = 9) (Fig. 2, C-F). In whole-cell recordings, the increase in GIRK1/GIRK4 currents at acidic pH_o 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 pH_o is likely to be produced by augmentation of the single channel conductance.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Effects of acidic pHo on single-channel properties and voltage dependence of heteromeric GIRK1/GIRK4 channels. A, single-channel currents were recorded from an outside-out patch using symmetric concentrations of K+ (140 mM) applied to both sides of the patch membrane. Two active channels are seen at pHo 7.4. The single channel conductance indicated by straight lines is 27 picoSiemens for both of these channels. B, at pHo 6.2, their single channel conductance becomes 37 picoSiemens. C-F, the channel Po was studied on a stretch of record of 20 s with a holding potential of 80 mV using the same solutions as A. The Po at pHo 6.2 (D) did not show significant difference from that at pHo 7.4 (C). Note that E and F are obtained from areas indicated by an arrow in C and D, respectively. Label C, closure; label 1, first opening; and label 2, second opening. G-I, the voltage dependence was studied in whole-cell recording. G, at base line (pHo 7.4), the heteromeric GIRK1/GIRK4 currents showed almost linear conductance at negative membrane potentials (Vm). H, the currents affected by low pH were isolated by subtracting base-line currents from those recorded during pHo 6.2. The isolated currents showed conductance similar to G at negative Vm. I, the I-V relationship of the isolated currents (triangle) is similar to that of the base-line currents (circle).

Effect of pH_o on Homomeric Channels-- 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 G-subunits, whereas the small currents are probably derived from heteromeric channels formed by Xenopus endogenous GIRK5 with the exogenous GIRK subunits (35, 36). 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 (35). This homomeric expression technique has been conducive to elucidating the modulation of GIRK channels by several modulators (11, 35, 37, 38). 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. (35). Consistent with their results, functional expressions of both these homomeric GIRK1 and GIRK4 channels were seen with the mutations (Fig. 3, A and B). When the channels were challenged with extracellular acidosis (pH_o 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. 3C). These data indicated that both GIRK1 and GIRK4 possess proton-sensing mechanism with the one in GIRK4 more prominent.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effects of acidic pHo on homomeric GIRK1 and GIRK4 channels. Homomeric expressions of GIRK1 and GIRK4 channels were achieved by F137S mutation in GIRK1 and S143T mutation in GIRK4. The GIRK1-F137S was moderately stimulated at pHo 6.2 (A), whereas the GIRK4-S143T currents were strongly augmented (B). C, summary of the pHo responses. I, current. Data are presented as the means ± S.E. (n = 6-8).

Identification of Potential Proton Sensors-- 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 pH_o-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 pH_o levels 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. 4A). 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. 4A). Thereafter, we site-specifically mutated this histidine to a neutral polar glutamine. We found that the histidine mutation totally abolished the pH_o sensitivity in both homomeric GIRK1-F137S and GIRK4-S143T channels (Figs. 4, B and C, and 5A), indicating that the histidine residue is the proton sensor in these homomeric channels.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Identification of proton sensors in the homomeric GIRK1 and GIRK4 channels. A, alignment of amino acid sequence of several inward rectifier K+ (Kir) channels in the extracellular domains and P-loop. A histidine residue is found in this region of GIRK1 and GIRK4. This histidine is conserved in all GIRK channels but absent in other Kir channels. B, mutation of the His-116 eliminated the pHo sensitivity of the homomeric GIRK1 channels. C, similarly, the His-120 mutation made the GIRK4 channels pHo-insensitive.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effects 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).

Because the homomeric GIRK4-S143T is more sensitive to pH_o than the GIRK1-F137S, it is possible that the His-120 in GIRK4 plays a more important role in the pH_o 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 pH_o-sensitive, although their pH_o sensitivity was significantly lower than the wild-type channels. Simultaneous mutations of the histidine in both GIRK1 and GIRK4 (GIRK1H116Q-GIRK4H120Q) completely eliminated the pH_o sensitivity of the heteromeric channels (Figs. 5B 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.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Elimination of the pHo sensitivity by mutations of the histidine residue in both GIRK1 and GIRK4. A, there was no change in the current amplitude in the GIRK1-H116Q/GIRK4-H120Q when pHo was reduced from 7.4 to 6.2. B, the GIRK1-H116Q/GIRK4-H120Q mutant channel did not respond to a range of pHo changes from 8.4 to 6.2. I, current. Data are presented as the means ± S.E. (n = 4-6).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This is the first demonstration of the pH_o 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 pH_o 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 (32, 33). 2) Our data indicate that augmentation of these currents is associated with a change in pH_o. 3) A decrease in pH_i 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 pH_o indeed. 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 (34, 39-45). In comparison with other Kir channels, the GIRK1/GIRK4 heteromeric channels are characterized by 1) sensitivity to pH_o, which make them similar only to the Kir2.3 (32, 40), and 2) stimulation by acidic pH, which renders them more like Kir6 channels (34). 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 (39-45).

The proton detection in the GIRK1/GIRK4 channels depends on a histidine residue in the extracellular protein domain. The pH_o 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 (46-48). 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. 32, 40, and 49), are insensitive to pH_o.

One interesting finding from our current studies is the graded pH_o 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 pH_o sensitivity, whereas the heteromeric channel loses the pH_o 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 pH_o 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 (26). 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 (27). As the GIRK channels are major targets of the neurotransmitters on the postsynaptic membranes, the pH_o 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 pH_o 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.

	ACKNOWLEDGEMENT

We thank Dr. Norman Davidson (California Institute of Technology, Pasadena, CA) for the GIRK1 and GIRK4 cDNA.

	FOOTNOTES

^* This work was supported by the National Institutes of Health Grant HL58410 and the American Diabetes Association Grant 1-01-RA-12.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.

Career Investigator of the American Lung Association. To whom correspondence should be addressed: Dept. of Biology, Georgia State University, 24 Peachtree Center Ave., Atlanta, Georgia 30302-4010. Tel.: 404-651-0913; Fax: 404-651-2509; E-mail: cjiang@gsu.edu.

Published, JBC Papers in Press, October 1, 2002, DOI 10.1074/jbc.M205438200

	ABBREVIATIONS

The abbreviations used are: GIRK, G-protein-coupled inward rectifier K⁺; pH_o, extracellular pH; pH_i, intracellular pH; PIPES, 1,4-piperazinediethanesulfonic acid; ANOVA, analysis of variance; P, pore.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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