Gbeta gamma Binding to GIRK4 Subunit Is Critical for G Protein-gated K+ Channel Activation*

Grigory Krapivinsky, Matthew E. Kennedy, Jan Nemec, Igor Medina, Luba Krapivinsky, and David E. ClaphamDagger

From the Howard Hughes Medical Institute, Childrens' Hospital, Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The cardiac G protein-gated K+ channel, IKACh, is directly activated by G protein beta gamma subunits (Gbeta gamma ). IKAChis composed of two inward rectifier K+ channel subunits, GIRK1 and GIRK4. Gbeta gamma binds to both GIRK1 and GIRK4 subunits of the heteromultimeric IKACh. Here we delineate the Gbeta gamma binding regions of IKACh by studying direct Gbeta gamma interaction with native purified IKACh, competition of this interaction with peptides derived from GIRK1 or GIRK4 amino acid sequences, mutational analysis of regions implicated in Gbeta gamma binding, and functional expression of mutated subunits in mammalian cells. Only two GIRK4 peptides, containing amino acids 209-225 or 226-245, effectively competed for Gbeta gamma binding. A single point mutation introduced into GIRK4 at position 216 (C216T) dramatically reduced the potency of the peptide in inhibiting Gbeta gamma binding and Gbeta gamma activation of expressed GIRK1/GIRK4(C216T) channels. Conversion of 5 amino acids in GIRK4 (226-245) to the corresponding amino acids found in the G protein-insensitive IRK1 channel, completely abolished peptide inhibition of Gbeta gamma binding to IKACh and Gbeta gamma activation of GIRK1/mutant GIRK4 channels. We conclude from this data that Gbeta gamma binding to GIRK4 is critical for IKACh activation.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Acetylcholine (ACh)1 secreted from the vagus nerve binds cardiac muscarinic receptors, initiating a sequence of events leading to slowing of heart rate (1-3). IKACh, an inwardly rectifying, K+-selective channel, mediates part of this process by hyperpolarizing pacemaker cells in sinoatrial and atrioventricular nodes of the heart (for review, see Ref. 4). Muscarinic, adenosine, and other receptors (5) all catalyze the release of Gbeta gamma subunits from pertussis toxin-sensitive heterotrimeric G proteins, which in turn directly activates the channel (6-9). Cardiac IKACh is composed of two homologous inward rectifier K+ channel subunits, GIRK1 (9, 10) and GIRK4 (11), which form a heterotetramer consisting of two GIRK1 and two GIRK4 subunits (12). Similar complexes comprised of GIRK1 and GIRK2 (13-17), or GIRK1 and GIRK3 (13) appear to form neuronal G protein-gated K+ channels. Both channel subunits bind Gbeta gamma with similar affinity (18-22).

An important finding that is crucial to understanding Gbeta gamma regulation of IKACh is that Gbeta gamma activates homomultimeric GIRK4 (11, 15) or homomultimeric GIRK2 (16) channels. This implies that the GIRK4 subunit alone contains the necessary elements required for activation. In contrast to GIRK2 and GIRK4, GIRK1 subunits are not functionally expressed as homomultimers (23-25), and this fact limits conclusions that can be drawn about GIRK1 contributions to Gbeta gamma regulation.

Initially, GIRK1 was thought to comprise the entire IKACh channel (9, 10), and initial studies on the mechanism of Gbeta gamma activation of IKACh focused on GIRK1. Chimeric channels were constructed from GIRK1 and non-G protein-sensitive K+ channel inward rectifier (Kir) subunits to examine this problem. Results from the expression of these chimeras in Xenopus oocytes were used to conclude that the GIRK1 C-terminal and/or N-terminal cytoplasmic domains (18, 26-28) conferred Gbeta gamma sensitivity on the channel complex. However, expression studies in Xenopus oocytes are complicated by the fact that GIRK1 forms a functional channel complex only by combining with an intrinsic oocyte GIRK4 homolog (XIR; Ref. 23). It should be noted that chimeras containing structural elements that promote interaction with XIR can be confused with elements responsible for Gbeta gamma regulation. Also, a consistent shortcoming of previous chimeric approaches has been the lack of direct biochemical determination of chimeric expression levels, plasma membrane targeting, and heterotetramer formation.

Several biochemical studies attempted to identify a Gbeta gamma binding site within channel subunits. These studies usually employed Gbeta gamma binding to glutathione S-transferase-fused GIRK1 fragments and demonstrated that Gbeta gamma bound fusion proteins containing regions of GIRK1 cytoplasmic N-terminal and/or C-terminal domains (18, 19, 21, 22).

Here we localize Gbeta gamma binding sites on the GIRK4 subunit of native cardiac IKACh using an in vitro Gbeta gamma binding assay and competition of binding with peptides derived from the subunit primary structure. We confirmed the functional significance of these binding sites using mutational analysis of regions implicated in Gbeta gamma binding and functional expression of mutated subunits in mammalian cells. We identified a single GIRK4 cysteine residue important for Gbeta gamma binding and demonstrated that Gbeta gamma binding to GIRK4 is critical for channel activation.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Protein Purification, Gbeta gamma Binding, and Peptide Preparation-- G proteins were isolated from bovine brain, separated into Galpha and Gbeta gamma subunits as described (29) and additionally purified by affinity chromatography over immobilized Galpha (30). Bovine atrial plasma membranes were isolated as described (31). Membranes were solubilized in 1.0% CHAPS-HEDN buffer (in mM: 10 HEPES, 1 EDTA, 1 dithiothreitol, and 100 NaCl) containing protease inhibitors and IKACh was purified by immunoprecipitation (12, 32). Immunoprecipitates were washed with solution containing 0.1% CHAPS-HEDN buffer and used for the Gbeta gamma binding assay (20). 20 amino acid length peptides derived from GIRK1 and GIRK4 amino acid sequences were synthesized in the Mayo Protein Core Facility (Mayo Clinic, Rochester, MN), high pressure liquid chromatography-purified, and their identity and purity verified by mass spectrometry. Peptide N and C termini were carboxymethylated or alpha -amidated, respectively. For competitive binding experiments, peptides were dialyzed against H2O for 48 h, their concentration measured by spectrophotometry (optical density at 205 nm), and the peptides were transferred into the binding assay solution. Gbeta gamma was preincubated with peptides for 20 min before incubation with the channel protein. Gbeta gamma was labeled with 125I using the Bolton-Hunter reagent (NEN Life Science Products) as described (20).

Cell Culture and Transfection-- CHO and COS-7 cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Sigma) at 37 °C, 5% CO2. Cells were plated at 3 × 106 or 2 × 106 cells/100-mm dish, respectively, 1 day prior to transfection. For electrophysiology experiments in CHO or COS-7 cells, each 100-mm plate was transfected with 5 µg of plasmid DNA, (2 µg of each plasmid containing either wild type or mutant GIRK1 or GIRK4 cDNA and 1 µg of pGREEN lantern-GFP (Life Technologies, Inc.) using TransIT LT-1 lipid transfection reagent (PanVera Corp, Madison, WI). For biochemical and immunofluorescence detection experiments in COS-7 cells, cells were transfected with 5 µg each of plasmids containing wild type and/or mutant subunit using TransIT LT-1 reagent (PanVera Corp, Madison, WI).

Site-directed Mutagenesis, Immunoflourescence Detection, and Subunit Coimmunoprecipitation-- Deletion and point mutants of GIRK1 and GIRK4 and the extracellular epitope-tagged GIRK1-Flag construct were generated by oligo-mediated mutagenesis using the site elimination method (Transformer Mutagenesis, CLONTECH). The pCDNA3 vector carrying the coding region of either C-terminally tagged GIRK1 (GIRK1-AU5) or GIRK4 (GIRK4-AU1) was used as a template for mutagenesis. Mutations were verified by DNA sequencing. Mutant subunits were evaluated for expression levels, cellular localization, and subunit interaction after transient expression in COS-7 cells as described (24). Triton X-100 was omitted in experiments designed to detect GIRK1-Flag cell surface expression.

Electrophysiological Analysis-- Transfected cells were visualized using an inverted fluorescence microscope to detect GFP expression. Electrophysiological recordings were performed from CHO-K1 and COS-7 cells using the patch clamp technique in the inside-out configuration. Pipette and bath solutions were identical and contained (in mM): 140 KCl, 5 EGTA, 10 HEPES-KOH, 2.0 MgCl2, pH 7.2. The resistance of the recording borosilicate glass pipettes was 2-4 megaohms. Single-channel currents were recorded at a holding potential of -80 mV using an Axopatch 200B amplifier (Axon Instruments, Inc.). Data were filtered at 5 kHz, digitized at 20 kHz through a 1200 DigiData A-D converter using pClamp 6 software (Axon Instruments, Inc.), and stored on disk for off-line analysis. The channel opening probability was calculated using pClamp 6 software (Axon Instruments, Inc.) from 5-s segments of inside-out patch recordings (33). Changes in concentrations of Gbeta gamma used in the dose-response experiments were achieved by transferring inside-out patches from the recording chamber to the Gbeta gamma -containing solution.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

To find the GIRK subunit locus responsible for Gbeta gamma binding to IKACh, we first studied in vitro binding of purified Gbeta gamma to purified cardiac IKACh. This approach included using peptides corresponding to stretches of the channel subunits in a competition binding assay. The cytoplasmic domains of GIRK4 and cytoplasmic domains of GIRK1 not strongly homologous to GIRK4, were screened by dividing them into contiguous nonoverlapping peptides of ~20-amino acid length. Our strategy assumed that peptides that correspond to Gbeta gamma -binding epitopes would interfere with the interaction of Gbeta gamma with GIRK channel subunits. Not every region of the GIRK subunit was examined. Peptides corresponding to transmembrane domains of GIRK were not used because it seemed unlikely that Gbeta gamma would interact with the channel's transmembrane domains. Also, we did not test amino acids 18-32 of GIRK4 because they comprise the epitope for the immunoprecipitating antibody, and would interfere with the binding assay. Finally, some synthesized peptides were insoluble and could not be evaluated. A summary of peptide influence on Gbeta gamma binding is shown in Fig. 1.


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Fig. 1.   Summary of GIRK1 and GIRK4 peptide competition. The diagram displays the regions of GIRK1 and GIRK4 that were synthesized as peptides to be used for competition experiments in an in vitro assay for Gbeta gamma binding to purified native atrial IKACh. In the lower panel, a two-dimensional plot of the GIRK1 and GIRK4 subunits highlights the two GIRK1 and two GIRK4 peptides that inhibit Gbeta gamma binding to native IKACh.

Almost all peptides tested at concentrations of 50-200 µM significantly inhibited Gbeta gamma binding to purified IKACh. However, inhibition was not consistent, varying from batch to batch of peptide. After peptides were dialyzed against distilled water, most peptides did not inhibit binding at concentrations less than 200 µM, but all weakly inhibited binding at higher concentrations. The simplest interpretation of this result was that peptide sample impurities inhibited Gbeta gamma interaction with the channel and that most of these impurities were removed by dialysis. All peptides we tested corresponding to the N terminus of both GIRK1 and GIRK4 were ineffective. Only four of the peptides tested against the C terminus inhibited in vitro Gbeta gamma binding to the channel in a concentration-dependent manner at concentrations significantly less than 200 µM (Fig. 2). The specificity of this inhibition was verified in experiments with scrambled and truncated peptides (Figs. 3, 4, and 6).


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Fig. 2.   Dose response curve for peptide inhibition of Gbeta gamma binding to native IKACh. Gbeta gamma binding was assayed in the presence of the peptides indicated. Binding activity was plotted as a function of peptide concentration and is given as percent of total binding in the absence of peptide. All data are shown as the mean of three independents assays (±S.E.).


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Fig. 3.   GIRK1 amino acids 364-367 participate in Gbeta gamma binding to IKACh but are not necessary for channel activation. A, sequence of peptides used to determine the specific amino acids within the 364-383 region of GIRK1 important for inhibition of binding. B, truncation of 4 amino acids (MLLM; bold in A) from the GIRK1(364-383) peptide eliminated its ability to inhibit Gbeta gamma binding. A peptide derived from adenylyl cyclase (QEHAQEPERQY) previously reported to inhibit Gbeta gamma activation of IKACh (34) did not inhibit Gbeta gamma binding to the channel (150 µM concentration). Binding activity was plotted as a function of peptide concentration and is given as percent of total binding in the absence of peptide (n = 6; ±S.E.). C, single-channel currents recorded from inside-out patches of COS-7 cells expressing either heteromultimeric wild type GIRK1/GIRK4 channels or mutant GIRK1(Delta 364-367)/GIRK4 channels, as indicated. Bath application of 20 nM Gbeta gamma similarly activated both wild type (n = 6) and mutant (n = 4) channels. Jumps to +80 mV confirmed that the channel was inwardly rectifying.


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Fig. 4.   Cysteine 216 of the GIRK4(209-225) inhibitory peptide is critical for inhibition of Gbeta gamma binding to IKACh. A, comparison of the amino acid sequence of the GIRK4(209-225) peptide with homologous sequences from other inward rectifier K+ channel subunits. Only 2 amino acids (Ala-202 and Trp-212) in the Gbeta gamma -insensitive IRK1 differ from the corresponding region of the GIRK subunits shown. GIRK1 contained a unique Thr at position 210, which corresponded to Cys-216 in GIRK4. B, the GIRK1 peptide containing the unique Thr residue did not inhibit Gbeta gamma binding in contrast to the GIRK4(209-225) peptide. The IRK1(202-218) peptide did inhibit Gbeta gamma binding. GIRK1(203-219) peptide was 50 µM, whereas other peptides were 10 µM. Binding activity is given as percent of total binding in the absence of peptide (n = 6; ±S.E.).

The GIRK1(364-383) peptide (Fig. 2) potently inhibited Gbeta gamma binding to native IKACh. Truncation of only 4 amino acids from this peptide (amino acids 364-367; MLLM) resulted in its complete inactivation (Fig. 3B), leading us to conclude that amino acids 364-367 in GIRK1 participated in Gbeta gamma binding to IKACh. However, heteromultimeric channels containing the mutant GIRK1 subunit in which amino acids 364-367 had been deleted were activated by Gbeta gamma indistinguishably from wild type GIRK1/GIRK4 channels (Fig. 3C). Because the peptide competition result suggested that the MLLM sequence was critical for inhibition of Gbeta gamma binding to the channel complex, and deletion of these amino acids from GIRK1 did not change channel activation, it is possible that GIRK1(364-383) inhibited IKACh activation by competition with a Gbeta gamma -binding domain on GIRK4. Indeed GIRK1(364-383) significantly inhibited Gbeta gamma binding to the recombinant GIRK4 subunit, which was expressed and purified from COS-7 cells. 6 of the 8 amino acids of the 268-275 stretch of GIRK4 were homologous to this region of GIRK1 (5/8 identical). Unfortunately we were not able to directly test this site for Gbeta gamma binding using peptide blockade because the GIRK4 peptide derived from this area was insoluble. The region from GIRK1 (364-383) also contains a motif previously implicated in Gbeta gamma activation of IKACh. Chen et al. (34) suggested that the putative Gbeta gamma binding region (motif (Q/N)XXER) of adenylyl cyclase type 2 (AC2 (956-982); Fig. 3A) had homology to GIRK1(378-382). Indeed, their experiments showed that AC2 (956-982) peptide blocked cardiac IKACh activation in excised inside-out patches. We assessed the functional role of GIRK1(378-382) by examination of whether AC2 (956-966) and homologous GIRK1(378-387) peptides inhibited Gbeta gamma binding to IKACh but failed to detect any inhibition (Fig. 3B). We also mutated and deleted the putative Gbeta gamma binding motif NSKER (GIRK1 378-382) and coexpressed the mutant GIRK1 with GIRK4 in COS-7 cells but found no difference in activation by Gbeta gamma (n = 4; data not shown).

As shown in Fig. 2, the most potent inhibitor of Gbeta gamma binding to IKACh was the GIRK4 (209-225) peptide. This peptide corresponded to the carboxyl-terminal region of GIRK4, lying just outside the second putative transmembrane domain (Fig. 1). GIRK4 (209-225) completely inhibited Gbeta gamma binding at concentrations of 3-5 µM. Deletion of amino acids 209-225 of the GIRK4 subunit (mutant GIRK4(Delta 209-225)) and expression with wild type GIRK1 abolished Gbeta gamma activation (Fig. 5C). However we failed to detect significant coimmunoprecipitation of GIRK1 with mutant GIRK4(Delta 209-225) nor did GIRK4(Delta 209-225) promote detectable cell surface localization of GIRK1 (Fig. 7) characteristic for wild type GIRK4 (24). The simplest interpretation of these data is that the region 209-225 in GIRK4 is critical for both Gbeta gamma binding and subunit interaction.

To refine the area responsible for the peptide inhibition, we compared the sequence of this peptide to homologous sequences of related Gbeta gamma binding GIRK subunits, as well as to corresponding sequences of the Gbeta gamma -insensitive IRK1 (Fig. 4A). Comparative analysis suggested that residues Ser-209, Phe-219, or Asp-224 might be involved in the interaction with Gbeta gamma . Mutated GIRK4 peptide (F219W) and homologous peptides derived from GIRK1(203-219) and IRK1(202-218) were then used to test the significance of these amino acids in Gbeta gamma binding. Surprisingly, the IRK1(202-218) peptide inhibited Gbeta gamma binding as well as the peptide GIRK4(209-225; Fig. 4B). Also, the GIRK1-derived peptide, GIRK1(203-219), did not inhibit Gbeta gamma binding at concentrations up to 50 µM (Fig. 4B). Sequence comparison suggests that the substitution of Cys-216 in GIRK4 for Thr at position 210 in the homologous GIRK1 peptide might be responsible for the observed difference in potency of inhibition between GIRK4 and GIRK1 peptides. To test this possibility, the GIRK4 (C216T) mutant subunit was coexpressed in CHO cells with GIRK1. As for COS-7 cells, CHO cells do not contain endogenous GIRK-like proteins (tested with GIRK-specific antibodies; data not shown) and do not support functional expression of GIRK1(11). Heteromultimeric channels containing the GIRK4(C216T) mutant produced functionally active IKACh channels but with a greater than 50-fold lower EC50 for Gbeta gamma (Fig. 5, A and C). The rightward shift in potency of Gbeta gamma activation was not because of changes in either surface localization or heteromultimerization with GIRK1 (Fig. 7), confirming that Cys-216 in GIRK4 is important for binding of Gbeta gamma to IKACh.


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Fig. 5.   Dose response relation for Gbeta gamma activation of wild type and mutant GIRK4 subunits coexpressed with wild type GIRK1. Single channel recordings from inside-out patches of CHO cells expressing either GIRK1/GIRK4 (A) or GIRK1/GIRK4(C216T) (B). Note the much lower Gbeta gamma -sensitivity in B. Single channel amplitudes were not affected by the C216T GIRK4 mutation. C, the GIRK1/GIRK4(C216T) mutant channel was significantly less sensitive to Gbeta gamma (>50-fold) than the GIRK1/GIRK4 wild type channel (n = 8 for mutant, n = 9 for wild type; ±S.E.). Deletion of GIRK4 amino acids 209-225 led to loss of Gbeta gamma -dependent GIRK1/GIRK4(Delta 209-225) channel activity (n = 11).

A peptide designed from an adjacent region of GIRK4, GIRK4(226-245), was also found to significantly inhibit Gbeta gamma binding to IKACh(Fig. 2). Again, sequence comparison between other Gbeta gamma binding GIRK subunits and the homologous, but G protein-independent inward rectifier, IRK1, led us to evaluate 5 amino acids potentially participating in Gbeta gamma binding (Fig. 6A). An IRK1 peptide amino acids 219-238, homologous to GIRK4(226-245), did not inhibit Gbeta gamma binding to IKACh. Mutation of these 5 amino acids in GIRK4 to the corresponding amino acids in IRK1 ablated Gbeta gamma activation after coexpression of this mutant GIRK4 with GIRK1 (Fig. 6B). These specific GIRK4 mutations did not influence assembly with GIRK1 or targeting to the plasma membrane (Fig. 7). These data support the hypothesis that the GIRK4(226-245) locus is also important for the Gbeta gamma binding and activation.


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Fig. 6.   Amino acids 226-245 of GIRK4 are necessary for Gbeta gamma binding to and activation of IKACh. A, comparison of amino acids 226-245 of GIRK4 with homologous sequences from other inward rectifier K+ channel subunits. 5 amino acids of GIRK4 not conserved in IRK1 were evaluated for their potential importance for Gbeta gamma binding (letters in bold at top). B, the IRK1 peptide homologous to the GIRK4(226-245) peptide did not inhibit Gbeta gamma binding. This suggests that these 5 amino acids were crucial for the inhibitory potency of the GIRK4(226-245) peptide. The scrambled GIRK4(226-245) peptide did not inhibit Gbeta gamma binding, demonstrating its specificity. C, single channel recordings from inside-out patches of GIRK1/mutant GIRK4(N226K, S233H, K237Q, Q243I and K245S) expressed in COS-7 cells. Gbeta gamma -induced channel activity was completely abolished (n = 21 for COS-7 and n = 5 for CHO cells).


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Fig. 7.   Immunolocalization and subunit interaction of GIRK1/GIRK4 subunits. Panels A, C, E, and G, immunofluorescence detection of wild type or mutant GIRK4 subunits expressed alone in COS-7 cells (Triton X-100 permeabilized cells; anti-AU1 monoclonal antibody); panels B, D, and F, mutant GIRK4 subunits promoted cell surface localization of the GIRK1-Flag epitope-tagged subunit when coexpressed in COS-7 cells (cells not permeabilized). In panels E and F the GIRK1 right-arrow IRK1 226-245 designation corresponds to the mutant GIRK4(N226K, S233H, K237Q, Q243I, and K245S) subunit. Panel H, coexpression of GIRK1 and the mutant GIRK4(Delta 209-225) did not result in cell surface expression of the GIRK1-Flag epitope-tagged subunit. Immunolocalization experiments were performed at least twice. Panel I, coimmunoprecipitation of wild type GIRK1-AU5 subunits (middle panel) with anti-AU1 antibody directed against the wild type or mutant GIRK4-AU1 subunit. The GIRK4(Delta 209-225) mutant protein displayed significantly lower levels of expression and coimmunoprecipitation of GIRK1 protein (right panel).

Finally, we tested a peptide derived from the GIRK1 region (220-239) homologous to GIRK4(226-245), which contained amino acids conserved in all GIRKs. The peptide inhibited Gbeta gamma binding to IKACh (Fig. 2) with a dose response similar to GIRK4(226-245). This result was not surprising because this region is highly conserved between GIRK4 and GIRK1. We suggest that the GIRK1(220-239) peptide also inhibited Gbeta gamma binding to the previously identified GIRK4(226-245). However, we cannot rule out the possibility that this region of GIRK1 is also important for interaction of channel with Gbeta gamma .

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have combined peptide competition and mutagenesis techniques to delineate domains within GIRK1 and GIRK4 that bind Gbeta gamma and play a role in Gbeta gamma -mediated activation of GIRK1/GIRK4 channels. In summary, we have identified a single GIRK4 region (amino acids 209-245) that appears to be crucial for Gbeta gamma binding. This region lies close to the putative second transmembrane domain of GIRK4.

We have identified several peptides derived from both GIRK1 and GIRK4 that block Gbeta gamma binding to immunopurified cardiac IKACh. In confirmation of these results, the mutation of regions that bound Gbeta gamma also disrupted Gbeta gamma -mediated channel activation. Deletion and point mutations in regions corresponding to blocking peptides from GIRK4 either completely disrupted or significantly reduced the potency of Gbeta gamma in activating GIRK1/mutant GIRK4 channels measured in inside-out patches exposed to purified Gbeta gamma subunits. Interestingly, deletion of GIRK1 amino acids corresponding to the peptide capable of inhibiting Gbeta gamma binding to IKACh had no effect on Gbeta gamma activation of GIRK1(Delta 364-367)/GIRK4 channels. This result suggests that either the GIRK1 peptide inhibited binding to homologous sites on the GIRK4 subunit or that Gbeta gamma binding to the GIRK1 site was not necessary for channel activation. The observation that amino acids 273-462 of GIRK1 bound Gbeta gamma subunits (19) supports our hypothesis that Gbeta gamma binding to GIRK1 is not required for channel activation. We found that Cys-216 in GIRK4 is important for high affinity binding of Gbeta gamma to the native channel. This same position is occupied by Thr in GIRK1. Because mutation of Cys-216 in GIRK4 to threonine dramatically reduced the potency of Gbeta gamma , we suggest that Gbeta gamma does not bind to this region of GIRK1. Combined with the known Gbeta gamma -dependent activation of GIRK4 homomultimers, we suggest that GIRK4 plays a necessary role in the Gbeta gamma -dependent activation of heteromultimeric IKACh.

Huang et al. (19) showed that a peptide corresponding to amino acids 434-462 of GIRK1 inhibited single channel activity in oocytes expressing GIRK1 and Gbeta gamma or GIRK1/GIRK4 and Gbeta gamma . In contrast, we found that peptides derived from this region of GIRK1 had no effect on Gbeta gamma binding. Furthermore truncation of GIRK1 at amino acid 373 and coexpression with GIRK4 in COS-7 cells did not alter channel acitivation.2 Although Huang et al. (22) found a role for N-terminal cytoplasmic domain (34-86 in GIRK1) binding to Gbeta gamma we could not confirm this result because peptides corresponding to these regions of GIRK1(67-83) or GIRK4(66-85) were insoluble and could not be tested in our binding assay.

We found that some peptides, which did not influence Gbeta gamma binding to the channel, nevertheless suppressed channel activation by Gbeta gamma when applied to inside-out patches. A reasonable explanation of these results is that the peptides interacted with the channel itself and interfered with intersubunit interaction. Such an explanation is supported indirectly by noncompetitive inhibition of the GIRK1/XIR channel by a C-terminal GIRK1 peptide (35). Similarly, the fact that an IRK1 peptide was also found to block Gbeta gamma binding to IKACh does not imply that IRK1 has a Gbeta gamma binding site, although this possibility cannot be excluded.

In conclusion, Gbeta gamma binding to IKACh channels involves a region of the carboxyl tail of GIRK4 in close proximity to the putative second transmembrane domain, and binding of Gbeta gamma to this region is critical for channel activation. Interestingly, these regions lie close to the putative pore domain of the tetrameric complex, perhaps providing an important clue as to why the weaver mouse GIRK2 pore mutant (a close relative of GIRK4) demonstrated loss of Gbeta gamma sensitivity (16). Although we have shown that Gbeta gamma binds to GIRK1, our attempts to demonstrate that this binding plays a role in Gbeta gamma -induced channel activation were not successful. However, it is likely that a combined GIRK1/GIRK4 conformation determines the Gbeta gamma binding site, and activation cannot be fully understood in the absence of detailed structural data.

    FOOTNOTES

* This work was supported by National Institutes of Health, National Heart, Lung, and Blood Institute Grant 54873 (to D. E. C.), National Research Service Award/Howard Hughes Medical Institute (to M. K.), the Mayo Foundation (to J. N.), and the Howard Hughes Medical Institute (to D. E. C.).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.

Dagger To whom correspondence should be addressed: Howard Hughes Medical Institute, Professor of Neurobiology, Pediatrics Childrens' Hospital, Harvard Medical School, 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: ACh, acetylcholine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CHO, Chinese hamster ovary.

2 M. E. Kennedy, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Loewi, O., and Navratil, E. (1926) Pflugers Arch. Eur. J. Physiol. 189, 239-242
  2. Hartzell, H. C., Kuffler, S. W., Stickgold, R., and Yoshikami, D. (1977) J. Physiol. 271, 817-846[Medline] [Order article via Infotrieve]
  3. Wickman, K., Nemec, J., Gendler, S., and Clapham, D. E. (1998) Neuron 20, 103-114[Medline] [Order article via Infotrieve]
  4. Ackerman, M. J., and Clapham, D. E. (1997) N. Engl. J. Med. 336, 1575-1586[Free Full Text]
  5. Kurachi, Y. (1995) Am. J. Physiol. 269, C821-C830[Abstract/Free Full Text]
  6. Logothetis, D. E., Kurachi, Y., Galper, J., Neer, E. J., and Clapham, D. E. (1987) Nature 325, 321-326[CrossRef][Medline] [Order article via Infotrieve]
  7. Wickman, K., 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[CrossRef][Medline] [Order article via Infotrieve]
  8. Clapham, D. E., and Neer, E. J. (1997) Ann. Rev. Pharmacol. 37, 167-204[CrossRef][Medline] [Order article via Infotrieve]
  9. Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y. (1993) Nature 364, 802-806[CrossRef][Medline] [Order article via Infotrieve]
  10. Dascal, N., Schreibmayer, W., Lim, N. F., Wang, W., Chavkin, C., DiMagno, L., Labarca, C., Kieffer, B. L., Gaveriaux, R. C., Trollinger, D., Lester, H. A., and Davidson, N. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10235-10239[Abstract]
  11. Krapivinsky, G., Gordon, E., Wickman, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141[CrossRef][Medline] [Order article via Infotrieve]
  12. Corey, S., Krapivinsky, G., Krapivinsky, L., and Clapham, D. E. (1998) J. Biol. Chem. 273, 5271-5278[Abstract/Free Full Text]
  13. Lesage, F., Duprat, F., Fink, M., Guillemare, E., Coppola, T., Lazdunski, M., and Hugnot, J. P. (1994) FEBS Lett. 353, 37-42[CrossRef][Medline] [Order article via Infotrieve]
  14. Kofuji, P., Davidson, N., and Lester, H. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6542-6546[Abstract]
  15. Velimirovic, B. M., Gordon, E. A., Lim, N. F., Navarro, B., and Clapham, D. E. (1996) FEBS Lett. 379, 31-37[CrossRef][Medline] [Order article via Infotrieve]
  16. Navarro, B., Kennedy, M. E., Velimirovic, B., Bhat, D., Peterson, A., and Clapham, D. E. (1996) Science 272, 1950-1953[Abstract]
  17. Liao, Y. J., Jan, Y. N., and Jan, L. Y. (1996) J. Neurosci. 16, 7137-7150[Abstract/Free Full Text]
  18. Kunkel, M. T., and Peralta, E. G. (1995) Cell 83, 443-449[Medline] [Order article via Infotrieve]
  19. Huang, C. L., Slesinger, P. A., Casey, P. J., Jan, Y. N., and Jan, L. Y. (1995) Neuron 15, 1133-1143[Medline] [Order article via Infotrieve]
  20. Krapivinsky, G., Krapivinsky, L., Wickman, K., and Clapham, D. E. (1995) J. Biol. Chem. 270, 29059-29062[Abstract/Free Full Text]
  21. Inanobe, A., Morishige, K. I., Takahashi, N., Ito, H., Yamada, M., Takumi, T., Nishina, H., Takahashi, K., Kanaho, Y., Katada, T., and Kurachi, Y. (1995) Biochem. Biophys. Res. Commun. 212, 1022-1028[CrossRef][Medline] [Order article via Infotrieve]
  22. Huang, C. L., Jan, Y. N., and Jan, L. Y. (1997) FEBS Lett. 405, 291-298[CrossRef][Medline] [Order article via Infotrieve]
  23. Hedin, K. E., Lim, N. F., and Clapham, D. E. (1996) Neuron 16, 423-429[Medline] [Order article via Infotrieve]
  24. Kennedy, M. E., Nemec, J., and Clapham, D. E. (1996) Neuropharmacology 35, 831-839[CrossRef][Medline] [Order article via Infotrieve]
  25. Woodward, R., Stevens, E. B., and Murrell-Lagnado, R. D. (1997) J. Biol. Chem. 272, 10823-10830[Abstract/Free Full Text]
  26. Takao, K., Yoshii, M., Kanda, A., Kokubun, S., and Nukada, T. (1994) Neuron 13, 747-755[Medline] [Order article via Infotrieve]
  27. Slesinger, P. A., Reuveny, E., Jan, Y. N., and Jan, L. Y. (1995) Neuron 15, 1145-1156[Medline] [Order article via Infotrieve]
  28. Tucker, S. J., Pessia, M., and Adelman, J. P. (1996) Am. J. Physiol. 40, 379-385
  29. Sternweis, P. C., and Robishaw, J. (1984) J. Biol. Chem. 259, 13806-13813[Abstract/Free Full Text]
  30. Ueda, N., Iñiguez-Lluhi, J. A., Lee, E., Smrcka, A. V., Robishaw, J. D., and Gilman, A. G. (1994) J. Biol. Chem. 269, 4388-4395[Abstract/Free Full Text]
  31. Slaughter, R. S., Sutko, J. L., and Reeves, J. P. (1983) J. Biol. Chem. 258, 3183-3190[Abstract/Free Full Text]
  32. Krapivinsky, G., Krapivinsky, L., Velimirovic, B., Wickman, K., Navarro, B., and Clapham, D. E. (1995) J. Biol. Chem. 270, 28777-28779[Abstract/Free Full Text]
  33. Logothetis, D. E., Kurachi, Y., Galper, J., Neer, E., and Clapham, D. E. (1987) Nature 325, 321-326[CrossRef][Medline] [Order article via Infotrieve]
  34. Chen, J., DeVivio, M., Dingus, J., Harry, A., Li, J., Sui, J., Carty, D. J., Blank, J. L., Exton, J. H., Stoffel, R. H., Inglese, J., Lefkowitz, R. J., Logothetis, D. E., Hildebrandt, J. D., and Iynegar, R. (1995) Science 268, 1166-1169[Medline] [Order article via Infotrieve]
  35. Luchian, T., Dacal, N., Dessauer, C., Platzer, D., Davidson, N., Lester, H. A., and Screibmayer, W. (1997) J. Physiol. 505.1, 13-22


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