The Stoichiometry of Gbeta gamma Binding to G-protein-regulated Inwardly Rectifying K+ Channels (GIRKs)*

Shawn CoreyDagger and David E. Clapham§

From the Dagger  Neuroscience Program, Mayo Foundation, Rochester, Minnesota 55905 and the § Howard Hughes Medical Institute, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115

Received for publication, January 3, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

G-protein-coupled inwardly rectifying K+ (GIRK; Kir3.x) channels are the primary effectors of numerous G-protein-coupled receptors. GIRK channels decrease cellular excitability by hyperpolarizing the membrane potential in cardiac cells, neurons, and secretory cells. Although direct regulation of GIRKs by the heterotrimeric G-protein subunit Gbeta gamma has been extensively studied, little is known about the number of Gbeta gamma binding sites per channel. Here we demonstrate that purified GIRK (Kir 3.x) tetramers can be chemically cross-linked to exogenously purified Gbeta gamma subunits. The observed laddering pattern of Gbeta gamma attachment to GIRK4 homotetramers was consistent with the binding of one, two, three, or four Gbeta gamma molecules per channel tetramer. The fraction of channels chemically cross-linked to four Gbeta gamma molecules increased with increasing Gbeta gamma concentrations and approached saturation. These results suggest that GIRK tetrameric channels have four Gbeta gamma binding sites. Thus, GIRK (Kir 3.x) channels, like the distantly related cyclic nucleotide-gated channels, are tetramers and exhibit a 1:1 subunit/ligand binding stoichiometry.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Roughly 2% of the human genome encodes G-protein-coupled receptors (1). Agonist binding to these G-protein-coupled receptors catalyzes the activation of Galpha and Gbeta gamma subunits of heterotrimeric G-proteins. The free Galpha and Gbeta gamma subunits can then interact independently or in concert with numerous effectors. Gbeta gamma regulates processes as diverse as the yeast pheromone response (2-4) and mammalian heart rate (5). The increasing list of Gbeta gamma effectors includes ion channels (6-12), phospholipase C beta  (13), adenylyl cyclases (14), G-protein-coupled receptor kinases (15), PI1 3-kinase (16), plasma membrane Ca2+ pumps (17), Bruton's tyrosine kinase (18), and calmodulin (19). Little is known about how Gbeta gamma interacts with its effectors. The repeating WD40 motif of Gbeta gamma gives it a rigid propeller-like structure, which does not appear to be altered upon its interaction with effectors (20-23).

Homotetrameric and heterotetrameric combinations of the four known mammalian GIRK subunits are activated by neurotransmitters in the nervous system, pancreas, and heart. Muscarinic (m2, m4), gamma -aminobutyric acid (GABAB), D2-dopamine, alpha 2-adrenergic, opiate, somatostatin, and adenosine all employ the Galpha i-Gbeta gamma signal transduction system to activate GIRK channels via direct Gbeta gamma binding to the tetrameric channel. GIRK4-knockout mice have irregularities in heart rate variability (5) and difficulties with spatial learning (24). GIRK2-knockout mice are prone to seizures (25). Weaver mice have a mutation in the pore domain of the GIRK2 subunit (26) that renders the channel nonselective (27) and results in the degeneration of cerebellar granule cells (28) and the dopaminergic neurons of the substantia nigra (29, 30).

The native atrial IKACh channel is composed of two GIRK1 subunits and two GIRK4 subunits (31-33) that comprise a channel that mediates neuronal regulation of heart rate. Biochemical studies indicate that Gbeta gamma binds the native IKACh complex with a Kd of 55 nM (9). Gbeta gamma binds both recombinant GIRK1 (Kd = 125 nM) and GIRK4 (Kd = 50 nM) (9). GIRK1 subunits are unable to form functional homomultimers (34), whereas GIRK4 homomultimers have been biochemically isolated from bovine atria (35). GIRK2/3 and GIRK1/2 heteromultimers have also been isolated from brain (1, 36). The C-terminal tail of GIRK1 and GIRK4 subunits bind Gbeta gamma (9, 32, and 37-46), but the detailed steps of how this binding leads to channel gating is not known. Furthermore, there is limited data about the areas of Gbeta gamma that bind GIRK channel subunits (43, 47) and about how many Gbeta gamma subunits can bind the tetrameric channel complex.

We have used a biochemical approach to determine how many Gbeta gamma subunits bind GIRK tetramers. By extending our previous chemical cross-linking studies (31), which indicated that GIRKs form tetramers, we demonstrate that GIRK4 homotetramers bind four Gbeta gamma subunits in their natural membrane environment.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation, Solubilization, and Purification of GIRK1/GIRK4 Heteromultimers (IKACh) from Native Atrial Membranes-- Bovine atrial plasma membranes were isolated (48) and solubilized as described (31). Native GIRK1/GIRK4 heteromultimers (IKACh) were purified to greater than 90% homogeneity as described (31). The protease inhibitors leupeptin (50 µg/ml Sigma-Aldrich Inc.), phenylmethylsulfonyl fluoride (100 µg/ml, Sigma-Aldrich Inc.), aprotinin (1 µg/ml, Sigma-Aldrich Inc.), and pepstatin (2 µg/ml, Sigma-Aldrich Inc.) were used during all steps of the purification.

Expression and Isolation of GIRKs from COS7 and CHO Cells-- Plasma membrane proteins containing GIRK1-AU5 and GIRK4-AU1 were isolated from COS7 cells and solubilized as described (49).

Gbeta gamma Purification-- G-proteins were isolated from bovine brain and separated into Galpha and Gbeta gamma subunits (50) and were further purified by affinity chromatography using immobilized Galpha (51).

Gbeta gamma Binding in Membranes-- Isolated COS7 cells or native atrial membranes were treated for 1 h with 100 mM dithiothreitol and then dialyzed against 20-50 mM HEPES, 100 mM NaCl, pH 7.4-7.5 (Gbeta gamma binding buffer). Individual membrane aliquots were preincubated with purified bovine brain Gbeta gamma and rotated for 20 min at room temperature prior to cross-linking. The Gbeta gamma stock solution was in Gbeta gamma binding buffer containing 0.1% CHAPS (0.1% Gbeta gamma binding buffer). The final CHAPS concentration was less than or equal to 0.1%.

Gbeta gamma Binding to Solubilized Protein-- Solubilized COS7 membrane proteins were treated for 1 h with 100 mM dithiothreitol and then dialyzed against 0.1% Gbeta gamma binding buffer. Individual aliquots were preincubated with purified brain Gbeta gamma (supplied in 0.1% Gbeta gamma binding buffer) and rotated for 20 min at room temperature prior to cross-linking.

Chemical Cross-linking-- 5 mM dithiobis[sulfosuccinimidylpropionate] (DTSSP, Pierce Chemical, Rockford, IL) was prepared as an 11× stock solution immediately prior to use in 100 mM HEPES-containing buffer, pH 7.5. Iodine was added only to solutions containing purified IKACh. Cross-linking reactions were allowed to proceed for 30 min at room temperature and then quenched with 50 mM Tris. Typically 5-10 µg of membrane proteins were used per reaction in a final volume of 15 µl.

SDS-PAGE and Immunoblotting-- Atrial membrane proteins or recombinant GIRK proteins were resuspended in Laemmli sample buffer containing 100 mM dithiothreitol (or 30 mM iodoacetamide when a cross-linking agent was used) for 15 min at 50 °C, 30 min at room temperature, and 15 min at 50 °C. 3-10% separating, 3% stacking, and pre-cast 2-15% (ISS) gels were utilized. Samples were analyzed by immunoblotting with anti-GIRK4 antibodies (generated against amino acids 19-32, Ref. 31) and/or anti-GIRK1 antibodies (generated against the last 156 amino acids of GIRK1, Ref. 31). Several antibodies were tested for use in the anti-Gbeta gamma immunoblotting experiments. Only one anti-Gbeta gamma antibody (anti-KTREGNVRVSREL, Chemicon International, Inc. Temecula, CA) reacted with Gbeta gamma after DTSSP treatment. Typically, DTSSP treatment reduced total antigenic signal by >90%, >60%, and >95% for anti-GIRK4, anti-GIRK1, and anti-Gbeta gamma antibodies, respectively. Transfer times for immunoblot analysis were extended to >2 h at 15 V to improve transfer of the high molecular weight complexes. A GS-700 imaging densitometer (Bio-Rad Laboratories, Hercules, California) was used to analyze the protein gels and immunoblots. Molecular masses were calculated using densitometry profiles from a combination of prestained high molecular mass markers (Bio-Rad) and low and high molecular mass markers (Amersham Pharmacia Biotech). In a portion of the gels, thyroglobulin (Amersham Pharmacia Biotech) was added to ensure linearity up to 330 kDa.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous chemical cross-linking studies demonstrated that GIRK subunits form tetrameric channels and that the native atrial channel IKACh is composed of 2 GIRK1 and 2 GIRK4 subunits (31). Complete cross-linking of purified atrial IKACh formed a single adduct with a total molecular mass that was most consistent with a tetramer. In addition, partial cross-linking of purified IKACh produced subsets of molecular weight adducts consistent with monomers, dimers, trimers, and tetramers. In this study, we extended our previous experiments to determine how many Gbeta gamma molecules can be cross-linked to GIRK tetramers.

Gbeta gamma Cross-linking to Purified Native IKACh-- To test whether GIRK1/GIRK4 heteromultimers could be directly and specifically cross-linked to Gbeta gamma , we used isolated native atrial GIRK1 and GIRK4 subunits (31) and bovine brain Gbeta gamma (9). Isolated GIRK1 and GIRK4 heterotetramers were preincubated with isolated Gbeta gamma , followed by cross-linking with DTSSP (Fig. 1). Although the predicted molecular masses of GIRK1 and GIRK4 subunits are 56 and 45 kDa, respectively, the glycosylated GIRK1 migrates in a broad band between 67-72 kDa (9). In the absence of Gbeta gamma , a band formed at 230 kDa, consistent with the total molecular mass of two GIRK1 (56, 67-72 kDa) and two GIRK4 (45 kDa) subunits. In the presence of Gbeta gamma , a band corresponding to a molecular mass of 390 kDa was detected. Because Gbeta gamma , GIRK1 and GIRK4 were the predominant proteins present, we interpreted the 160-kDa shift as the result of direct cross-linking of Gbeta gamma to GIRK channels. The molecular mass of Gbeta gamma is 42 kDa, suggesting that the 160-kDa shift was because of cross-linking of several Gbeta gamma molecules to the GIRK1/GIRK4 heterotetramers. Because GIRK4 can form homotetramers, we repeated the previous cross-linking experiment using recombinant GIRK4 subunits. In the absence of Gbeta gamma , cross-linking of recombinant GIRK4 resulted in a band at 170 kDa, corresponding to GIRK4 homotetramers. When Gbeta gamma was added to recombinant GIRK4, cross-linking yielded a band at ~320 kDa (not shown). This banding pattern is most consistent with four specific and saturable Gbeta gamma binding sites per GIRK4 homotetramer.



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Fig. 1.   Purified native atrial GIRK1 and GIRK4 heterotetramers can be directly and specifically cross-linked to purified Gbeta gamma . A, schematic depicting procedure used to generate B. Squares represent GIRK subunits. B, 2-15% SDS-PAGE and anti-GIRK1 immunoblotting of solubilized GIRK1 and GIRK4 heterotetramers. Lane 1, pure solubilized native atrial GIRK1 and GIRK4 heterotetramers treated with 1 mM DTSSP. Lane 2, pure solubilized GIRK1 and GIRK4 heterotetramers preincubated with 3 µM Gbeta gamma and treated with 1 mM DTSSP. Exposure times for lanes 1 and 2 were not identical.

Cross-linking of Membrane-confined GIRK4 Homotetramers-- It is important to study GIRK-Gbeta gamma binding in its membrane environment because phosphatidylinositol bisphosphate (PIP2) (52, 53) is involved in the Gbeta gamma -mediated activation of GIRK channels. Our previous GIRK cross-linking studies employed isolated solubilized channels (31). In this study, we tested whether GIRK subunits could be cross-linked into tetramers in membranes. After DTSSP cross-linking of membranes from COS7 cells expressing either recombinant GIRK4, GIRK1, or GIRK1 and GIRK4, SDS-PAGE yielded 180-220-kDa bands (Fig. 2B, lanes 1-3). These bands are similar in molecular mass to those produced when solubilized GIRKs are cross-linked into tetramers (31). Of the GIRK tetramers, the chemically cross-linked GIRK4 homotetramers produced the narrowest band, around 190 kDa (Fig. 2B, lane 1). In addition, the GIRK4 band cross-linked directly in membranes (Fig. 2B, lane 1) was narrower than that of GIRK4 that had been solubilized before cross-linking (31).



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Fig. 2.   Membrane-confined GIRK channels can be completely chemically cross-linked into tetramers. A, schematic depicting procedure used to generate B. B, CHO membrane proteins were cross-linked with DTSSP and analyzed by 2-15% SDS-PAGE and immunoblotting. Lanes 1-3, membranes from CHO cells transfected with the indicated GIRK subunit and cross-linked with 2.5 mM DTSSP.

Partial Gbeta gamma Cross-linking to Membrane-confined GIRK Tetramers-- GIRK4 homotetramers were used in our membrane-confined GIRK-Gbeta gamma binding experiments because cross-linking of GIRK4 homotetramers in membranes yielded the narrowest bands. We altered our cross-linking conditions to verify that there were indeed four Gbeta gamma binding sites in the GIRK tetramer. DTSSP and Gbeta gamma concentrations were adjusted so that variable numbers of Gbeta gamma molecules were cross-linked to the GIRK4 homotetramers. COS7 cells were transiently transfected with GIRK4, and their membranes were divided into separate aliquots. Each aliquot was treated with variable amounts of Gbeta gamma and DTSSP and then analyzed by SDS-PAGE and immunoblotting. Untreated GIRK4, migrated as a 47-kDa trichloroacetic acid-disruptable monomer (Fig. 3B, lane 1). GIRK4 cross-linked with DTSSP migrated as a 170-kDa tetramer (Fig. 3B, lane 2). GIRK4, preincubated with Gbeta gamma and then cross-linked with DTSSP, resulted in a laddering pattern of four main adducts (in addition to the GIRK4 homotetramer adduct) with consistent 40-45-kDa increments between bands (Fig. 3B, lanes 3 and 4). The proportion of high molecular weight adducts increased with increasing Gbeta gamma concentrations. Unlike our previous experiments that used solubilized GIRK protein, a population of the membrane-confined GIRK4 homotetramers (166 kDa) remained resistant to any Gbeta gamma binding. One possible explanation for this observation is that a subpopulation of GIRK4 homotetramers may not have been accessible to the exogenously applied Gbeta gamma . In five independent trials, four GIRK-Gbeta gamma adducts consistently appeared. In some trials, high molecular mass, lower intensity smears formed, but these bands were not consistently reproducible. A laddering pattern was not formed when Gbeta gamma was boiled prior to its addition to membranes (data not shown). We hypothesize that the five adducts formed by treatment of Gbeta gamma and GIRK4-containing solutions with DTSSP represent the binding of zero, one, two, three, and four Gbeta gamma molecules to GIRK4 homotetramers.



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Fig. 3.   DTSSP treatment of membrane-confined GIRK4 homotetramers in the presence of Gbeta gamma creates a laddering pattern consistent with four Gbeta gamma sites per GIRK tetramer. 3-10% SDS-PAGE analysis, anti-GIRK4 immunoblotting and cross-linking of membranes from COS7 cells that were transfected with GIRK4. A, schematic depicting procedure used to generate B. B, lane 1, GIRK4-containing membranes that were trichloroacetic acid-precipitated prior to SDS-PAGE. Lane 2, GIRK4-containing membranes cross-linked with 5 mM DTSSP. Lanes 3 and 4, GIRK4-containing membranes pretreated with 0.13 µM and 0.63 µM Gbeta gamma , respectively, followed by cross-linking with 5 mM DTSSP. Squares represent GIRK subunits. Circles represent Gbeta gamma molecules. The molecular mass markers at left correspond to panel 1. The molecular mass markers on the right correspond to adducts in panel 2. C, GIRK4-containing membranes pretreated with 3.1 µM Gbeta gamma and then cross-linked with 5 mM DTSSP, enlarged view. D, in a separate trial, densitometry profiles were created by scanning each lane of the gel top to bottom, which is now represented as top to bottom on the graph. Profile 1, GIRK4-containing membranes cross-linked with 5 mM DTSSP (0.0 µM Gbeta gamma ). Profile 2, GIRK4-containing membranes preincubated with 0.6 µM Gbeta gamma and cross-linked with DTSSP (0.6 µM Gbeta gamma ). Profile 3, GIRK4-containing membranes preincubated with 3.1 µM Gbeta gamma and cross-linked with 5 mM DTSSP (3.1 µM Gbeta gamma ).

Multiple lines of evidence suggest that Gbeta gamma is directly cross-linked to GIRK channels in our experiments. Gbeta gamma has been coimmunoprecipitated with GIRK subunits under the conditions used in our experiments (9). The ~45-kDa increments between cross-linked GIRK-Gbeta gamma adducts are consistent with the stepwise addition of 42-kDa Gbeta gamma subunits to the channel. Finally, similar results were obtained even when IKACh and Gbeta gamma were purified to >95% homogeneity prior to cross-linking. Because, IKAch and Gbeta gamma are the predominant proteins in solution, the molecular mass shift with Gbeta gamma addition strongly suggests that Gbeta gamma is being directly cross-linked to the channel. As a final precaution, we tested whether the putative GIRK-Gbeta gamma adducts are recognized by anti-Gbeta gamma antibodies. COS7 cells were transiently transfected with GIRK4 and their membranes were isolated. The membranes were treated with Gbeta gamma and DTSSP, followed by SDS-PAGE analysis. Immunoblots were probed with anti-GIRK4 antibodies then stripped and reprobed with anti-Gbeta gamma antibodies (Fig. 4, lanes 1 and 2, respectively). The anti-Gbeta gamma antibodies recognized bands at molecular masses that correspond to the putative GIRK-Gbeta gamma adducts.



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Fig. 4.   Anti-Gbeta gamma antibodies recognize bands at molecular masses that correspond to the putative GIRK-Gbeta gamma adducts. 3-10% SDS-PAGE analysis and immunoblotting of membranes from COS7 cells that were transfected with GIRK4, pretreated with Gbeta gamma , and then treated with DTSSP. Lane 1, GIRK4-containing membranes pretreated with 6.3 µM Gbeta gamma and cross-linked with 4 mM DTSSP probed with anti-GIRK4 antibodies. Lane 2, (lane 1) stripped and reprobed with anti-Gbeta gamma antibodies.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study of Gbeta gamma binding to channel proteins has several advantages over other approaches. First, we ensured that we were using intact tetramers throughout our Gbeta gamma binding experiments. In addition, we purposely studied Gbeta gamma binding in membranes to approximate physiological conditions. This is especially important because PIP2, a component of the cell membrane, plays a role in Gbeta gamma -mediated activation of GIRKs (52, 53). Nonprenylated Gbeta gamma mutants do not activate GIRK channels (54, 55), indicating that Gbeta gamma association with cell membranes may be a prerequisite for Gbeta gamma binding. We have paid careful attention to detergent concentrations, because low detergent conditions can potentially expose hydrophobic patches on GIRKs, producing nonspecific binding. Indeed, we found it difficult to prevent GIRK and Gbeta gamma aggregation in low detergent concentrations.

The stoichiometry of the IKACh-Gbeta gamma interaction has been repeatedly estimated by using the Hill equation to fit the Gbeta gamma -IKACh dose-response curve. Estimates of the Hill coefficient for IKACh activation varied from 1.5 (9) to 3 (56, 57) whereas it was ~1 in the study of the direct binding of purified IKACh and Gbeta gamma proteins (9). Although often used to infer binding stoichiometry of Gbeta gamma with GIRK subunits, the Hill coefficient is a measure of cooperativity, not the number of binding sites. For the Hill coefficient to equal the Gbeta gamma binding stoichiometry, two criteria need to be met or approximated. The Gbeta gamma molecules must bind the channel simultaneously and Gbeta gamma must bind with infinite cooperativity (58). In addition, the Hill equation does not take into account the increasing open probability of the channel with each ligand molecule bound. Thus, the stoichiometry of Gbeta gamma binding to IKACh is not adequately determined by fits of the Hill equation to the Gbeta gamma dose-response relations. Even the more complicated Monod, Wyman, and Changeux (MWC) formula does not properly describe the subunit gating of the cyclic nucleotide-gated channel (59). Nevertheless, the cooperativity in IKACh activation (9, 56, 57) and the Gbeta gamma -dependent shifts in its gating modes (60, 61) suggest that GIRK channels have multiple Gbeta gamma binding sites.

Given the inadequacy of available models, a direct biochemical approach was used to determine GIRK-Gbeta gamma binding stoichiometry. Solutions containing purified GIRK1 and GIRK4 were treated with the cross-linking reagent DTSSP in the presence or absence of Gbeta gamma . A 230-kDa band was observed in the absence of Gbeta gamma , compared with a 390-kDa band when Gbeta gamma was present. We concluded that the 160-kDa shift was the result of covalent linkage of multiple 42-kDa Gbeta gamma molecules to the channel. Next, solubilized recombinant GIRK4 homotetramers were treated with DTSSP in the presence and absence of Gbeta gamma . A 170-kDa band formed without Gbeta gamma in contrast to the 320-kDa band in the Gbeta gamma -containing experiment. The 150-kDa shift in the presence of Gbeta gamma is most consistent with the chemical cross-linking of four 42-kDa Gbeta gamma molecules to the GIRK4 homotetramers with complete Gbeta gamma binding site saturation and cross-linking. Finally, a variety of membrane-associated GIRK channels were treated with DTSSP and analyzed by SDS-PAGE. In each case, completely cross-linked GIRK tetramers resulted. To verify that there were four Gbeta gamma binding sites in GIRK tetramers, we altered our cross-linking conditions. DTSSP and Gbeta gamma concentrations were adjusted so that variable numbers of Gbeta gamma molecules were cross-linked to the GIRK4 homotetramers. Five adducts, representing zero, one, two, three, and four Gbeta gamma molecules cross-linked to the channel, were detected. We were unable to cross-link more than four Gbeta gamma molecules to the channel, even with Gbeta gamma concentrations two orders of magnitude higher than the Kd for Gbeta gamma binding to GIRK subunits. We conclude that four Gbeta gamma subunits can bind to a GIRK tetramer.

Currently, the Gbeta gamma binding site on GIRK subunits is thought to reside primarily on the cytoplasmic C-terminal region shortly after the second transmembrane domain. But a detailed description of the GIRK/Gbeta gamma binding site will undoubtedly require direct structural determination. For example, it is not possible to determine with our experiments whether the Gbeta gamma binding pockets were formed within subunits or between subunits. Short of direct structural determination, in future experiments it may be possible to cross-link Gbeta gamma to GIRKs during patch clamp recording. Such a technique has proven valuable in evaluating cyclic nucleotide binding to cyclic nucleotide-gated channels (59).


    ACKNOWLEDGEMENTS

We would like to thank Matt Kennedy for his expertise and for providing epitope-tagged GIRK1 and GIRK4, Yiping Chen for providing technical assistance, Grigory Krapivinsky and Luba Krapivinsky for purified Gbeta gamma , anti-GIRK1 and anti-GIRK4 antibodies, Heidi Chial for critical reading of the manuscript and Dr. Eva Neer for helpful discussions.


    FOOTNOTES

* 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.

To whom correspondence should be addressed: Howard Hughes Medical Inst., Professor of Neurobiology, Pediatrics, Harvard Medical School, Children's Hospital, 1309 Enders, 320 Longwood Ave., Boston, MA 02115. Tel.: 617-355-6163; Fax: 617-731-0787; E-mail: clapham@rascal.med.harvard.edu.

Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M100058200


    ABBREVIATIONS

The abbreviations used are: PI, phosphatidylinositol; GIRK, G-protein-gated inwardly rectifying K+ channel; DTSSP, dithiobis[sulfosuccinimidylpropionate]; PAGE, polyacrylamide gel electrophoresis; Kir, inwardly rectifying K+-selective channel; IKACh, native atrial G-protein-gated K+ channel; CHO, Chinese hamster ovary cells; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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