©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
G Binds Directly to the G Protein-gated K Channel, I(*)

(Received for publication, September 22, 1995; and in revised form, October 19, 1995)

Grigory Krapivinsky Luba Krapivinsky Kevin Wickman David E. Clapham (§)

From the Department of Pharmacology, Mayo Foundation, Rochester, Minnesota 55905

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cardiac G protein-gated K channel, I, is activated by application of purified and recombinant beta and subunits (Gbeta) of heterotrimeric G proteins to excised inside-out patches from atrial membranes (Logothetis, D. E., Kurachi, Y., Galper, J., Neer, E., and Clapham, D. E.(1987) Nature 325, 321-326; Wickman, K., [Medline] Iniguez-Lluhi, J., Davenport, P., Taussig, R. A., Krapivinsky, G. B., Linder, M. E., Gilman, A., and Clapham, D. E. (1994) Nature 368, 255-257). Cardiac I [Medline] is composed of two inward rectifier K channel subunits, GIRK1 and CIR (Krapivinsky, G., Gordon, E., Wickman, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E.(1995) Nature 374, 135-141). We show that Gbeta directly binds to immunoprecipitated cardiac I as well as to recombinant CIR and GIRK1 subunits, with dissociation constants (K) of 55, 50, and 125 nM, respectively. In each case, binding appeared specific as judged by competition of unlabeled Gbeta with radiolabeled Gbeta and inhibition of binding by antigenic peptide or Galpha-GDP, but not Galpha-GTPS (guanosine 5`-3-O-(thio)triphosphate). In contrast, Galpha (GTPS- or GDP-bound) did not bind to the native channel. We conclude that Gbeta binds directly and specifically to I via interactions with both CIR and GIRK1 subunits to gate the channel.


INTRODUCTION

Acetylcholine (ACh) (^1)secreted from the vagus nerve binds cardiac muscarinic receptors, initiating a sequence of events leading to slowing of the heart rate. I, an inwardly rectifying, potassium-selective channel stimulated by the beta and subunit (Gbeta) of pertussis toxin-sensitive heterotrimeric G proteins, mediates part of this process by hyperpolarizing pacemaker cells in sinoatrial and atrioventricular nodes of the heart(4, 5, 6, 7) . All evidence indicates that the critical components involved in I activation are confined to the membrane. However, it is unclear whether Gbeta binds directly to I to elicit the stimulatory effect or acts via an unknown intermediate step(s)(4, 5) .

Cardiac I is a heteromultimer of two homologous inward rectifier K channel subunits, GIRK1 (8, 9) and CIR(3) . Recent evidence also suggests that a similar complex comprised of GIRK1 and GIRK2(10) , a structural and functional CIR homolog, forms a neuronal G protein-gated K channel(11) . We tested whether Gbeta binds directly to cardiac I and, if so, to which subunit(s). By immunoprecipitation with subunit-specific antibodies, we were able to effectively purify whole cardiac I channel and individual recombinant subunits and study binding of I-labeled Gbeta to these immunoprecipitated species. Here we show that Gbeta binds directly and specifically to the whole channel and to each I subunit.


MATERIALS AND METHODS

Electrophysiological experiments with atrial myocytes were performed as described(2) . The pipette and bath solutions were identical (in mM): 140 K, 144 Cl, 5 EGTA, 2 Mg, 10 HEPES, pH 7.2. Holding potential was -80 mV. Spaces in the upper trace (see Fig. 1A) represent only the time required to add the indicated substance to the bath. The lower, expanded trace in Fig. 1A was filtered (8-pole Bessel) at 2.5 kHz. The concentration-response analysis in Fig. 1B was performed as described(2) . The data were fit to the sigmoid function f(x) = (a - d)/(1 + (x/c)) + d using the Marquardt-Levenberg least squares curve-fitting algorithm (a and d represent the asymptotic maximum and minimum, respectively; c is K, the value of x at the inflection point; b is the Hill coefficient).


Figure 1: Gbeta activates I in inside-out patches from rat atrial myocytes. A, 10 nM bovine brain Gbeta consistently activated I. Subsequent addition of 30-100 nM bovine brain Galpha-GDP completely inhibited I activity elicited by Gbeta. Channel activity was restored by the addition of excess Gbeta. B, dependence of I activity (relative Np) on Gbeta concentration. The cumulative K and the Hill coefficient determined by averaging data obtained for bovine brain Gbeta and five recombinant Gbeta complexes (includes data reported in (2) ) were 5 nM and 1.5, respectively. The table inset shows the relevant parameters determined for each type of Gbeta preparation. Np values are normalized to subsequent GTPS stimulation (Np= 1).



G-proteins were isolated from bovine brain, separated into Galpha and Gbeta subunits as described(12) , and additionally purified by affinity chromatography over immobilized Galpha (13) or Gbeta(14) . Bovine atrial plasma membranes were isolated as described(15) . Membranes were solubilized in 1.0% CHAPS-HEDN buffer (in mM: 10 HEPES, 1 EDTA, 1 dithiothreitol, and 100 NaCl) containing protease inhibitors. Two different antipeptide affinity-purified antibodies were used for immunoprecipitation experiments: anti-CIR (aCIRN2, amino acids 19-32, 0.5 µg/assay) and anti-GIRK1 (aCsh, amino acids 356-501, 0.3 µg/assay of atrial membrane protein and 0.7 µg/assay of Sf9 membrane protein). aCIRN2 did not immunoprecipitate in vitro translated GIRK1, and aCsh did not immunoprecipitate in vitro translated CIR (3) . (^2)Proteins were immunoprecipitated for 1.5 h at 4 °C with corresponding antibody and PrA FF-Sepharose (Pharmacia Biotech Inc.). Immunoprecipitates were washed four times in the same buffer, followed by two washes with 0.1% CHAPS-HEDN. Anti-Gbeta antibody was purchased from Calbiochem.

For radiolabeling of G protein subunits, 20 µg of purified protein was labeled with I using 250 µCi of I-Bolton-Hunter reagent (DuPont-NEN) yielding 1 mol of I/3 mol of G-protein subunit. I-Bolton-Hunter reagent, at this stoichiometry, does not prevent formation of a functional heterotrimer by labeled subunits. Both labeled Galpha and Gbeta were able to bind their unlabeled immobilized counterparts, and incubation with AlF(4) led to their dissociation (data not shown). For each binding assay, immunoprecipitates were obtained from 50 µg of atrial membrane protein and 50 or 500 µg of Sf9 membrane protein containing rCIR or rGIRK1, respectively. Immunoprecipitated proteins were incubated with 1.25 nMI-Gbeta (10^5 cpm) and unlabeled competitors in 0.1% CHAPS-HEDN and rotated for 15 min at room temperature (75 µl, total volume). Subsequently, the Immunobeads were washed four times by centrifugation, each time using 0.5 ml of the same ice-cold buffer. Total washing time was 7 min. In control experiments, the amount of bound Gbeta did not increase after 15 min at room temperature, and tfor dissociation of bound Gbeta was 90 min at 4 °C. Bound Gbeta was counted using a counter. Data were fit to a competition equation with a single binding site(17) .

Recombinant baculoviruses containing the 5`-untranslated region and coding region of GIRK1 and the coding region of CIR (3) were produced using the non-fusion baculovirus transfer vector pBlueBac III. The viruses were generated, isolated, and amplified as described (MaxBac, Invitrogen). Five days after infection, cells were harvested and homogenized in a hypotonic buffer. Membranes were then collected, solubilized, and immunoprecipitated as described for atrial membranes.


RESULTS

The functional interaction between Gbeta and I has been well studied in inside-out membrane patches from atrial myocytes. Bovine brain Gbeta reproducibly activates I, and as expected for a Gbeta-dependent process, channel activity is inhibited by excess Galpha-GDP (Fig. 1A; see also Refs. 1, 2, 6, and 7). Inhibition by Galpha-GDP is overcome by excess Gbeta (Fig. 1A; see also (2) ). Because there was no statistically significant difference between the potency of bovine brain Gbeta and the potencies of all Gbeta recombinant subunits tested previously (except transducin Gbeta11; (2) ), these data were averaged to generate the cumulative concentration-response relation shown in Fig. 1B. The resultant K and the Hill coefficient, as determined by the best fit of the cumulative data, were 5 nM and 1.5, respectively.

This type of functional study cannot address whether there is a direct interaction between Gbeta and I. To examine this issue, we studied Gbeta binding to the channel. An anti-peptide antibody (aCIRN2) directed against a unique amino-terminal domain of the CIR subunit immunoprecipitated CIR and coimmunoprecipitated GIRK1 from bovine atrial membranes.^2 Endogenous cardiac Gbeta bound the native channel (Fig. 2A). Significantly, native cardiac Gbeta associated with the aCIRN2-immunoprecipitated channel complex only when atrial sarcolemmal membranes were treated with GTPS to activate endogenous G proteins. This suggests that prior to activation, G protein heterotrimers do not complex with I. Although the association of native cardiac Gbeta with the channel was clear, the signal was inadequate for accurate quantitation of binding.


Figure 2: Gbeta binds to native atrial I. A, Gbeta binds to I only after dissociation of endogenous heterotrimeric G proteins by GTPS. Bovine atrial plasma membranes (1 mg) were treated with 100 µM GTPS, solubilized in 1.0% CHAPS-HEDN, and immunoprecipitated by aCIRN2. Immunoprecipitated proteins were Western blotted with an anti-Gbeta antibody. B, I-Gbeta binds to cardiac I immunoprecipitated by the anti-CIR antibody, aCIRN2. Binding of 1.25 nMI-Gbeta was inhibited by 200 µM CIRN2 antigenic (Ag) peptide, 1.3 µM unlabeled Gbeta, and 125 nM Galpha-GDP. In contrast, virtually no inhibition was observed with 125 nM Galpha-GTPS. For this and Fig. 3, A and B, 100 on the y axis refers to full binding of I-Gbeta in the absence of competing proteins. C, competition of unlabeled Gbeta and I-Gbeta for binding sites on I was used to evaluate the equilibrium binding constant. Binding of I-Gbeta to aCIRN2-precipitated I in 0.1% CHAPS (circle) or 0.1% Lubrol PX (bullet) and binding of I-Gbeta to aCsh-precipitated I in 0.1% CHAPS (down triangle) is shown.. All data points represent the average of three separate experiments. The data were fit well to a model consisting of a single type of binding site.




Figure 3: Gbeta binding to recombinant CIR and GIRK1 subunits. A and B demonstrate the specificity of Gbeta binding to recombinant CIR (rCIR) or to recombinant GIRK1 (rGIRK1). Recombinant CIR and recombinant GIRK1 were immunoprecipitated with aCIRN2 and aCsh, respectively. Conditions were identical to those described in the legend to Fig. 2. Nonspecific binding for aCsh immunoprecipitates was determined using 500 µg of wild type Sf9 cell membranes. C, binding affinity of Gbeta to recombinant CIR and recombinant GIRK1. Nonspecific binding was subtracted, and each point was normalized to maximal binding in absence of competitor.



To quantify binding of Gbeta to the channel, we measured the binding of I-labeled, purified bovine brain Gbeta to the immunoprecipitated atrial GIRK1-CIR complex (I; Fig. 2B). The observed binding was due to an interaction between Gbeta and the channel as determined by competition with antigenic peptide. As shown in Fig. 2B, the presence of this peptide resulted in a significant decrease in Gbeta binding. Unlabeled Gbeta similarly decreased the level of binding of labeled Gbeta, demonstrating specificity of I-Gbeta binding in the concentration range under study. Finally, consistent with results from electrophysiological experiments, the presence of excess Galpha-GDP, but not Galpha-GTPS, prevented an interaction between Gbeta and I (Fig. 2B). We conclude that Gbeta binds directly and specifically to native cardiac I. Given the previous reports of I stimulation by Galpha-GTPS(18) , we tested whether I-Galpha (GDP- or GTPS-bound) interacted physically with immunoprecipitated I. The amount of I-Galpha that bound to immunoprecipitates was insignificant (100-fold less) in relation to the amount of bound Gbeta (data not shown).

The binding constant for the interaction between Gbeta and I was determined by competition between unlabeled and labeled Gbeta for channel binding sites(17) . The binding data were most simply and adequately fit by a model with a single type of binding site. The Gbeta binding constant to cardiac I immunoprecipitated by aCIRN2 was 55 nM (Fig. 2C). Thus, the affinity of the channel for Gbeta determined in this binding assay was 10-fold lower than that suggested by results from electrophysiological experiments (Fig. 1B). Since this discrepancy could potentially be explained by antibody interference with Gbeta binding, we also determined the Gbeta binding constant to the channel using an immunoprecipitating antibody targeting the carboxyl terminus of the GIRK1 subunit of I instead of the CIR subunit (aCsh(3) ). The Gbeta binding affinity to aCsh and aCIRN2 immunoprecipitates were virtually identical (Fig. 2C), indicating that Gbeta binding was not affected by the immunoprecipitating antibody. Note that Lubrol PX, even at low concentrations (0.1%), significantly inhibited Gbeta binding, consistent with the inhibition by Lubrol PX of Gbeta-induced I activation observed in patch-clamp experiments(19) .

Given the results of the binding studies between Gbeta and native cardiac I, the next logical step was to determine whether GIRK1, CIR, or both subunits interacted with Gbeta. Since the subunit-specific antibodies did not affect the binding of Gbeta to cardiac I, they were used to study binding of Gbeta to GIRK1 and CIR subunits expressed and isolated from Sf9 cells. The control experiments used to confirm specificity of Gbeta binding to cardiac I (see Fig. 2B) were also performed for the individually expressed subunits to assess the significance of any observed interactions. Both recombinant GIRK1 and CIR subunits demonstrated specific binding to Gbeta (Fig. 3, A and B). There was no evidence for cooperativity in the binding of Gbeta to either recombinant subunit; the data were well fit to a model with a single type of binding site. The apparent affinity of Gbeta for CIR was almost identical to that for cardiac I (K = 50 nM; Fig. 3C) and slightly lower for GIRK1 (K = 125 nM).


DISCUSSION

Despite the widespread electrophysiological evidence for membrane-delimited G protein activation of ion channels(20) , no biochemical evidence has been presented for a direct interaction between G protein subunits and channel proteins for two major reasons. First, in comparison to other G protein effectors such as adenylyl cyclase, cGMP phosphodiesterase, and phospholipase Cbeta, channel proteins are of lower abundance in cells. Ion channels of specific subtypes number only a few thousand per cell. Second, the most dramatic example of a G protein-regulated ion channel is I, a member of a class of ion channels only recently cloned(8, 21) . The generation of immunoprecipitating antibodies to the I channel subunits, GIRK1 and CIR, enabled us to develop an assay for Gbeta binding. The present work shows that Gbeta binds CIR, GIRK1, and the native cardiac channel with similar affinities.

There are two findings in the current study that are discrepant with the electrophysiological data. The concentration of Gbeta eliciting half-maximal I activity was 5 nM in inside-out atrial patches, while the calculated Gbeta binding constant for the solubilized channel was 50 nM. This difference could be simply due to the presence of the higher concentration of CHAPS in the binding reaction than in the electrophysiological experiments (1.6 versus 0.13 mM). On the other hand, Gbeta is hydrophobic and when added to an inside-out patch might concentrate in the membrane, giving a higher Gbeta concentration in the vicinity of the channel compared with the bath concentration. Thus, such functional studies might overestimate the real affinity of Gbeta for I. Our electrophysiologic data were fit with a Hill coefficient of 1.5, suggesting mild cooperativity (but see (7) ). However, we did not observe cooperativity in Gbeta binding to the channel or to its individual subunits. It is possible that detergent solubilization of the channel could influence interactions between Gbeta binding sites on the channel. Our current hypothesis is that one Gbeta binds each GIRK1 or CIR subunit of the I heteromultimer to activate the channel.

We have shown that I is gated by Gbeta, not Galpha(1, 2) , that I is a heteromultimer of GIRK1 and CIR inward rectifier K channel subunits(3) , and that Gbeta, not Galpha, directly binds both subunits of cardiac I. We have not localized the binding sites for Gbeta on the individual GIRK1 or CIR subunits, but Gbeta has been shown to associate with a fusion protein containing the full carboxyl-terminal residues of GIRK1(16) . Interestingly, our studies indicate that the antibodies raised against this entire region do not interfere with the observed binding of Gbeta to cardiac I. Initially, the obvious candidate region for Gbeta binding in GIRK1 was the extreme 150 carboxyl-terminal amino acids, since this domain was not present in the G protein-insensitive IRK (Kir 2.0) or ROMK (Kir 1.0) families. However, CIR, which binds Gbeta even better than GIRK1, has no corresponding region. Comparisons of the GIRK (Kir 3.0) family (including CIR) carboxyl-terminal regions do not reveal any unique GIRK family similarities, which might suggest a functional Gbeta-binding domain. In contrast, the amino termini of the GIRK family contain scattered conserved amino acids not found in ROMK and IRK subfamilies. Additional structure/function studies will address these issues.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant 53483 (to D. E. C.). 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.

§
To whom correspondence should be addressed. Tel.: 507-284-5881; Fax: 507-284-9111; clapham@mayo.edu.

(^1)
The abbreviations used are: ACh, acetylcholine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GTPS, guanosine 5`-3-O-(thio)triphosphate).

(^2)
Krapivinsky, G., Krapivinsky, L., Velimirovic, B., Wickman, K., Navarro, B., and Clapham, D. E.(1995) J. Biol. Chem.270, 28777-28779.


ACKNOWLEDGEMENTS

KGA (GIRK1) cDNA was a generous gift from Dr. H. Lester. Recombinant Gbeta subunits were made in the laboratory of Dr. Alfred Gilman(2) .


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.