©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Specificity of Coupling of Muscarinic Receptor Isoforms to a Novel Chick Inward-rectifying Acetylcholine-sensitive K Channel (*)

(Received for publication, December 19, 1995)

Albert P. Gadbut (1)(§) Daniela Riccardi (2)(¶) Leeying Wu (1) Steven C. Hebert (2) Jonas B. Galper (1)(**)

From the  (1)The Cardiovascular Division and (2)Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The G-protein-gated inward-rectifying K channel GIRK1 has been demonstrated in heart and brain. These tissues also both express the M(2), M(3), and M(4) muscarinic acetylcholine receptors (mAChR) (Gadbut, A. P., and Galper, J. B.(1994) J. Biol. Chem. 269, 25823-25829). Only the M(2) mAChR has been demonstrated to couple to GIRK1 (Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y.(1993) Nature 264, 802-806). In this study we determined the specificity of coupling of the M(3) and M(4) mAChR to a new GIRK1 cloned from a chick brain cDNA library. This clone codes for a 492-amino acid protein that is 93% identical to rat GIRK1 and is expressed in brain, atrium, and ventricle, but not skeletal muscle. In Xenopus laevis oocytes co-expression of GIRK1 with either the chick M(2) or M(4) mAChR gave carbamylcholine (10 µM)-stimulated K currents of 308 ± 26 nA and 298 ± 29 nA, respectively, which were both Ba- and pertussis toxin-sensitive. Activation of the M(3) receptor produced 2382 ± 478 nA of current which was insensitive to Ba and pertussis toxin, but was 85% inhibitable by the Cl channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (10-20 µM) consistent with coupling to an endogeneous Ca-activated Cl channel via a phosphatidylinositol-dependent mechanism. Co-expression of the cardiac inward rectifier CIR with chick M(2) or M(4) mAChR and GIRK1 increased currents more than 10-fold, but had no effect on specificity of coupling. These data demonstrate a new function for the M(4) mAChR and a high degree of specificity for coupling of each receptor subtype to GIRK1.


INTRODUCTION

Muscarinic acetylcholine receptors (mAChR) (^1)have been demonstrated to play an important role in both the heart and the central nervous system. The M(2) isoform of the mAChR has been shown to be coupled to an inward-rectifying K channel in the atrium that is responsible for mediating the negative chronotropic response of the heart (for review, see (1) and (2) ). The M(4) mAChR isoform is expressed at high levels in the basal ganglia(3, 4) , and more recently M(4) mRNA has been demonstrated in the heart(5, 6) . The physiological role of M(4) receptors in these tissues is not well understood. M(2) and M(4) isoforms of the muscarinic receptor expressed in Chinese hamster ovarian cells were both coupled by a pertussis toxin-sensitive G-protein to inhibition of adenylate cyclase activity and to a modest stimulation of phospholipase C activity. M(1) and M(3) isoforms of the muscarinic receptor expressed in Chinese hamster ovarian (CHO) cells were coupled via a pertussis toxin-insensitive G-protein, such as Galpha(q), to a robust stimulation of inositol trisphosphate production and were not coupled to inhibition of adenylate cyclase(7) . Whether the similarity in M(2) and M(4) coupling extends to stimulation of the inward-rectifying acetylcholine-stimulated K channel has not been determined.

Studies of inside-out patches from membranes of cultured chick and rat atrial cells have demonstrated that GTP-dependent release of beta from heterotrimeric G-proteins activates inward-rectifying K channels(8) . Recently the G-protein-coupled inward-rectifying K channel (GIRK1) has been cloned from rat and shown to be activated by acetylcholine in Xenopus laevis oocytes co-expressing the M(2) muscarinic receptor, alpha, beta(1), and (2)(9, 10) . Activation of this channel by acetylcholine has been mimicked by adding various combinations of different beta and subunits to inside-out patches of rat atrial myocytes(11) .

Recently Krapivinsky et al.(12) have demonstrated that antibodies to GIRK1 co-immunoprecipitated two proteins from atrial myocytes. One of the co-immunoprecipitating proteins was GIRK1, and the second protein, when purified and cloned, was determined to be a cardiac inward rectifier (CIR), which was strikingly similar to the rcK-1 reported by Ashford et al.(13) . Co-expression of CIR and GIRK1 in Xenopus oocytes resulted in an enhanced inward rectifier channel with properties more closely resembling the atrial acetylcholine-sensitive K channel. In the present study various muscarinic receptor isoforms are co-expressed in frog oocytes with a new GIRK1 from chick brain that is present in atrium and ventricle. Experiments demonstrate that both M(2) and M(4) mAChR are coupled via a similar mechanism to activation of GIRK1. The data suggest a high degree of specificity of a given mAChR isoform for activation of GIRK1 and the lack of an effect of co-expression of CIR on the specificity of coupling.


EXPERIMENTAL PROCEDURES

Cloning of Chick GIRK1

A cDNA library from chick brain 17 days in ovo constructed in the -Zap vector (Stratagene, a gift of K. Takeyasu), was screened at low stringency with the full-length cDNA coding for the rat G-protein-gated inward-rectifying K channel (GIRK1, a gift of L. Jan). The probe was labeled with [alpha-P]dCTP by the random primer method(14) . The hybridization and washing conditions were as described previously(5) ; nitrocellulose membranes were hybridized to the probe in 10% dextran sulfate, 40% formamide, 1 times SSC, 7 mM Tris, pH 7.6, 0.8% 100 times Denhardt's, and 1% salmon sperm DNA (2 mg/ml) for 24 h at 55 °C. The membranes were washed three times in 2 times SSC, 0.1% SDS for 5 min at 25 °C followed by three washes in 0.2 times SSC, 0.1% SDS for 15 min at 48 °C. From the primary screening 12 clones were isolated, and nine clones were plaque-purified. One clone was completely sequenced on both strands using a combination of subcloned restriction fragments and synthetic oligonucleotide primers, by the dideoxy chain termination method(15) . This clone contained an open reading frame for a full-length K channel protein.

RNase Protection Analysis

Total RNA was isolated from 17 day in ovo chick tissues using guanidinium CsCl centrifugation(16) . For the generation of chick GIRK1 RNase protection probes, a SalI-EcoRI fragment (nucleotides 993-1310) was subcloned into pBluscript II (Stratagene) and linearized with SalI. Using T3 RNA polymerase (Boehringer Mannheim) in the presence of [P]UTP, this linearized template generated a 307-nucleotide antisense riboprobe to an area 150 bases from the 3` end of coding region. RNase protection was performed as described(17) . Following hybridization of the probes to total RNA, the samples were treated with ribonuclease and analyzed by polyacrylamide gel electrophoresis on 6% gels containing urea followed by autoradiography for 24 h.

X. laevis Oocytes Culture and Electrophysiology

Preparation, handling, and injection of oocytes have been described previously(18) . In brief, defolliculated oocytes were injected with 50 nl of H(2)O (control) or with the following cRNAs: M(2) mAChR, 1.5-15 ng; M(3) mAChR, 0.5-1.5 ng; M(4) mAChR, 1.5-15 ng; G alpha subunit, 1-15 ng; G(q) alpha subunit, 1-15 ng generated from a cDNA cloned from a chick brain cDNA library (data not shown); chick GIRK1, 0.5-15 ng; and rat CIR (gift of D. Clapham), 1-2.5 ng. Oocytes were incubated 3-6 days at 18 °C in ND96 ``standard'' solution (96 mM NaCl, 2 mM KCl, 1 mM CaCl(2), 1 mM MgCl(2), 5 mM Hepes, pH 7.4-7.6), supplemented with gentamicin (50 µg/ml), and 2.5 mM sodium pyruvate. Where indicated, oocytes were incubated for 24-72 h with pertussis toxin (1 µg/ml) prior to experiment. Two-electrode voltage clamp was performed with a GeneClamp 500 amplifier (Axon Instruments, Inc., Foster City, CA) using 0.2-1 M microelectrodes filled with 3 M KCl. Each oocyte was placed in the chamber initially perfused with ND96 standard solution, pH 7.4-7.6, and kept at a holding potential of -80 mV. Oocytes were then perfused with a high potassium solution (HK) containing 96 mM KCl, 2 mM NaCl, 1 mM CaCl(2), 1 mM MgCl(2), 5 mM Hepes, pH 7.4.-7.5. Current-voltage (IV) characteristics were recorded using voltage ramps (from -120 to +40 mV) lasting 1 s. The ``ND96 without Ca'' solution had the following composition: 96 mM NaCl, 5 mM KCl, 0.5 mM MgCl(2), 5 mM Hepes, pH 7.4-7.5. The ``HK without Ca'' solution had the following composition: 96 mM KCl, 2 mM NaCl, 0.5 mM MgCl(2), 5 mM Hepes, pH 7.4-7.5. Carbamylcholine (10 µM) and Ba (1 mM), were added directly to the HK solution. The chloride channel blocker, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10-30 µM) was dissolved in dimethyl sulfoxide (Me(2)SO) and then added to the ND96 or HK solutions as indicated. The Me(2)SO in both control and NPPB treated cells was 0.1%. Where indicated, oocytes were incubated with 10 µM NPPB for 10 min prior to experiment.


RESULTS

Cloning of Chick GIRK1

A chick brain cDNA library was screened using full-length rat GIRK1 cDNA. Screening of 3.5 times 10^5 recombinant plaques with the full-length rat GIRK1 gave 12 positive plaques. Nine clones were purified and analyzed by restriction endonuclease mapping and nucleotide sequencing. One of those clones contained an open reading frame coding for a predicted 492-amino acid protein beginning at the first in frame ATG that was 93% identical to the rat GIRK1 (Fig. 1). The translational initiation codon is flanked by a consensus Kozak sequence (19) . This clone termed chick GIRK1 was 2.5 kilobases in length and contained 40 base pairs of 5`-untranslated region and 1 kilobase of 3`-untranslated region. GIRK1 belongs to a family of ion channels containing two putative transmembrane domains and an H5-like segment that is homologous to the pore-forming region of voltage- and ligand-gated channels(20) . Of the 38 amino acid substitutions in chick GIRK1 compared with rat 13 were in the amino terminus 5 were in the M(1)-H5-M(2) region (four of the five were in the M(1)-H5 linker segment), and 20 were in the carboxyl terminus. Excluding a conserved substitution of valine for isoleucine in the first predicted transmembrane-spanning region, the chick GIRK1 is identical to the rat peptide in the two transmembrane-spanning regions and the H5 region. In addition, the predicted amino terminus is 8 amino acids shorter than that in the rat.


Figure 1: Restriction endonuclease map and amino acid comparison of chick GIRK1. A, restriction map of the 2.5-kilobase pair cDNA coding for the chick GIRK1. Crossbars represent the coding region. B, amino acid comparison between the chick and rat GIRK1. The asterisk indicates amino acids that are identical to the chick GIRK1, and dots signify gaps introduced to align sequences. The predicted transmembrane domains and the H5 region are indicated by rectangular boxes.



Tissue Distribution of Chick GIRK1

Because chick GIRK1 mRNAs are expressed in low levels we used RNase protection to assess tissue distribution. The GIRK1 riboprobe protected a fragment of the predicted size from total RNA isolated from chick brain, atrium, ventricle, but not from skeletal muscle of embryos 17 days in ovo. Although chick GIRK1 mRNA was expressed in highest abundance in atrium, Fig. 2shows that chick GIRK1 mRNA is also expressed in ventricle at approximately the same level as in brain.


Figure 2: RNase protection analysis of the expression of chick GIRK1 in chick tissues. Total RNA from the brain, atrium, ventricle, and skeletal muscle of chick embryos 17 days in ovo were subjected to RNase protection analysis to yield a 307-base pair product. The full-length riboprobe undigested with ribonuclease is shown in the left-hand lane. The probe contained an additional 72 nucleotides of Bluescript II on the 5` end of the GIRK1 transcript, which was not protected. DNA sequencing ladders run as standards in each experiment (not shown) were used to determine fragment sizes indicated on the left of the panel. Antisense riboprobes were hybridized to 15 µg of total RNA or tRNA as control. Autoradiograms represent exposures of 24 h.



Specificity of Coupling of GIRK1 to Different Muscarinic Receptor Subtypes

To determine whether chick GIRK1 encodes a functional K channel it was co-expressed with the M(2) mAChR in X. laevis oocytes. Treatment of these cells with 10.0 µM carbamylcholine in HK solution evoked an inward-rectifying current of 308 ± 26 nA (Table 1) that was sensitive to Ba. Significant carbamylcholine-induced currents were not detected in either H(2)0 injected oocytes or in cells expressing M2 or GIRK1 alone. The carbamylcholine-stimulated current in HK buffer was inhibited 75% by pretreatment with 1 µg/ml pertussis toxin 308 ± 26 nA to 76 ± 26 nA, n = 16, p < 0.05 consistent with a muscarinic coupled K channel(8, 21) . The current-voltage relationship of the carbamylcholine-stimulated current in HK buffer was characteristic for flux through a strong inward-rectifying K channel with a zero slope at positive potentials and an intercept at 0 mV (Fig. 3C). Recently, Krapivinsky et al.(12) demonstrated that co-expression of an M(2) mAChR with a combination of inward-rectifying K channels, GIRK1 and CIR, resulted in acetylcholine-stimulated K currents that were 8-fold greater than in the absence of CIR. In the present study oocytes co-expressing the M(2) mAChR, chick GIRK1 and rat CIR gave a carbamylcholine-stimulated increase in Ba-sensitive current that was 15-fold higher than that in the absence of CIR (Table 1). This current was reduced by 94% from 4842 ± 548 nA to 290 ± 94nA (n = 8, p < 0.05) by pertussis toxin. The current-voltage relationships for muscarinic-stimulated currents measured in the oocytes co-expressing GIRK1 and CIR, although greater in magnitude, were identical in shape to those recorded in the absence of CIR (Fig. 3C). Overexpression of chick Galpha in these oocytes had no effect on either the magnitude or characteristics of the carbamylcholine-stimulated current, indicating that levels of endogenous G-proteins in these oocytes were not limiting in the functional coupling of M(2) to GIRK1.




Figure 3: Functional characterization of the coupling of different mAChR subtypes to GIRK1 ± CIR in X. laevis oocytes by two-electrode voltage clamp. Inward currents were evoked by 10 µM carbamylcholine in a 96 mM K-containing solution. A, B, D, E, G, H, and J represent individual oocytes injected with cRNAs coding for M(1) mAChR and GIRK1 (A); M(2) mAChR, GIRK1, and CIR (B); M(3) mAChR and GIRK1 (D); M(3) mAChR, GIRK1, and CIR (E); M(4) mAChR and GIRK1 (G); M(4) mAChR, GIRK1, and CIR (H); M(2) and GIRK1 (J). C, F, and I show IV relationships for carbamylcholine-stimulated currents in oocytes co-injected with M(2), mAChR, GIRK1 ± CIR (C); M(3), GIRK(1) ± CIR (F); M(4) mAChR, GIRK1 ± CIR (I); GIRK1 alone (); GIRK1 and CIR (bullet). indicates the addition of carbamylcholine (10 µM); indicates the addition of 1.0 mM Ba. As shown in E, we occasionally observed a small Ba-sensitive current in oocytes injected with M(3), GIRK1, and CIR cRNA. This Ba-sensitive current was observed with or without co-expression of CIR.



Although M(3) mAChR have been shown to activate different second messenger systems than M(2) and M(4) mAChR (7) , stimulation of M(3) receptors does result in release of beta subunits from heterotrimeric G-proteins, which could stimulate GIRK1 in the absence of the M(2) receptor. Furthermore, since chick GIRK1 has a significantly different amino terminus than rat, this could effect the specificity of coupling to muscarinic receptors. Phosphatidylinositol-coupled receptors like M(3) have been shown to produce agonist-dependent stimulation of Ca-activated Cl channels in Xenopus oocytes(23) . We used this system to assess whether the M(3) mAChR might couple to the chick GIRK1 isoform and/or activate the endogeneous Cl channel. In oocytes expressing only the M(3) mAChR, carbamylcholine (10.0 µM) stimulated large rapidly activating and inactivating inward currents of 3417 ± 1500 nA. In oocytes expressing both M(3) and GIRK1 carbamylcholine stimulated a similar large rapidly activating and inactivating inward current of 2382 ± 478 nA (n = 18) (Fig. 3D and Table 1), which was not significantly different from that with M(3) alone. Both these currents were Ba- and pertussis toxin-insensitive. This distinctive current profile, the sensitivity of the current to inhibition by NPPB, a Cl channel blocker (from 2,382 ± 478 nA to 364 ± 100 nA, n = 5, p < 0.05), together with the nonrectifying nature of the IV curve (Fig. 3F), provided strong evidence that M(3) AChR coupled only to the endogeneous Cl channel rather than a K channel(23) . To determine whether the lack of carbamylcholine stimulation of GIRK1 in oocytes expressing M(3) was due to the inability of endogenous G-proteins to couple M(3) to GIRK1, we assessed the effect of overexpressing chick Galpha on the coupling of the M(3) receptor to GIRK1. Even with the overexpression of chick Galpha, carbamylcholine did not stimulate M(3) mAChR-sensitive inward-rectifying K currents. Furthermore, co-expression of M(3) mAChR and GIRK1 with Galpha(q) had no effect on either the magnitude or characteristics of these carbamylcholine-stimulated Cl currents.

To determine whether the M(4) mAChR coupled to GIRK1, M(4) and GIRK1 were co-expressed in X. laevis oocytes. Carbamylcholine (10 µM) evoked Ba-sensitive currents of 298 ± 29 nA (Table 1) that were decreased by 99% to 4 ± 4 nA (n = 5, p < 0.05) in response to pretreatment with pertussis toxin. The magnitude of this current was not significantly different from that observed in oocytes expressing M(2) and GIRK1 (Table 1). In cells expressing the M(4) receptor alone, no significant current could be detected in response to carbamylcholine. The current-voltage relationship of carbamylcholine stimulation of oocytes co-expressing GIRK1-M(4) mAChR demonstrated a strong inward rectification (Fig. 3I) characteristic of GIRK1 currents.

As seen with M(2) mAChR, carbamylcholine stimulation of oocytes co-expressing CIR, M(4) mAChR, and GIRK1 was 13-fold higher than in the absence of CIR ( Table 1and Fig. 3H) and had no effect on the shape of the current-voltage relationship (Fig. 3I). Pertussis toxin reduced the carbamylcholine-stimulated current in oocytes expressing CIR and GIRK1 by 75% from 4010 ± 803 nA to 544 ± 170 nA (n = 9, p < 0.05). Furthermore the magnitude of the carbamylcholine-stimulated current was not significantly different from that stimulated in oocytes expressing the M(2) mAChR, CIR, and GIRK1 (Table 1). As in the case of the M(2) mAChR, overexpression of Galpha with M(4) and GIRK1 with or without CIR had no effect on the magnitude or characteristics of the carbamylcholine-stimulated currents.

Others have demonstrated the ability of M(2) mAChR to activate Ca-stimulated Cl currents in oocytes(22) . We observed in some, but not all oocytes co-expressing M(2) or M(4) mAChR with GIRK1, small oscillatory spike-like currents superimposed on the carbamylcholine-stimulated K currents (Fig. 3J). It has been suggested that these spike-like currents represent the weak activation of endogenous oocyte Cl channels(23, 24) .

In order to determine whether expression of chick Galpha(q) might enhance PI coupling of M(2) or M(4) to these Ca-activated Cl channels, we assessed the characteristics of carbamylcholine-stimulated currents in oocytes expressing chick Galpha(q). In oocytes expressing chick Galpha(q) with either M(2) or M(4) mAChR and GIRK1, we observed no enhancement of coupling to the Ca-activated Cl currents.


DISCUSSION

These data demonstrate that we have cloned a G-protein-gated inward-rectifying K channel cDNA from chick with a high degree of identity to the rat GIRK1. The mRNA coding for this channel is expressed in brain, atrium, and at surprisingly high levels in ventricle. The channel is coupled to M(2) and M(4) isoforms of the muscarinic receptor via a pertussis toxin-sensitive G-protein, but not to the M(3) receptor. When M(2) and M(4) receptors and GIRK1 are co-expressed with CIR in oocytes, the carbamylcholine-stimulated current increased markedly without affecting the specificity of receptor-effector coupling.

Recently using COOH-terminal deletion mutants of rat GIRK1, Reuveny et al.(25) found that the COOH terminus was involved in activation by beta. Truncation of GIRK1 at Leu-403 or Pro-355 resulted in the loss of response in oocytes expressing Gbeta(1) and G(2). Takao et al.(26) made chimeras of GIRK1 and the inward-rectifying K channel (IRK1) in which the COOH-terminal segment of IRK1 (residues 261-428) and GIRK1 (residues 339-401) were exchanged. Only the chimera expressing the carboxyl terminus of GIRK1 was responsive to muscarinic stimulation. Studies with inside-out patches from rat atrial myocytes suggested that six different combinations of recombinant beta subunits were capable of activating the inwardly rectifying potassium channel(11) . The present data suggest that differences in amino acid sequences between chick and rat GIRK1 had no effect on the characteristics of carbamylcholine-M(2) mAChR-stimulated currents (20) or the ability of CIR to interact with GIRK1(12) , leading to an increase in carbamylcholine induced K currents.

The novel finding in the present study is that the M(4) muscarinic receptor is coupled to GIRK1. This demonstrates that M(2) and M(4) are coupled to similar effectors in all cases so far examined. This finding, coupled with the previous observation that M(4) mRNA is found in high levels in the brain (5, 27) and that M(4) receptor protein is expressed throughout the brain, but at high levels in the basal ganglia(3, 4) , suggest that in brain muscarinic agonist activation of M(4) may function at least in part through stimulation of this inward-rectifying K channel.

Although beta subunits have been implicated in the activation of a number of effector systems, including beta-adrenergic receptor kinase, phospholipase C, certain isoforms of adenylate cyclase, and mitogen-activated protein kinase (for review, see (28) ), factors that determine the specificity of beta released following agonist receptor binding for activation of a specific effector system are not well understood. Although no direct studies of the role of beta in activating the Cl channel or GIRK1 are presented here, experiments described here do demonstrate that three different isoforms of the muscarinic receptor all of which are known to mediate the release of beta following agonist binding have a very high degree of specificity for activation of either GIRK1 or the Ca-stimulated Cl channel in Xenopus oocytes. Furthermore, expression of chick Galpha with the M(3) receptor had no effect on coupling of M(3) to GIRK1, and expression of chick Galpha(q) with the M(2) receptor had no effect on the low level of coupling of M(2) mAChR to Cl channel activation. Although these studies do not rule out the possibility that levels of beta may be limiting in the oocyte, the large size of the M(3)-activated Cl currents should be sufficient to affect activation of GIRK1 if such coupling existed. Since in vitro studies suggest that various beta subunits can activate GIRK1, it seems unlikely that the specific beta subunits expressed in oocytes can account for the lack of coupling of M(3) mAChR to GIRK1. Other factors such as compartmentalization of beta released in response to receptor activation or precoupling of receptor G-protein and effectors may account for the remarkable specificity of receptor GIRK1 coupling.


FOOTNOTES

*
This work was supported in part by grants from the National Institutes of Health, NHLBI Grant HL36014 (to J. B. G.) and NIDDK Grants DK48330 and DK37605, as well as a grant from NPS Pharmaceutical Inc. (to S. C. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L35555[GenBank].

§
Recipient of a National Research Service Award HL08463 from The National Institutes of Health.

Recipient of a National Kidney Foundation Fellowship.

**
To whom correspondence should be addressed: Dept. of Medicine, Cardiovascular Division, Brigham and Women's Hospital and Harvard medical School, Thorn 1109, 75 Francis St., Boston MA 02115. Tel.: 617-732-5679; Fax: 617-732-5132.

(^1)
The abbreviations used are: mAChR, muscarinic acetylcholine receptor(s); CIR, cardiac inward rectifier; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid.


ACKNOWLEDGEMENTS

We thank D. Clapham for the kind gift of CIR, Y. N. Jan for rat GIRK1, K. Takeyasu for the cDNA library, and M. Goutas for typing the manuscript.


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