Number and Stoichiometry of Subunits in the Native Atrial G-protein-gated K+ Channel, IKACh*

Shawn CoreyDagger , Grigory Krapivinsky§, Luba Krapivinsky§, 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

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The G-protein-regulated, inwardly rectifying K+ (GIRK) channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK channels are distributed predominantly throughout the heart, brain, and pancreas. In recent years, GIRK channels have received a great deal of attention for their direct G-protein beta gamma (Gbeta gamma ) regulation. Native cardiac IKACh is composed of GIRK1 and GIRK4 subunits (Krapivinsky, G., Gordon, E. A., Wickman, K. A., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). Here, we examine the quaternary structure of IKACh using a variety of complementary approaches. Complete cross-linking of purified atrial IKACh protein formed a single adduct with a total molecular weight that was most consistent with a tetramer. In addition, partial cross-linking of purified IKACh produced subsets of molecular weights consistent with monomers, dimers, trimers, and tetramers. Within the presumed protein dimers, GIRK1-GIRK1 and GIRK4-GIRK4 adducts were formed, indicating that the tetramer was composed of two GIRK1 and two GIRK4 subunits. This 1:1 GIRK1 to GIRK4 stoichiometry was confirmed by two independent means, including densitometry of both silver-stained and Western-blotted native atrial IKACh. Similar experimental results could potentially be obtained if GIRK1 and GIRK4 subunits assembled randomly as 2:2 and equally sized populations of 3:1 and 1:3 tetramers. We also show that GIRK subunits may form homotetramers in expression systems, although the evidence to date suggests that GIRK1 homotetramers are not functional. We conclude that the inwardly rectifying atrial K+ channel, IKACh, a prototypical GIRK channel, is a heterotetramer and is most likely composed of two GIRK1 subunits and two GIRK4 subunits.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Since the initial cloning of the Shaker K+ channel in 1987 (1-3), a wealth of K+-selective channels have been cloned and their electrophysiologic properties characterized. Detailed structural information on K+-selective channels, however, has lagged considerably behind. Typically, K+-selective channels exist at low densities of 1-10 channels/µm2 (4-6), compared with ~ 10,000 channels/µm2 for the nicotinic receptor (7-9). Because they are membrane proteins, they require detergents to keep them solubilized. This has made purification, crystallization, and biochemical characterization difficult. To date, there is no crystal structure or high resolution electron micrographic image of a K+-selective channel.

Of the K+-selective channels, the most structural information is known about the voltage-gated K+-selective channels (Kv).1 Kv channels have been proposed to be tetramers of four identical or highly homologous subunits, based on a wide variety of methods: toxin binding studies (10), covalently linked constructs (11), low resolution electron microscopic imaging (12), and cross-linking studies (13). Each subunit is thought to consist of 6 transmembrane domains with a loop contributing to the pore (P-loop) region located between the fifth and sixth transmembrane domains. Less is known about the inwardly rectifying K+-selective (Kir) channels. Kir channels are distantly related to the Kv channels and contain regions equivalent only to the fifth transmembrane domain, the P-loop, and the sixth transmembrane domains of the Kv channels. Despite these similarities, the regions critical for assembly are likely to differ between Kir and Kv channels . Although the N-terminal domain has been implicated in Kv assembly (14), the second transmembrane domain and the proximal C-terminal domains have been implicated in Kir channel assembly (15). Several groups have suggested that, like Kv channels, Kir channels are tetramers (16-18).

We are interested in the unique subfamily of Kir channels that are ligand-gated by direct Gbeta gamma binding (19, 20), the G-protein-regulated, inwardly rectifying K+ channels (GIRKs). Using a combination of size exclusion chromatography and sucrose density gradients, it was originally proposed that GIRK1, in combination with an unknown subunit, contained three to five subunits of unknown stoichiometry (21). Later, by studying the biophysical properties of concatenated subunits, Silverman et al. (22) suggested that GIRK1 and GIRK4 formed a tetramer in a 1:1 stoichiometry. However, Silverman et al. were unable to determine a preferred subunit arrangement around the pore and suggested that more than one arrangement may be viable. Tucker et al. (23) found that the GIRK1-GIRK4-GIRK1-GIRK4, rather than the GIRK1-GIRK1-GIRK4-GIRK4 arrangement, produced a higher ratio of agonist-induced to basal current.

To date, all of the studies on the stoichiometry of Kir channels have relied upon the formation of multimeric concatemers. Briefly, a single protein is formed by the translation of a single mRNA encoding several artificially concatenated subunits. Several combinations of concatenated subunits are examined, and conclusions are drawn from their varying properties. These studies have an advantage in that they define the smallest functional unit, whereas biochemical studies can define only the smallest physically associating unit. However, concatemer-based studies assume that: 1) combinations of tandemly linked subunits that yield the most current are the most representative of the native channel configuration, 2) tandemly linked subunits do not coassemble with other tandemly linked subunits, and 3) tandemly linked subunits are completely translated and are not proteolytically cleaved between linked subunits. Unfortunately, there are instances in which each of these assumptions has been proven to be incorrect (18, 22, 24, 25). Silverman et al. (22) were forced to assume that two trimeric constructs combine to form a functional channel to explain the high currents produced when only trimeric constructs were expressed. In addition, these studies have been complicated by the presence of endogenous oocyte subunits. Despite these drawbacks, in recent years, the use of tandemly linked subunits has dominated the field of channel stoichiometry and has remained virtually unchecked by other independent methods. Clearly, alternative means must be used to test the validity of this approach.

In this report, we present the first purification of a native mammalian K+ channel to homogeneity, the first cross-linking study examining inwardly rectifying K+ channel quaternary structure and stoichiometry, and the first densitometry study examining GIRK stoichiometry. Using multiple independent methods, our data indicate that GIRK channels are tetramers. We hypothesize that native IKACh is composed of two GIRK1 subunits and two GIRK4 subunits.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Purification of Native IKACh and Recombinant GIRK1-GIRK4 Heteromultimers-- Bovine atrial plasma membranes were isolated as described (26). Membranes were solubilized in 1.0% CHAPS-HEDN buffer, pH 7.5 (in mM: 10 HEPES, 1 EDTA, 1 dithiothreitol, and 100 NaCl). The protease inhibitors leupeptin (50 mg/ml, Sigma-Aldrich Inc.), phenylmethylsulfonyl fluoride (100 mg/ml, Sigma-Aldrich Inc.), aprotinin (1 mg/ml, Sigma-Aldrich Inc.), and pepstatin (2 mg/ml, Sigma-Aldrich Inc.) were used in all steps of the purification. Approximately 150 mg of solubilized atrial proteins were loaded onto a Toyopearl RedTM affinity column. Flow rates were 0.1 ml/min and 1 ml/min during the binding and elution steps, respectively. Bound protein was eluted with the same buffer containing 1 M NaCl. Fractions were assayed for IKACh subunit content by Western blot. Fractions containing both IKACh subunits were pooled and dialyzed against 400 mM NaCl containing 1.0% CHAPS-HEDN buffer (pH 7.5). The equilibrated fractions were concentrated in Centriprep-50TM (Amicon, Inc., Beverly, MA) concentrators to <2.0 ml and loaded onto a HighLoad 16/60 SuperdexTM (Pharmacia Biotech Inc., Uppsala, Sweden) size exclusion chromatography column at a flow rate of 0.4 ml/min. Pooled IKACh fractions were dialyzed against immunoprecipitation (IP) buffer (1% CHAPS, 10 mM HEPES, 100 mM NaCl, 5 mM EDTA at pH 7.5), loaded onto a wheat germ agglutinin (WGA, Sigma-Aldrich Inc.) affinity column, and eluted with 0.25 M N-acetylglucosamine. Finally, the eluate was immunoprecipitated for 150 min at 4 °C with anti-GIRK4 peptide antibody (anti-CIRN2 was generated against amino acids 19-32; Refs. 19 and 27) and washed three times (1-ml amounts) for 1 min each, three times (1 ml) for 5 min each, and eluted with 1 mg/ml antigenic peptide three times (100 µl) for 30 min each at 22 °C.

Plasma membrane proteins containing epitope-tagged GIRK1-AU5 and GIRK4-AU1 were isolated from COS7 cells as described previously (28). Samples were precleared for 1 h at 4 °C with 20 µl of Protein A-Sepharose (Pharmacia Biotech Inc.). The two-step purification consisted of sequential immunoprecipitations in which samples were first immunoprecipitated with anti-GIRK4 antibodies and were then immunoprecipitated with anti-GIRK1 antibodies. For the anti-GIRK4 IP, Protein A-Sepharose was preincubated with 3 µg of anti-CIRN2 (27, 28). After a 30-min preincubation, solubilized proteins from a single 100-mm dish were added and incubated at 4 °C for 150 min. The Protein A-Sepharose-antibody (Ab)-GIRK complexes were washed five times (1-ml amounts) with IP buffer. The GIRK heteromultimers were eluted with 1 mg/ml CIRN2 antigenic peptide for 150 min at 22 °C with three eluate exchanges. For the anti-GIRK4 IP, the eluate was added to Protein A-Sepharose and a 1:50 dilution of ascites fluid containing AU5 monoclonal antibodies (BabCO, Berkeley Antibody Co., Richmond, CA) was incubated at 4 °C for 150 min. The Protein A-Sepharose-Ab-GIRK complexes were washed five times (1-ml amounts) with IP buffer. GIRK heteromultimers were eluted with 0.25 mg/ml AU5 antigenic peptide at 22 °C with three 100-µl eluate exchanges for 1 h each.

Chemical Cross-linking-- Protein to be cross-linked was treated for 1 h on ice with 100 mM dithiothreitol and dialyzed against 1% CHAPS, 10 mM HEPES, 400 mM NaCl, pH 8.5 (cross-linking buffer). To block free sulfhydryl groups, dialyzed aliquots were treated with 25 mM iodoacetamide (Sigma-Aldrich Inc.) for 1 h on ice. Aliquots to be cross-linked via disulfides were not treated with iodoacetamide. Typical reaction volumes were 15 µl for the pure, atrial IKACh and 75 µl for the recombinant protein. Increasing the reaction volumes >10-fold had no effect on the adducts formed. Approximately 0.1 ng of pure IKACh, 10 µg of crude solubilized atrial protein, or 10 µg of solubilized COS-7 membrane protein was used in each reaction.

For complete cross-linking, solutions containing 3 mM dithiobis(sulfosuccinimidylpropionate) (DTSSP, Pierce), 1 mM disuccinimidyl suberate (DSS, Pierce), or 3 mM sulfosuccinimidyl (4-azidophenyldithio)propionate (SSADP, Pierce) were used. Immediately prior to use, cross-linking reagents were prepared as 10 × stock solutions in cross-linking buffer. The water-insoluble DSS was prepared as a 9 × stock solution in dimethyl sulfoxide (Me2SO). Unless specified otherwise, the reactions were allowed to proceed for 1 h on ice. Reactions were terminated for 30 min with 50 mM Tris, pH 7.5, or 15 mM iodoacetamide, when either H2O2 or iodine was used. The hetero-bifunctional SSADP was first used like the other reagents, and then the azide was photoactivated by a 1-min exposure in a CL-1000 ultraviolet cross-linker (UVP, Upland, CA).

For partial cross-linking, dimethyl adipimidate·2HCl (DMA, Pierce) and dimethyl suberimidate·2HCl (DMS, Pierce) were also used. These reagents were used at 10 mM final concentrations in 100 mM HEPES containing cross-linking buffer. For DTSSP, SSADP, and iodine, a 10-fold dilution over what was used to completely cross-link the channel generally created a laddering pattern. When necessary, trichloroacetic acid (Sigma-Aldrich Inc.) precipitation was used to concentrate the samples prior to SDS-PAGE analysis.

Electrophoresis and Immunoblotting-- Native IKACh or recombinant GIRK protein was resuspended in Laemmli sample buffer containing either 50 mM dithiothreitol (reducing conditions) or 25 mM iodoacetamide (non-reducing conditions) for 30 min at 50 °C. 10% separating and 3% stacking, 3-10% separating and 3% stacking, and precast 2-15% (ISS) gels were all utilized. Samples were analyzed by fluorography with Amplify (Amersham Corp.), Gel CodeTM silver staining (Pierce), or by immunoblotting with anti-GIRK1 antibodies and/or anti-GIRK4 antibodies. Transfer times for Western blot analysis were extended to >2 h at 15 V to ensure transfer of the larger cross-linked complexes. When sequential probing with antibodies was necessary, polyvinylidene fluoride membranes (Millipore, Bedford, MA) were stripped with 62.5 mM Tris-HCl, 2% SDS, 100 mM 2-mercaptoethanol for 45 min at 50 °C. When quantitation was required, X-Omat AR film (Eastman Kodak Co.) was preflashed (SensitizeTM, Amersham Corp.) to 0.15 OD above background and exposed at -70 °C. A GS-700 imaging densitometer (Bio-Rad) was used to analyze the protein gels and immunoblots. Molecular weights were calculated using densitometry profiles from a combination of prestained high molecular weight markers (Bio-Rad) and low and high molecular weight markers (Pharmacia Biotech Inc.). In a portion of the gels, thyrogloblin (Pharmacia Biotech Inc.) was added to ensure linearity through at least 330 kDa. When lanes from several gels were presented together (Figs. 4-6), the molecular weight markers corresponded to lanes 1 and 2. The remaining lanes were approximately aligned with the molecular weight markers. In addition, the molecular weights of all the adducts are presented in Tables I and II.

Antibody Standardization-- Purified [35S]methionine-labeled recombinant GIRK1 and/or GIRK4 subunits were purified to homogeneity, as described previously. The pure GIRK1-GIRK4 heteromultimers were divided into two aliquots, which were analyzed separately by SDS-PAGE. The first lane was fixed and exposed to film (Fig. 3B), and the second lane was transferred to a polyvinylidene fluoride membrane and Western-blotted along with several lanes of decreasing quantities of solubilized atrial sarcolemma membranes (Fig. 3A). Autoradiography (Fig. 3B) revealed a GIRK1:GIRK4 band intensity ratio of 1.8:1.0 and 1.5:1.0 after a correction for the methionine content of each protein (see Equation 1). The intensity ratio represents the stoichiometry of the recombinant protein, but not necessarily the stoichiometry of the subunits in native atrial IKACh.
<UP>Recombinant stoichiometry</UP>=<FR><NU><UP>GIRK1 radiometric counts</UP></NU><DE><UP>GIRK4 radiometric counts</UP></DE></FR>×<FR><NU><UP>number of methionines in GIRK</UP>4</NU><DE><UP>number of methionines in GIRK</UP>1</DE></FR> (Eq. 1)
The aliquot of recombinant GIRK1-GIRK4 that had been Western-blotted was then analyzed by densitometry. The GIRK1:GIRK4 band intensity ratio was 8:1 and will be referred to as the Ab stoichiometry (apparent stoichiometry as detected by antibody). The Ab stoichiometry is measured from the ratio of the intensity of Western blot bands and is a product of the number of moles of each individual subunit and the number of Abs bound to each epitope.

Dividing the recombinant stoichiometry by the Ab stoichiometry yields a useful term, which we will refer to as the Ab standardization factor (Equation 2).
  <UP>Ab standardization factor</UP>=<FR><NU><UP>recombinant stoichiometry</UP></NU><DE><UP>Ab stoichiometry</UP></DE></FR> (Eq. 2)
In our case, the Ab standardization factor was 0.19 (0.19 = 1.5/8). The Ab standardization factor multiplied by the GIRK1:GIRK4 band intensity ratio of the Western-blotted native channel yields the true stoichiometry of the native channel (Equation 3).
<UP>True stoichiometry of native I<SUB>KACh</SUB></UP>=<UP>measured Ab stoichiometry</UP>×<UP>Ab standardization factor</UP> (Eq. 3)
In our case, the true stoichiometry of native IKACh was ~1:1 (0.19 × 6 = 1.1).

The above procedure assumes that there is a direct linear relationship between the Western blot intensity and the total amount of protein on the blot and that this relationship is maintained for both antibodies over the range of the protein concentrations to be tested. We found that our antibodies satisfied this criteria; the intensity of the lanes from Western-blotted atrial sarcolemma membrane varied in a direct and linear manner with the amount of protein.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Purification of Native IKACh and Recombinant GIRK1-GIRK4 Heteromultimers-- We have purified native bovine IKACh and recombinant GIRK1-GIRK4 heteromultimeric channels to near homogeneity. Both purification procedures were specifically designed to purify only heteromultimeric channels composed of GIRK1 and GIRK4 subunits (Fig. 1). Potential homomultimeric channels, or dissociated monomers, were not purified with these procedures.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1.   Purification schemes designed to select for heteromultimeric channels. A, purification of native IKACh. A combination of WGA (wheat germ agglutinin) chromatography and immunoprecipitation with anti-GIRK4 antibodies was used to purify native heteromultimeric channels. B, purification of recombinant GIRK1-GIRK4 heteromultimers. Sequential immunoprecipitation with anti-GIRK4 and anti-GIRK1-AU5 tag antibodies was used to purify recombinant heteromultimeric channels. A tetrameric complex is assumed for the purpose of the diagram. Question mark (?), either GIRK1 or GIRK4; branched tails on circles, glycosylated.

Native IKACh was purified from isolated bovine atrial plasma membranes (Fig. 1A). The membranes were solubilized in 1% CHAPS and subjected to the following purification steps: 1) Toyopearl RedTM affinity chromatography, 2) size exclusion chromatography, 3) WGA affinity chromatography, and 4) IP with anti-GIRK4 antibodies followed by elution with antigenic peptide. WGA affinity chromatography was specific for GIRK1, because GIRK4 is not glycosylated (39). Thus, the combination of WGA affinity chromatography and immunoprecipitation with anti-GIRK4 antibodies ensured that no homomultimeric channels or dissociated monomers were purified. The final product, purified to greater than 95% homogeneity, was native bovine atrial IKACh. Aliquots of purified native IKACh were analyzed by SDS-PAGE and silver-stained (Fig. 2, lane 1) or immunoblotted with anti-GIRK4 antibodies (Fig. 2, lane 2) and then stripped and reimmunoblotted with anti-GIRK1 antibodies (Fig. 2, lane 3). The predominant bands in the silver-stained lane were also recognized by anti-GIRK1 and anti-GIRK4 antibodies when Western-blotted. The bands correspond to GIRK4 (48 kDa), GIRK1 (54 kDa), and glycosylated GIRK1 (56-76 kDa). Interestingly, the WGA affinity chromatography enhanced the proportion of glycosylated to unglycosylated GIRK1 when compared with unpurified channels, but did not completely eliminate unglycosylated GIRK1 (Fig. 2A, 54-kDa band). Presumably, some proportion of the native IKACh heteromultimers contain both glycosylated and unglycosylated GIRK1 subunits.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 2.   Purified native IKACh and recombinant GIRK1-GIRK4 heteromultimers. Purified native IKACh and recombinant GIRK1-GIRK4 heteromultimers were analyzed by 10% SDS-PAGE. A, purified native IKACh was silver-stained or immunoblotted with anti-GIRK4 antibodies (alpha GIRK4) or anti-GIRK1 antibodies (alpha GIRK1). Presumably, some channel complexes contained at least one glycosylated (56-76 kDa) and one unglycosylated GIRK1 (54 kDa) subunit, which allowed the unglycosylated GIRK1 subunit to be co-purified. Densitometry analysis of lane 1 was consistent with a 1:1 GIRK1:GIRK4 stoichiometry. B, purified recombinant GIRK1-GIRK4 heteromultimers were immunoblotted simultaneously with anti-GIRK1 and anti-GIRK4 antibodies (alpha GRIK1/4, lane 1) or autoradiographed (lane 2). The majority of bands labeled by silver staining or [35S]methionine ([35S]Met) were also recognized by antibody. gly, glycosylated.

Recombinant GIRK1-GIRK4 heteromultimeric channels were purified from transiently transfected COS7 cells using sequential anti-GIRK1 and anti-GIRK4 immunoprecipitations (see Fig. 1B and "Experimental Procedures"). The sequential immunoprecipitations assured that only heteromultimeric channels were purified. The purified protein was analyzed by SDS-PAGE, followed by either Western blotting or autoradiography (Fig. 2B). All of the protein bands that appeared on the autoradiogram were also recognized by anti-GIRK1 or anti-GIRK4 antibodies.

Densitometry of Silver-stained Native IKACh and [35S]Methionine-labeled Recombinant Proteins Suggests a 1:1 GIRK1 to GIRK4 Subunit Stoichiometry for Native IKACh-- Two independent methods based on densitometry were used to examine GIRK1:GIRK4 stoichiometry. In the first method, silver-stained SDS-PAGE gels of purified native IKACh were analyzed by densitometry (Fig. 2A, lane 1). The ratio of band intensities for GIRK1:GIRK4 was 1.2:1. When this ratio was corrected by multiplying it by the predicted unglycosylated molecular weight of GIRK4(47 kDa)-GIRK1(56 kDa), a molar ratio of 1:1 (n = 3) for GIRK1:GIRK4 was obtained. The advantage of this procedure is that it examines GIRK1:GIRK4 stoichiometry as it exists in native atrial tissue. GIRK1 and GIRK4 share 57% overall amino acid identity, and the silver-stained bands representing GIRK1 and GIRK4 proteins varied by less than 3-fold in intensity. These GIRK1 and GIRK4 similarities minimize the potential inaccuracies of protein quantification by silver staining.

The second independent method used to examine GIRK1:GIRK4 stoichiometry was based on the [35S]methionine labeling of purified recombinant COS7 GIRK1 and GIRK4 heteromultimers. Because the number of counts emitted by a radiolabeled subunit is directly proportional to its methionine content, this method more accurately quantifies GIRK1 and GIRK4. Ideally, we could determine the native atrial GIRK1:GIRK4 stoichiometry by comparing 35S-labeled GIRK1 and GIRK4 (Fig. 2B). However, the stoichiometry may vary with the amount of RNA injected into oocytes (29, 30). If the stoichiometry varied in COS-7 cells, as well, this approach would be inadequate for determining native stoichiometry. Indeed, we found that the GIRK1:GIRK4 stoichiometry of heteromultimeric channels did vary directly with the ratio of GIRK1:GIRK4 DNA used to transfect the COS-7 cells (data not shown). We were unable to force the GIRK1:GIRK4 stoichiometry beyond 1:3 or 3:1 by varying the ratio of GIRK1:GIRK4 DNA transfected by 30-fold, supporting the conclusion that recombinant GIRK1 and GIRK4 subunits form tetramers.

Because the recombinant system does not necessarily reflect native stoichiometry, we developed a hybrid approach that combined the accurate qauntitation achievable with [35S]methionine labeling of recombinant proteins with the ability of our antibodies to detect native protein. This method involved standardizing the relative blotting sensitivities of our anti-GIRK1 and anti-GIRK4 antibodies with a known ratio of labeled recombinant protein. The standardized antibodies were then used to probe native atrial protein and the true native stoichiometry was computed as ~1:1 (see "Experimental Procedures" and Fig. 3). Previously, Huang et al. (31) showed that the ratio of two antigens could be estimated with confidence using a similar antibody-blotting method.


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 3.   Immunoblotting native IKACh with standardized antibodies yields 1:1 GIRK1:GIRK4 stoichiometry. [35S]Methionine-labeled recombinant GIRK1 and GIRK4 were used to standardize anti-GIRK1 and anti-GIRK4 antibodies. The standardized antibodies were then used to immunoblot native IKACh. A, lane 1, [35S]methionine-labeled, recombinant GIRK1 and GIRK4; lanes 2-5, 2.0, 1.0, 0.5, 0.25 µg, respectively, of solubilized atrial membrane protein. All lanes were analyzed by 10% SDS-PAGE and immunoblotted with anti-GIRK1 and anti-GIRK4 antibodies. B, [35S]methionine-labeled recombinant GIRK1 and GIRK4 (rGIRK1 and rGIRK4) were analyzed by 10% SDS-PAGE followed by autoradiography.

Complete Cross-linking of Native Purified IKACh-- We extensively cross-linked native bovine IKACh (Fig. 4), recombinant GIRK1 homomultimers (Fig. 5), and recombinant GIRK4 homomultimers (Fig. 5) using a wide variety of cross-linking reagents. First, purified native IKACh was treated with the highly reactive, amine-specific, N-hydroxysuccinimide (NHS) ester, DTSSP. This reaction produced a multimeric protein detected as a single 234 ± 7-kDa band (n = 8). This cross-linked product was recognized by both anti-GIRK1 (Fig. 4, lane 4) and anti-GIRK4 (Fig. 4, lane 3) antibodies, indicating that both GIRK1 and GIRK4 were cross-linked. The lipid-soluble NHS ester, disuccinimidyl suberate (DSS), yielded a nearly identical 235-kDa band (Fig. 4, lane 5). Finally, the entire complex was cross-linked through simple oxidation with iodine (Fig. 4, lane 6), and similar results were obtained.


View larger version (77K):
[in this window]
[in a new window]
 
Fig. 4.   Complete cross-linking of native IKACh yields products that are most consistent with tetrameric channel formation. Native IKACh was treated with a wide variety of cross-linking reagents. Cross-linked products were then analyzed by SDS-PAGE and immunoblotted. Lane 1, no cross-link control immunoblotted with anti-GIRK1 and anti-GIRK4 antibodies (alpha GIRK1 and alpha GIRK4). Lane 2, solubilized atrial membrane proteins treated with 3 mM SSADP followed by photolysis for 1 min and immunoblotted with anti-GIRK1 antibodies. Lane 3, pure IKACh treated for 1 h with 3 mM DTSSP and immunoblotted with anti-GIRK1 antibodies. Lane 4, lane 3 stripped and reimmunoblotted with anti-GIRK4 antibodies. Lane 5, pure IKACh treated for 1 h with 1 mM DSS and immunoblotted with anti-GIRK4 antibodies. Lane 6, pure IKACh treated for 1 h with 50% saturated iodine and immunoblotted with anti-GIRK4 antibodies. Molecular weight markers were run with lanes 1 and 2; lanes 3-6 were derived from separate gels and aligned based on corresponding molecular weight standards. See Table I for a summary of averaged molecular weights.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 5.   Native IKACh heteromultimers and recombinant GIRK homomultimers form similar oligomeric structures. Despite the inability of GIRK1 subunits to produce functional channels alone, GIRK1 subunits form oligomeric structures similar to native IKACh heteromultimers and GIRK4 homomultimers. A, native IKACh and recombinant GIRK1 or GIRK4 homomultimers were chemically cross-linked. Cross-linked products were then analyzed by SDS-PAGE and immunoblotted. The molecular weights of the cross-linked native IKACh, recombinant GIRK1, and recombinant GIRK4 homomultimers were all consistent with tetrameric complex formation. Lane 1, no cross-link control immunoblotted with anti-GIRK1(alpha GIRK1) and anti-GIRK4(alpha GIRK4) antibodies. Lane 2, solubilized atrial membrane proteins treated with 3 mM SSADP followed by photolysis for 1 min and immunoblotted with anti-GIRK1 antibodies. Lane 3, recombinant GIRK1 homomultimers treated for 1 h with 3 mM DTSSP and immunoblotted with anti-GIRK1 antibodies. Lane 4, recombinant GIRK4 homomultimers treated for 1 h with 3 mM DTSSP and immunoblotted with anti-GIRK4 antibodies. Molecular weight markers were run with lanes 1 and 2; lanes 3 and 4 were derived from separate gels and aligned based on corresponding molecular weight standards. See Table I for a summary of averaged molecular weights. B, native IKACh and recombinant GIRK1 elute at similar volumes during size-exclusion chromatography. The portion of the channel that eluted with the void volume (Vo) varied between trials. No attempt was made to estimate the molecular weight of the channel due to the complications caused by detergent binding.

To address the potential concern that the native complex is composed of an integral multiple of the ~235-kDa complex, or that associating proteins were lost, native IKACh protein was treated with an even more highly reactive cross-linking agent. The heterobifunctional NHS ester/aryl azide SSADP was used to cross-link native IKACh immediately after solubilization and prior to any further purification to prevent potential subunit degradation or dissociation. SSADP cross-linked protein (like DTSSP, DSS, and iodine) was detected as a single 224 ± 3-kDa (n = 4) band (Fig. 4, lane 2). Monomers were detected only after extended exposure to film, if at all (data not shown). This indicates that the entire GIRK1-GIRK4 complex was intact after purification because dissociated monomers would have remained at the bottom of the gel. Cross-linking of recombinant GIRK1 (Fig. 5A, lane 3) and GIRK4 (Fig. 5A, lane 4) homomultimers also produced a single unique complex of 216 ± 22 kDa (n = 4) and 212 ± 13 kDa (n = 3), respectively. If significant amounts of interchannel cross-linking rather than intrachannel cross-linking had occurred, a smear would have appeared at the top of the gel. A tetramer made up of equal numbers of GIRK1 subunits (approximately 65 kDa with glycosylation) and GIRK4 subunits (approximately 48 kDa) would have had a molecular weight of ~226 kDa. Thus, the total molecular weight of the native cross-linked IKACh complex, ~234 kDa, is consistent with a tetramer. Similarly, the total molecular weights of completely cross-linked homomultimeric GIRK1 ~216-kDa channel (222 kDa, predicted) and GIRK4 ~211-kDa channel (188 kDa, predicted) are consistent with a tetramer. Finally, we found that recombinant GIRK1 homomultimers eluted to a position similar to that for native IKACh during size exclusion chromatography (Fig. 5B). The size exclusion chromatography and complete cross-linking experiments support a similar oligomeric structure for GIRK1 homomultimers and the native IKACh heteromultimer. A summary of the various cross-linking reactions is given in Table I.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Complete cross-linking of GIRK channels

Partial Chemical Cross-linking of Native IKACh Reveals Monomeric, Dimeric, Trimeric, and Tetrameric Complexes-- Another approach to testing the tetrameric channel hypothesis involves analysis of partially cross-linked native IKACh. Previously, it was shown that the molecular weight of partially cross-linked proteins increases in a linear fashion with the number of cross-linked subunits (32). In this experiment, native IKACh was cross-linked with DMA, an imidoester, or with DTSSP, the more reactive NHS ester. The electrophoretic pattern produced following SDS-PAGE and Western blotting is shown in Fig. 6. The blot was first probed with anti-GIRK4 antibodies (Fig. 6A, lanes 1-3) and then completely stripped and reprobed with anti-GIRK1 antibodies (Fig. 6A, lanes 4-6). Partial cross-linking of native IKACh produced a laddered pattern consisting of four main adducts (Fig. 6A). The four adducts represent (from bottom to top) monomeric (41-61 kDa), dimeric (94-138 kDa), trimeric (~185 kDa), and tetrameric (~231 kDa) forms of the channel (Fig. 6A). The adducts formed in a manner in which the proportion of higher molecular weight adducts increased with increasing cross-linking times (Fig. 6, lane 1 versus lane 2) or cross-linking agent concentrations (data not shown). By densitometric scanning of Western blots (Fig. 6A, lanes 1 and 4), GIRK1 and GIRK4 antigenicity profiles were developed (Fig. 6B). Cross-linking ladders were also created when native IKACh was treated with DMS, iodine, or DSS (Fig. 6, C, D, and inset to D). In addition, partial cross-linking of recombinant homomultimeric GIRK4 and GIRK1 channels yielded a laddered pattern which was most consistent with homotetrameric proteins. The mean molecular weights of the various adducts produced by cross-linking of native atrial IKACh and recombinant homomultimeric channels are summarized in Table II.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 6.   Partial cross-linking of pure, native IKACh is most consistent with a tetramer composed of two GIRK1 subunits and two GIRK4 subunits. Partial cross-linking produces adducts that represent monomers, dimers, trimers, and tetramers. A, pure, native IKACh was treated with 10 mM DMA for 15 min (lanes 1 and 4), 2 h (lanes 2 and 5), or DTSSP for 1 h (lanes 3 and 6). The products were analyzed by 3-10% SDS-PAGE and immunoblotted. Lanes 1-3 were immunoblotted with anti-GIRK4 antibodies; lanes 4-6 correspond to lanes 1-3 when stripped and immunoblotted with anti-GIRK1 antibodies. B, anti-GIRK1 (alpha GIRK1) and anti-GIRK4 (alpha GIRK4) antigenicity profiles were created by densitometry scanning of lanes 2 and 4 of A. Likewise, profiles created when native IKACh was treated with either iodine (C), DMS (D), or DSS (D, inset) are shown. gly, glycosylated.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Partial cross-linking of GIRK channels
1, GIRK1; 4, GIRK4. Molecular weights are given as a mean in kDa ± standard deviation (n, number of trials).

An Examination of Specific Dimer, Trimer and Tetramer Adducts Confirms That Native Atrial IKACh Is a Heterotetramer Composed of Two GIRK1 and Two GIRK4 Subunits-- Examination of dimers formed by partial cross-linking of purified native atrial ICh protein reveals GIRK4-GIRK4, GIRK1-GIRK4, and GIRK1-GIRK1 adducts (Fig. 6). As expected, the relative proportions of the specific dimeric adducts that formed depended on the side chain specificity, cross-linking span, and lipid solubility of the cross-linking agent. The formation of the GIRK1-GIRK1 and GIRK4-GIRK4 adducts demonstrated that native atrial IKACh heterotetramers are composed of two GIRK1 and two GIRK4 subunits and corroborates the previous densitometry experiments. The trimeric peak in Fig. 6C is composed of GIRK1-GIRK1-GIRK4 and GIRK4-GIRK4-GIRK1 adducts. As expected, the trimeric peak is broad and the GIRK4 peak antigenicity is shifted toward the lower molecular weights (~179 kDa), whereas the GIRK1 peak antigenicity is shifted toward the higher molecular weights (~188 kDa). On no occasion did such an antigenicity shift occur in the tetrameric adduct. The simplest interpretation of this pattern is that the native IKACh tetramer is composed of a single population of channels with two GIRK1 and two GIRK4 subunits.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We report the purification of a native mammalian K+ channel to near homogeneity and provide direct biochemical evidence for IKACh channel stoichiometry and quaternary structure. Using numerous independent methods, we have shown that GIRK proteins form tetramers and that native IKACh is most likely a tetramer composed of two GIRK1 subunits and two GIRK4 subunits.

After purification of native IKACh to greater than 95% homogeneity, we found that the channel was comprised of GIRK4 (48 kDa), GIRK1 (54 kDa), and glycosylated GIRK1 (56-76 kDa) subunits. The complex tightly bound WGA during purification, indicating that it contained terminal sialic acid residues. The purified product cross-linked into a single high molecular weight complex, indicating that the purified channel was an intact tetramer. The high degree of native channel protein purity was the key to our experiments because it eliminated potential nonspecific cross-linking between native IKACh and other unrelated membrane proteins. We cannot rule out the possibility that other populations of GIRK1-GIRK4 heteromultimers with alternate stoichiometries did not copurify. However, immunodepletion experiments illustrate that greater than 90% of GIRK4 is associated with GIRK1 (27) and that greater than 90% of GIRK1 is associated with GIRK4 (data not shown). These immunodepletion experiments verify the lack of significant quantities of native homomultimeric complexes, if they exist at all.

Chemical cross-linking has been widely used in the nearest neighbor analysis of membrane proteins and to study subunit organization (see Refs. 32-34 for review). The total number of subunits in both the glycine receptor (25) and Shaker channels (13) have been estimated by cross-linking approaches. Here, we have demonstrated that GIRK channels are tetrameric complexes by using two chemical cross-linking approaches. In the first approach, we cross-linked purified native IKACh heteromultimers, recombinant GIRK1 homomultimers, and recombinant GIRK4 homomultimers. Four reagents with different side chain specificities, lipid solubilities, and cross-linking spans all produced a single unique adduct, strongly indicating that the channels were purified in an intact state and then completely cross-linked. Native IKACh heteromultimers, recombinant GIRK1 homomultimers, and recombinant GIRK4 homomultimers formed complexes of ~234, 216, and 211 kDa, respectively, consistent with predicted molecular weights of ~226, 222, and 188 kDa, respectively. The use of cross-linking agents and iodoacetamide potentially complicates the interpretation of our results, because both of these reagents can covalently bind the protein and therefore might increase its apparent molecular weight. These agents may also alter the protein's mobility characteristics by changing its charge, hydrodynamic properties, or SDS binding. Nonetheless, others have shown that the molecular weight of cross-linked complexes determined by SDS-PAGE closely approximated the true molecular weight of heteromultimeric proteins (25, 32, 35, 36). On balance, the internal consistency of our results suggests that native IKACh components were purified to homogeneity and cross-linked into one complete complex. To verify that the products resulting from complete cross-linking were tetramers, we partially cross-linked native atrial IKACh, homomultimeric GIRK4, and homomultimeric GIRK1 channels. Partial cross-linking formed four adducts representing monomers, dimers, trimers, and tetramers. The molecular weight of the adducts increased with the total number of subunits cross-linked in a linear fashion, again supporting the conclusion that native atrial IKACh heteromultimers, recombinant GIRK1 homomultimers, and recombinant GIRK4 homomultimers all formed tetrameric complexes.

Three species of dimers, GIRK1-GIRK1, GIRK1-GIRK4, and GIRK4-GIRK4, were detected when native IKACh was partially cross-linked, based upon interpretation of molecular weights and Western blotting. The formation of GIRK1-GIRK1 and GIRK4-GIRK4 cross-linked subunits within the native IKACh tetramer indicated that the tetramer is composed of two GIRK1 subunits and two GIRK4 subunits in ~1:1 stoichiometry. The 1:1 stoichiometry was supported by two additional experimental approaches. First, silver-stained gels of purified IKACh yielded a 1:1 GIRK1:GIRK4 staining intensity ratio after correction for their respective molecular weights. Both the purification scheme and cross-linking experiments assured that only complete tetrameric heteromultimers were examined by densitometry. Second, immunoblotting of native IKACh was consistent with 1:1 stoichiometry provided that the blotting antibodies were first standardized against a known ratio of recombinant GIRK1 and GIRK4 proteins. Thus, three different experimental methods support the conclusion that GIRK1:GIRK4 stoichiometry is 1:1, and rule out a fixed 3:1 or 1:3 GIRK1:GIRK4 stoichiometry.

Experimental results similar to those shown here might have resulted from a random assembly of GIRK1 and GIRK4 subunits. This is an intriguing possibility, considering that in our heterologous COS7 expression system, the stoichiometry of GIRK1:GIRK4 varied directly with the ratio of DNA transfected, and would provide yet another way to contribute to K+ channel diversity. However, this interpretation would require that the 3:1 and 1:3 GIRK1:GIRK4 pools were of equal size to be compatible with the 1:1 stoichiometry determined by densitometry. Furthermore, large pools of complexes with 1:3 and 3:1 stoichiometries would not be consistent with the observed nearly identical anti-GIRK1 and anti-GIRK4 tetrameric adduct profiles. Thus, we currently favor the simplest interpretation of the data, which is a fixed 2:2 GIRK1:GIRK4 stoichiometry rather than a random association model.

Previous work has demonstrated that GIRK4 in expression systems may form homomultimeric ion channels (27, 37, 39), whereas putative GIRK1 homomultimers are not functional (37-39 ). Moreover, GIRK1, by itself, does not localize to the membrane (28). One possible explanation for these findings is that GIRK1 is unable to assemble with itself to form a tetramer, and is instead shuttled into a degradative pathway as monomers or aggregates upon translation. It thus appears that GIRK1 must form heteromultimers with another GIRK family member to function. Wischmeyer et al. (30) suggest that GIRK1 homomultimers may not exist in vivo due to the spatial conflict of bulky phenylalanines in the pore structure. In this study, we show that recombinant GIRK1 subunits can form homotetrameric complexes, although all evidence to date suggests they are not functional.

In summary, the experiments presented here demonstrate that GIRK channels form tetramers and that IKACh is most likely made up of two GIRK1 subunits and two GIRK4 subunits. Although we cannot rule out the possibility that there exist multiple pools of channels with varying stoichiometries, the consistency of channel conductances and kinetics from single-channel recordings in numerous species make this unlikely. This study lays the foundation for future biochemical studies on Gbeta gamma binding stoichiometry, and determination of K+ channel subunit arrangement around the pore.

    ACKNOWLEDGEMENTS

We thank Matt Kennedy and Kevin Wickman for critically reading the manuscript, Matt Kennedy for expertise and for providing epitope-tagged GIRK1 and GIRK4, and Yiping Chen for providing technical assistance.

    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: Cardiovascular Division, Children's Hospital, 1309 Enders, 320 Longwood Ave., Boston, MA 02115. Tel.: 617-355-6163; Fax: 617-730-0692; E-mail: clapham{at}rascal.med.harvard.edu.

1 The abbreviations used are: Kv, voltage-gated K+-selective channel; GIRK, G-protein-regulated, inwardly rectifying K+ channel; DTSSP, dithiobis(sulfosuccinimidylpropionate); DSS, disuccinimidyl suberate; SSADP, sulfosuccinimidyl (4-azidophenyldithio)propionate; DMA, dimethyl adipimidate·2HCl; DMS, dimethyl suberimidate·2HCl; WGA, wheat germ agglutinin; NHS, N-hydroxysuccinimide; PAGE, polyacrylamide gel electrophoresis; Kir, inwardly rectifying K+-selective channel; IP, immunoprecipitation; Ab, antibody; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Papazian, D., Schwarz, T., Tempel, B., Jan, Y., and Jan, L. (1987) Science 237, 749-753[Medline] [Order article via Infotrieve]
  2. Pongs, O., Kecskemethy, N., Muller, R., Krah-Jentgens, I., Baumann, A., Koltz, H., Canal, I., Llamazares, S., and Ferrus, A. (1988) EMBO J. 7, 1087-1096[Abstract]
  3. Iverson, L., Tanouye, M., Lester, H., Davidson, N., and Rudy, B. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5723-5727[Abstract]
  4. Soejima, M., and Noma, A. (1984) Pflugers Arch. 400, 424-431[Medline] [Order article via Infotrieve]
  5. Koumi, S. J., and Wasserstrom, J. A. (1994) Am. J. Physiol. 266, H1812-H1821[Abstract/Free Full Text]
  6. Ito, H., Hosoya, Y., Inanobe, A., Tomoike, H., and Endoh, M. (1995) Naunyn-Schmiedebergs Arch. Pharmocol. 351, 610-617
  7. Land, B. R., Salpeter, E. E., and Salpeter, M. M. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3736-3740[Abstract]
  8. Matthews-Bellinger, J., and Salpeter, M. (1978) J. Physiol. 279, 197-213 [Abstract]
  9. Fertuck, H. C., and Salpeter, M. M. (1976) J. Cell Biol. 69, 144-158[Abstract/Free Full Text]
  10. MacKinnon, R. (1991) Nature 350, 232-235[CrossRef][Medline] [Order article via Infotrieve]
  11. Liman, E., Tytgat, J., and Hess, P. (1992) Neuron 9, 861-871[Medline] [Order article via Infotrieve]
  12. Li, M., Unwin, N., Stauffer, K., Jan, Y., and Jan, L. (1994) Curr. Biol. 4, 110-115[Medline] [Order article via Infotrieve]
  13. Schulteis, C., Naomi, N., and Papazian, D. (1996) Biochemistry 35, 12133-12140[CrossRef][Medline] [Order article via Infotrieve]
  14. Li, M., Jan, Y. N., and Jan, L. Y. (1992) Science 257, 1225-1230[Medline] [Order article via Infotrieve]
  15. Tinker, A., Jan, Y., and Jan, L. (1996) Cell 87, 857-868[Medline] [Order article via Infotrieve]
  16. Yang, J., Jan, Y., and Jan, L. (1995) Neuron 15, 1441-1447[Medline] [Order article via Infotrieve]
  17. Clement, J., Kunjilwar, K., Gonzalez, G., Schwanstecher, M., Panten, U., Aguilar-Bryan, L., and Bryan, J. (1997) Neuron 18, 827-838[Medline] [Order article via Infotrieve]
  18. Pessia, M., Tucker, J., Lee, K., Bond, C., and Adelman, J. (1996) EMBO J. 15, 2980-2987[Abstract]
  19. Krapivinsky, G., Krapivinsky, L., Wickman, K., and Clapham, D. E. (1995) J. Biol. Chem. 270, 29059-29062[Abstract/Free Full Text]
  20. Wickman, K., Iniguez-Lluhi, J., Davenport, P., Taussig, R. A., Krapivinsky, G. B., Linder, M. E., Gilman, A., Clapham, D. E. (1994) Nature 368, 255-257[CrossRef][Medline] [Order article via Infotrieve]
  21. Inanobe, A., Ito, H., Ito, M., Hosoya, Y., and Kurachi, Y. (1995) Biochem. Biophys. Res. Commun. 217, 1238-1244[CrossRef][Medline] [Order article via Infotrieve]
  22. Silverman, S. K., Lester, H. A., and Dougherty, D. A. (1996) J. Biol. Chem. 271, 30524-30528[Abstract/Free Full Text]
  23. Tucker, S. J., Pessia, M., and Adelman, J. P. (1996) Am. J. Physiol. 271, H379-H385[Abstract/Free Full Text]
  24. McCormack, K., Lin, L., Iverson, L., Tanouye, M., and Sigworth, F. (1992) Biophys. J. 63, 1406-1411[Abstract]
  25. Langosch, D., Thomas, L., and Betz, H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7394-73988[Abstract]
  26. Slaughter, R. S., Sutko, J. L., and Reeves, J. P. (1983) J. Biol. Chem. 258, 3183-3190[Abstract/Free Full Text]
  27. Krapivinsky, G., Krapivinsky, L., Velimirovic, K., Wickman, B., Navarro, B., and Clapham, D. E. (1995) J. Biol. Chem. 270, 28777-28779[Abstract/Free Full Text]
  28. Kennedy, M., Nemec, J., and Clapham, D. E. (1996) Neuropharmacology 35, 831-839[CrossRef][Medline] [Order article via Infotrieve]
  29. Slesinger, P., Reuveny, E., Jan, Y., and Jan, L. (1995) Neuron 15, 1145-1156[Medline] [Order article via Infotrieve]
  30. Wischmeyer, E., Doring, F., Wischmeyer, E., Spauschus, A., Thomzig, A., Veh, R., and Karschin, A. (1997) Mol. Cell. Neurosci 9, 194-206[CrossRef][Medline] [Order article via Infotrieve]
  31. Huang, D., and Maero, S. (1997) BioTechniques 22, 454-458 [Medline] [Order article via Infotrieve]
  32. Davies, G., and Stark, G. (1970) Proc. Natl. Acad. Sci. U. S. A. 66, 651-656[Abstract]
  33. Gaffney, B. (1985) Biochim. Biophys. Acta 822, 289-317[Medline] [Order article via Infotrieve]
  34. Richards, F., and Peters, K. (1977) Annu. Rev. Biochem. 46, 523-551[CrossRef][Medline] [Order article via Infotrieve]
  35. Aris, J. P., and Simon, R. D. (1983) J. Biol. Chem. 258, 14599-14609[Abstract/Free Full Text]
  36. Mourrain, P., Lasa, I., Guatreau, A., Gouin, E., Pugsley, A., and Cossart, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10034-10039[Abstract/Free Full Text]
  37. Velimirovic, B., Gordon, E., Lim, N., Navarro, B., and Clapham, D. E. (1996) FEBS Lett. 379, 31-37[CrossRef][Medline] [Order article via Infotrieve]
  38. Hedin, K., Nancy, L., and Clapham, D. (1996) Neuron 16, 423-429[Medline] [Order article via Infotrieve]
  39. Krapivinsky, G., Gordon, E. A., Wickman, K. A., Velimirovic, B., Krapivinsky, L., Clapham, D. E. (1995) Nature 374, 135-141[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.