Identification of a Potassium Channel Site That Interacts with G Protein beta gamma Subunits to Mediate Agonist-induced Signaling*

Cheng He, Hailin ZhangDagger §, Tooraj MirshahiDagger , and Diomedes E. Logothetisparallel

From the Department of Physiology and Biophysics, Mount Sinai School of Medicine, City University of New York, New York, New York 10029

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of heterotrimeric GTP-binding (G) proteins by their coupled receptors, causes dissociation of the G protein alpha  and beta gamma subunits. Gbeta gamma subunits interact directly with G protein-gated inwardly rectifying K+ (GIRK) channels to stimulate their activity. In addition, free Gbeta gamma subunits, resulting from agonist-independent dissociation of G protein subunits, can account for a major component of the basal channel activity.

Using a series of chimeric constructs between GIRK4 and a Gbeta gamma -insensitive K+ channel, IRK1, we have identified a critical site of interaction of GIRK with Gbeta gamma . Mutation of Leu339 to Glu within this site impaired agonist-induced sensitivity and decreased binding to Gbeta gamma , without removing the Gbeta gamma contribution to basal currents. Mutation of the corresponding residue in GIRK1 (Leu333) resulted in a similar phenotype. Both the GIRK1 and GIRK4 subunits contributed equally to the agonist-induced sensitivity of the heteromultimeric channel. Thus, we have identified a channel site that interacts specifically with Gbeta gamma subunits released through receptor stimulation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Signaling through GTP-binding (G) proteins depends on dissociation of the heterotrimer Galpha beta gamma into the Galpha -GTP and Gbeta gamma subunits. Direct interactions of Galpha or Gbeta gamma (or both) with effector proteins transduces the external signal into an intracellular response. Atrial potassium (K+) channels, the first example of a Gbeta gamma effector (1), are responsible for the acetylcholine(ACh)1-induced reduction in heart rate during vagal activity (2).

Five members of the G protein-gated inwardly rectifying K+ (GIRK1-5) channel subfamily have been reported thus far (3-8). The presumed topology of these channels includes a cytoplasmic N terminus (~90 amino acids), followed by two transmembrane domains with the "ion selectivity" P-region in between (~100 amino acids) and ending with a long cytoplasmic C terminus (over 200 amino acids) (3, 9). GIRK channels can function as highly active heteromultimers (pairing of GIRK1 with any other subtype) or low to moderately active homomultimers (GIRK2-5) (for review, see Ref. 10). Mutations at a specific position within the P-region of these channels ("P-region mutants", e.g. GIRK4-S143T) greatly enhance the activity of homomultimers (11, 12). Use of these highly active point mutants simplifies the experimental design of structure-function studies and allows assessment of the relative contributions of each of the two subunits in the heteromultimeric complex (12).

Several studies have demonstrated direct binding of Gbeta gamma subunits to entire GIRK proteins (13) or to segments of channel subunits (14-19). Although Gbeta gamma subunits can interact directly with both N and C termini, interactions with the C terminus of the channel were shown to be the strongest (14, 15). In addition, the N terminus also binds to Galpha subunits alone (14) or to the Galpha beta gamma heterotrimer (14, 18).

The beta gamma subunits of G proteins activate not only native GIRK heteromultimers (1, 6), but also recombinant hetero- or homomultimeric GIRK channels (7, 20). There is no qualitative difference in the Gbeta gamma sensitivity of P-region homomultimeric mutants versus heteromultimeric channels (12). In contrast, the inwardly rectifying K+ channel IRK1 (21) is Gbeta gamma insensitive (22), despite its high degree of similarity to the five members of the GIRK subfamily.

We sought to identify those residues of GIRK critical for transducing effects of the Gbeta gamma subunits. Our strategy was to generate chimeras between the GIRK4(S143T) (referred to as GIRK4*) and IRK1 channels, and screen for differences in Gbeta gamma -dependent function and binding to Gbeta gamma . Mutagenesis at a single site, namely GIRK4(L339E), reduced binding to Gbeta gamma and impaired agonist-induced activity, but left intact the Gbeta gamma dependence of the basal activity. Thus, we have identified a site on an effector protein that interacts specifically with Gbeta gamma released through receptor stimulation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Human homologs of GIRK1 and GIRK4 (GenBankTM accession numbers U39195 and U39196) (7) or their point mutated active counterparts (GIRK1-F137S or GIRK1* and GIRK4-S143T or GIRK4*), subcloned in the pGEMHE plasmid vector (23), were used as described previously (11, 12). The chimeric cDNA constructs were produced by splicing by overlapping extension polymerase chain reaction (24). Polymerase chain reactions, using Vent DNA polymerase, were performed for only 15 cycles to avoid errors. Point mutations were generated using the Quickchange site-directed mutagenesis kit (Stratagene). The sequence of all constructs was confirmed by automated DNA sequencing (Sequencing facility, Cornell University, Ithaca, NY). The beta ARK-PH construct (amino acids 452-689) was altered to incorporate the 15 N-terminal residues of Src for membrane targeting. This construct, generously provided by Dr. Eitan Reuveny, was altered and subcloned into pGEMHE.

All constructs were linearized with Nhel and cRNAs were transcribed in vitro using the "message machine" kit (Ambion). RNAs were electrophoresed on formaldehyde gels and concentrations were estimated from two dilutions using an RNA marker (Life Technologies, Inc.) as a standard.

Xenopus oocytes were surgically extracted, dissociated, defolliculated by collagenase treatment, and microinjected with 50 nl of a water solution containing the desired cRNA. Unless otherwise indicated, we used the following approximate quantities: GIRK channel subunits, 1.0 ng/species; IRK1 channel, 0.25 ng; m2 receptor, 1.0 ng; beta 2-adrenergic receptor, 2.0 ng; Galpha and Gbeta subunits, 1.0 ng; Ggamma subunit, 1.0 ng; beta ARK-PH, 1.0 ng. Gbeta 2 and Ggamma 2 were used in all Gbeta gamma coexpression experiments, unless otherwise indicated. Oocytes were incubated for 3 days at 19 °C. Whole oocyte currents were then measured by conventional two-microelectrode voltage clamp with a GeneClamp 500 amplifier (Axon Instruments). Agarose cushion microelectrodes were used with resistances between 0.1 and 1.0 megaohms (25). Oocytes were constantly superfused with a high potassium solution having (in mM): 91 KCl, 1 NaCl, 1MgCl2, 5 KOH/HEPES (pH 7.4). To block or activate currents, the oocyte chamber was perfused with solutions of the same composition with 3 mM BaCl2 or 5 µM ACh. Typically oocytes were held at 0 mV (EK), and currents were constantly monitored by 500 ms pulses to a command potential of -80 mV for 200 ms followed by a step to +80 mV for another 200 ms, and the cycle was repeated every 2 s. Periodically, a protocol was applied with a command potential from -100 to +100 mV with 10 mV increments. Current amplitudes were measured at the end of the 200 ms pulse at each potential. In such manner, control currents were evaluated 2-5 min after impaling the oocytes just before application of ACh, ACh-activated currents were evaluated at the peak of the response to ACh, and Ba2+-insensitive currents were evaluated once steady-state inhibition was achieved, 1-3 min after application of 3 mM Ba2+. Basal current is the difference between control and Ba2+-insensitive currents, and ACh-induced (or ACh-sensitive) current the difference between ACh-activated and control currents. Error bars in the figures represent mean ± S.E. Each experiment shown or described was performed on 3-5 oocytes of the same batch. A minimum of 2-3 batches of oocytes were tested for each experiment shown.

Single-channel activity was recorded on devitellinized oocytes under the cell-attached mode of standard patch-clamp methods (26), as described previously (27). The pipette solution contained 96 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.4. The bath solution was composed of 96 mM KCl, 1 mM MgCl2, 5 mM EGTA, and 10 mM HEPES, pH 7.4. 100 µM gadolinium was also included in the pipette solution. Gbeta gamma purified from bovine brain was used in the inside-out experiments (gift from Dr. John D. Hildebrandt). Recordings were performed at a holding membrane potential of -80 mV. Recordings were performed using the EPC-9 patch-clamp amplifier and the PULSE/PULSEFIT (v. 7.6) data acquisition software (Heka Electronik, Lambrecht, Germany). Data were stored on the hard disk of a PC compatible computer, and single channel analysis made use of TAC (v 2.6.1) software (Skalar Instruments, Inc., Seattle, WA). The sampling rate was 4 kHz for most recordings. Activity expressed as NPo (N, number of channels in the patch; Po, probability of opening) was calculated by integrating the current traces over 30-60 s intervals and dividing by the unitary current.

Recombinant bovine Gbeta 1gamma 2 subunits were purified from Sf9 cells infected with baculoviruses encoding for beta 1, gamma 2, and His6-alpha i1 as described by Kozasa and Gilman (32). cDNAs encoding the C termini of GIRK4, GIRK4(L339E), or IRK1, and the PH domain of beta ARK were generated by polymerase chain reaction and cloned in frame with the GST coding sequence in pGEX-4T-3 (Amersham Pharmacia Biotech). The resulting polymerase chain reaction fragments coded for: amino acids 184-419 for GIRK4, GIRK4(L339E), amino acids 177-428 for IRK1; and amino acids 546-670 for beta ARK. Expression of fusion proteins was induced by 0.1 mM isopropyl-1-thio-b-D-galactopyranoside at 37 °C for 2 h, and the fusion proteins were purified using glutathione 4B-Sepharose beads. The binding assay of Gbeta gamma to the fusion proteins was performed as described by Huang et al. (14). Briefly, 1.0 µM GST fusion protein and 0.1 µM Gbeta gamma were incubated in phosphate-buffered saline, 0.1% lubrol and glutathione-Sepharose beads at 4 °C for 30 min. After washing three times with 200 volume of phosphate-buffered saline with 0.01% lubrol, the bound proteins were eluted from beads by heating in protein sample buffer at 70 °C for 10 min and were then electrophoresed in a 12% SDS-polyacrylamide gel electrophoresis. GST fusion proteins were visualized by Coomassie staining, and Gbeta 1 was detected by immunoblotting using a Gbeta antibody (Santa Cruz Biotechnology) and visualized with ECL (Amersham). Densitometry was used to quantify the relative amounts of bound Gbeta gamma .

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GIRK4* Unlike IRK1 Is Gbeta gamma -sensitive-- We compared Gbeta gamma sensitivities of basal and agonist-induced currents between the highly active GIRK4* and IRK1 channels. All experiments were carried out by expression in Xenopus oocytes. In whole-cell experiments, Gbeta gamma sensitivity, in the presence of coexpressed G protein-coupled receptor, was assessed by (a) K+ current responses to agonist stimulation, (b) coexpression of channels with proteins such as Gialpha subunits or the PH domain of beta ARK that can act as "sinks" for endogenous Gbeta gamma subunits, and (c) coexpression of channels with exogenous Gbeta gamma subunits.

Expression of GIRK4* in oocytes led to large basal and ACh-induced currents (Fig. 1, A and C). Coexpression of either beta ARK-PH or Gialpha 1 led to a significant reduction in basal currents. However, oocytes coexpressing Gialpha 1, rather than beta ARK-PH, displayed ACh-induced currents. This result is consistent with the interpretation that Gialpha 1, and not beta ARK-PH, bound to endogenous Gbeta gamma may be available for receptor-mediated activation. Coexpression of GIRK4 with exogenous Gbeta gamma -enhanced agonist-independent K+ currents while preserving the ACh-induced response (as in Ref. 12). These results indicate that both the GIRK4* basal and agonist-induced currents are largely mediated by the Gbeta gamma subunits. In contrast, oocytes injected with IRK1 exhibited no ACh-induced currents and did not respond to coexpression with Gialpha 1, beta ARK-PH, or Gbeta gamma (Fig. 1, B and C).


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Fig. 1.   Gbeta gamma sensitivity of the GIRK4* and IRK1 inwardly rectifying channels. A, current-voltage (I-V) relationships of basal and agonist-induced currents of GIRK4(S143T), designated as GIRK4*, coexpressed with human muscarinic receptor 2 (hm2). Coexpression with beta ARK-PH or Gialpha 1 or the stimulatory Gbeta gamma subunits is indicated. Squares represent basal currents, whereas circles indicate total currents in the presence of ACh. Points in the I-V curves represent mean ± S.E. for 3-5 oocytes within this batch of oocytes. B, current-voltage relationships of IRK1 under identical experimental conditions, as shown in A. C, basal and agonist-induced currents of the experiments described in A and B at -80 mV. Bars represent mean ± S.E. for three to five separate experiments.

A Minimal Chimera between GIRK4* and IRK1 with a Defect in Agonist-induced Responses-- We constructed chimeras between GIRK4* and IRK1 (Fig. 2, left). We screened for minimal segments of GIRK4* that when replaced by the corresponding IRK1 regions impaired sensitivity to Gbeta gamma .


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Fig. 2.   Chimeras between GIRK4* and IRK1 channels reveal a region important in agonist-induced stimulation of K+ currents. Left, schematic of chimeric constructs between GIRK4* and IRK1. Specific segments of IRK1 (black regions) were used to replace corresponding segments of GIRK4* (white regions). Each chimera was named according to the IRK1 segment (identified by the first and last IRK1 amino acid of the segment), which replaced the corresponding segment of GIRK4*. Note that the corresponding numbers, defining the limits of the grafted segments, are shifted (6 to 7 numbers) as determined by the alignment of the two sequences. Right, basal and agonist-induced currents at -80 mV of GIRK4*, IRK1, and the five chimeras. Asterisks note significant differences in ACh-induced currents of the particular chimeras from the GIRK4* control (GIRK4*, -10.5 ± 2.98 µA, n = 4; GIRK4*(IRKL316-Y341), -0.62 ± 0.52 µA, n = 4; p < 0.05).

Chimeras were named for the IRK1 segment replacing the corresponding GIRK4 region. We first replaced the full C terminus of GIRK4* with that of IRK1 (GIRK4*(IRKV179-I428)). This chimera showed intact basal but impaired agonist-induced currents, consistent with a previous report (22). Huang et al. (15) found the GIRK1(Glu318-Pro462) segment to be a minimal Gbeta gamma binding region. From an alignment of the GIRK1 and GIRK4 primary amino acid sequences, residue GIRK1(Glu318) corresponds to GIRK4(Asp324). Thus, we tested the response of the chimera GIRK4*(IRKL316-I428) that replaced the GIRK4(Met323-Val419) region with the corresponding IRK1 segment. Again this chimera exhibited intact basal but impaired agonist-induced currents. To narrow the region responsible for the aberration of the GIRK4* agonist-induced currents, we constructed and tested three additional chimeras GIRK4*(IRKL316-Y341), GIRK4*(IRKS342-K365), and GIRK4*(IRKY366-I428). All three chimeras displayed intact basal currents. However, the response of GIRK4*(IRKL316-Y341) was impaired to agonist. These results suggest that differences between the two channels in this region, GIRK4(Met323-Tyr348) and IRK1(Leu316-Tyr341), may be important in their differential sensitivity to Gbeta gamma . This is unlike the downstream regions where differences were without effects on Gbeta gamma sensitivity.

An Agonist-insensitive Chimera with Intact Gbeta gamma -mediated Basal Currents-- The current resulting from expression of the chimera GIRK4*(IRKL316-Y341) had intact basal currents but impaired agonist-induced responses. To test its sensitivity to Gbeta gamma , the GIRK4*(IRKL316-Y341) chimera was coexpressed with Gialpha 1 or beta ARK-PH. A significant reduction of basal currents was obtained, similar to the GIRK4* control (Fig. 3A). Yet, this chimera differed from GIRK4* (see Fig. 1) in that its expression alone or with Gialpha or Gbeta gamma resulted in impaired ACh-induced currents. This result further supports the conclusion that this chimeric channel is defective in producing agonist-induced currents. Coexpression with Gbeta gamma did not stimulate basal levels of activity. Because the Gbeta gamma dependence of the basal currents was intact in the GIRK4*(IRKL316-Y341) chimera, it is likely that other regions may be involved in Gbeta gamma mediation of basal currents.


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Fig. 3.   Gbeta gamma sensitivity of basal currents of the agonist-insensitive chimera and point mutants. A, effects on the basal currents (at -80 mV) of the agonist-insensitive chimera GIRK4*(IRKL316-Y341) coexpressed with Gbeta gamma , beta ARK-PH, or Gialpha 1. ACh responses were impaired in all groups p < 0.005, n = 3-5. The basal currents of GIRK4*(IRKL316-Y341) coexpressed with beta ARK-PH or Gialpha 1 were also significantly reduced p < 0.005, n = 3-5. B, currents (at -80 mV) from mutants resulting from substitution of amino acids within GIRK4*(M323-Y348) (corresponding to GIRK4*(IRKL316-Y341)), which differ between GIRK4 and IRK1. Each of eleven mutations were introduced into the context of the GIRK4* backbone. Asterisks denote significant reduction in ACh-induced currents (p < 0.005, n = 3).

A Point Mutation Sufficient to Specifically Impair Agonist-induced Currents without Affecting the Gbeta gamma Contribution to Basal Activity-- We proceeded to test which of the distinct residues within the identified region of the GIRK4* and IRK1 channels were responsible for their differences in sensitivity to Gbeta gamma . Eleven point mutations were made in which residues in the Met323-Tyr348 region of GIRK4* were mutated to the corresponding residues found in the Leu316-Tyr341 region of IRK1 (Fig. 3B). Mutant names refer to the position and amino acid of GIRK4 that was mutated to the corresponding IRK1 residue. Only GIRK4*(L339E) showed impaired agonist-induced responsiveness, mimicking the responses obtained with the GIRK4*(IRKL316-Y341) chimera.

We next tested the Gbeta gamma sensitivity of the basal currents of GIRK4*(L339E), and compared them with that of the GIRK4* control in the same batch of oocytes.

Oocytes coexpressing GIRK4*(L339E), Gbeta gamma , Gialpha 1, or beta ARK-PH behaved similar to the chimera GIRK4*(IRKL316-Y341), demonstrating an intact Gbeta gamma -mediated basal current component. (Fig. 4A). Inside-out patch recordings from oocytes expressing the mutant and control channels were performed to test their responses to Gbeta gamma subunits. Fig. 4B (left) compares activity from one batch of oocytes expressing GIRK4* and GIRK4*(L339E) channels. Perfusion of inside-out patches with purified Gbeta gamma was ineffective in stimulating GIRK4*(L339E) activity compared with control GIRK4*. Stimulation of currents by endogenous G proteins through GTPgamma S application gave similar results as the application of purified Gbeta gamma (data not shown, n > 3). Regardless of their sensitivity to Gbeta gamma , control or mutant channels responded to a similar degree to intracellular Na+ ions (27), thus providing a positive control for gating by Na+ ions. These inside-out patch responses were consistent with the whole-cell data for GIRK4*(L339E). Perhaps the lack of stimulation of whole-cell currents by Gbeta gamma coexpressed with GIRK4*(L339E) reflects maximal basal currents for this mutant. To examine this possibility, we coexpressed beta ARK-PH in oocytes (same batch as the experiments in Fig. 4B, left) with GIRK4* or GIRK4*(L339E) channels. We perfused inside-out patches from such oocytes with Gbeta gamma purified from bovine brain (Fig. 4B, right). Inside-out patches of oocytes coexpressing GIRK4*(L339E) and beta ARK-PH convincingly responded to perfusion with exogenous Gbeta gamma , presumably recovering the beta ARK-PH inhibition of the basal currents seen in the whole-cell experiments. However, these responses were significantly smaller than those of the control GIRK4*. In all cases, GTPgamma S application failed to stimulate channel activity by activating endogenous G proteins, serving as a positive control for beta ARK-PH effectiveness (data not shown, n > 3). Through these experiments we conclude that the GIRK4(L339E) mutation selectively impairs agonist-induced Gbeta gamma -mediated responses.


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Fig. 4.   Comparison of Gbeta gamma sensitivity of GIRK4* and GIRK4*(L339E) in whole-cell and inside-out patch experiments. A, two-electrode voltage clamp experiments plotting currents (at -80 mV) of GIRK4* and GIRK4*(L339E) channels coexpressed with Gbeta gamma , beta ARK-PH, or Gialpha 1. ACh responses were impaired in the GIRK4*(L339E) groups, p < 0.005, n = 3-6. The basal currents of GIRK4*(L339E) coexpressed with beta ARK-PH or Gialpha 1 were also significantly reduced, p < 0.005, n = 3-6. B, (left) inside-out patches from oocytes expressing the control or point mutant GIRK4* channels. Responses to patch perfusion with Gbeta gamma or Na+ are shown for a representative patch and a number of patches tested within this batch of oocytes. Gbeta gamma increased channel activity significantly compared with control for GIRK4* (p < 0.005, n = 3) but not for GIRK4*(L339E) (n = 4); (right) inside-out patches from oocytes coexpressing the control or point mutant GIRK4* channels and beta ARK-PH. Responses to patch perfusion with Gbeta gamma or Na+ are shown for a representative patch and a number of patches tested within this batch of oocytes. Gbeta gamma increased channel activity significantly compared with control for GIRK4* and GIRK4*(L339E) (p < 0.005, n = 4). The increase in channel activity in response to Gbeta gamma was significantly less in GIRK4*(L339E) compared with GIRK4* (p < 0.005, n = 3-4).

The C Terminus of GIRK4(L339E) Shows Decreased Binding to the Gbeta Subunit-- To determine the effects of the GIRK4(L339E) mutation of the C terminus on Gbeta gamma binding, we constructed and purified GST fusion proteins containing the C termini of GIRK4 (GIRK4C), GIRK4(L339E), (GIRK4- (L339E)C) and IRK1 (IRK1C), or beta ARK-PH. GST fusion proteins were expressed in bacteria and purified (Fig. 5, top). In vitro binding assays were performed with the recombinant bovine Gbeta 1gamma 2 subunits purified from Sf9 cells.


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Fig. 5.   The Leu339 mutation of the GIRK4 C terminus causes a significant reduction in Gbeta gamma binding. GST fusion proteins were purified using glutathione 4B-Sepharose beads and were detected by Coomassie staining (top). Purified GST fusion protein were incubated with Gbeta gamma and glutathione-Sepharose beads. Following wash, the bound proteins were released from the beads by heating in protein sample buffer and were separated by SDS-polyacrylamide gel electrophoresis. Gbeta 1 was detected by immunoblotting with anti-Gbeta antibody. The position of the beta  subunit of Gbeta gamma is indicated by the arrow. GST was used as negative control, and GST-beta ARK-PH as positive control (middle). Density scanning was used to quantify the relative amounts of bound Gbeta gamma (bottom). The results shown represents mean ± S.E. for four separate experiments. *, p < 0.01 compared with the GST-GIRK4C group.

As shown in Fig. 5 (middle and bottom panels), the C termini of GIRK4 and GIRK4(L339E) were able to bind Gbeta gamma as compared with negative controls (GST and IRK1C) and a positive control (beta ARK-PH). GIRK4(L339E)C binding to Gbeta gamma was significantly reduced. These results suggest that the critical Leu of GIRK channels, and perhaps neighboring residues, directly interacted with Gbeta gamma subunits. Additionally, because GIRK4(L339E)C has reduced but measurable binding to Gbeta gamma , it is likely that additional C-terminal Gbeta gamma binding sites exist, which contribute to the Gbeta gamma dependence of basal currents.

Wild-type GIRK1/GIRK4 Subunits Contribute Equally to the Agonist-induced Activity of Heteromultimeric Channels-- To determine whether the effect seen with the L339E mutant was specific to the GIRK4* subunit, we mutated the corresponding amino acid residue in GIRK1, L333E. We tested for Gbeta gamma sensitivity of this point mutant in the context of the highly active homomultimer GIRK1(F137S) (see Refs. 11 and 12; referred to as GIRK1*).

Similar results were obtained with the GIRK1*(L333E) mutant as with the GIRK4*(L339E). Again, although ACh-induced currents were impaired by the mutation, the basal currents were reduced by Gialpha 1 and beta ARK-PH (Fig. 6A, right).


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Fig. 6.   Mutation of the critical Leu residue in GIRK1* or wild-type heteromultimers GIRK1/GIRK4 also impairs agonist-induced responses. A, two-electrode voltage clamp experiments plotting currents (at -80 mV) of GIRK1* and GIRK1*(L333E) coexpressed with Gbeta gamma , beta ARK-PH, or Gialpha 1. B, representative records of inside-out patches show the response of GIRK1* and GIRK1*(L333E) currents to GTPgamma S application. C, Leu to Glu substitutions of the wild-type GIRK1 or GIRK4 (not in the context of GIRK1* or GIRK4*) were performed. Coexpression of each mutant with the complementary wild-type subunit and of both mutant subunits. The responses to ACh were reduced significantly for all mutant bearing heteromultimers GIRK1(L333E)/GIRK4, GIRK1/GIRK4(L339E), and GIRK1(L333E)/GIRK4(L339E) compared with wild-type GIRK1/GIRK4, p < 0.005, n = 4.

Fig. 6B shows that GTPgamma S application to inside-out patches expressing GIRK1* or GIRK1*(L333E) resulted in ~42-fold increase in GIRK1* activity but caused no increase in the current of GIRK1*(L333E) (n = 5).

We next sought to determine the relative contribution of wild-type GIRK subunits to agonist-induced activation in heteromultimeric channels (Fig. 6C). We introduced the Leu to Glu mutations at the 333 and 339 positions of the wild-type GIRK1 and GIRK4 subunits, respectively. We compared basal and agonist-induced currents of GIRK1/GIRK4 heteromultimeric channels, composed of both wild-type, both Leu to Glu mutants, and each wild-type to mutant combination. Our results suggest that each of the wild-type subunits contribute equally to agonist-induced activity, because heteromultimeric channels containing either Leu to Glu mutants displayed reduced agonist-induced sensitivity. Moreover, heteromultimeric channels, where both the subunits contained the Leu to Glu substitution showed significantly impaired agonist-induced currents. Thus, these results confirmed the importance of the residue for receptor-stimulated currents in heteromultimeric channels.

Mutation of the Critical Leu Residue Does Not Distinguish among Channel Interactions with Specific Gbeta Subunits or Signaling through Specific Receptors-- Yeast two-hybrid experiments have shown that Gbeta 1 and Gbeta 2 interact with the N terminus of GIRK1 more strongly than do Gbeta 3-5 (19). To determine whether C-terminal mutation of the critical Leu residue could have altered the ability of the channel to interact with specific Gbeta subunits, we coexpressed Gbeta 1, Gbeta 2, or Gbeta 3 with Ggamma 2 and GIRK1/GIRK4 or GIRK1(L333E)/GIRK4(L339E) heteromultimers. All Gbeta gamma combinations stimulated wild-type basal currents (2-4-fold, n = 3). When tested with the mutated channel subunits, Gbeta 1-3gamma 2 subunits failed to stimulate basal currents (n = 3). These results suggest that mutation of the critical Leu residue does not exert its effects by altering the specificity of channel/Gbeta 1-3 interactions. However, possible changes in the specificity of Leu mutant channels with the Gbeta 4 or Gbeta 5 subunits that were not tested cannot be ruled out.

Gbeta gamma subunits released from Gsalpha subunits by beta 2-adrenergic receptor stimulation activate GIRK channels expressed in Xenopus oocytes (28). To test whether the critical Leu residue is involved in Gbeta gamma signaling by receptors other than m2, we coexpressed beta 2-adrenergic receptor and Gsalpha subunits with GIRK1/GIRK4 or GIRK1(L333E)/GIRK4(L339E) heteromultimers. Isoproterenol-induced currents were obtained after expression of wild-type heteromultimers (-7.65 ± 2.17 µA at -80 mV, n = 3) but not with mutants (-0.17 ± 0.09 µA at -80 mV, n = 3). These results suggest that the Gbeta gamma released after activation of these two different receptors interact in a similar fashion with the critical GIRK Leu residue.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Since Soejima and Noma (29) first reported the membrane-delimited nature of the atrial muscarinic K+ channel, the mechanism of G protein gating of ion channels has received great attention. Over a decade ago, G protein-gated inwardly rectifying K channels provided the first example of a Gbeta gamma controlled signaling pathway. Yet, despite intense efforts, many questions remain unanswered regarding specific sites of interaction between the channel and Gbeta gamma .

Biochemical studies from several groups have pointed to interaction of Gbeta gamma with the C and N termini of these channels. Specifically, Huang et al. (14), using deletion mutagenesis, found that deletion of the GIRK1(Val273-Pro354) segment reduced Gbeta gamma binding of the remaining C-terminal fragment. In subsequent studies, Huang et al. (15) determined the GIRK1(Glu318-Pro462) segment as a minimal Gbeta gamma binding region. Kunkel and Peralta (16), using a combination of chimeras and deletion mutations, reported the GIRK1(Thr290-Tyr356) region to be important in interactions with Gbeta gamma .

In this study, we screened the C terminus of GIRK4* for residues that control channel activity. We made chimeras that replaced specific sections of GIRK4* with those from the G protein-insensitive channel, IRK1. Expression of a minimal chimera, the GIRK4*(IRKL316-Y341), resulted in normal basal currents that did not respond to ACh when coexpressed with hm2 receptors. Expression of exogenous Gbeta gamma did not enhance basal currents in this chimera. Yet, basal currents were inhibited by coexpression of Gialpha 1 and beta ARK-PH.

Of all the amino acid differences between IRK1 and GIRK4* contained in this chimera, only the mutant GIRK4*(L339E) retained all properties of the chimera. This mutant displayed Gbeta gamma -sensitive basal currents that were not ACh-sensitive and did not respond to exogenous Gbeta gamma . Inside-out patch currents from oocytes expressing GIRK4*(L339E) and hm2 receptors were significantly smaller in response to Gbeta gamma or GTPgamma S. Binding of the Gbeta gamma subunits to the GIRK4 C terminus bearing the L339E mutation was significantly reduced. Within the broader boundaries suggested by others (14-16), the region surrounding Leu339 is a Gbeta gamma binding site with critical functional consequences.

Basal currents from the GIRK4*(L339E) channel were inhibited by coexpression of Gbeta gamma sinks, such as the beta ARK-PH. Because binding of the L339E mutant of the GIRK4 C terminus was not abolished and because basal currents of GIRK4*(L339E) could be inhibited by beta ARK-PH or Gialpha 1, it is likely that additional Gbeta gamma binding sites contribute to basal channel activity. Moreover, because Gbeta gamma perfusion of inside-out patches activated GIRK4*(L339E) channels only when they were coexpressed with beta ARK-PH, it is likely that this activation reflected reversal of the beta ARK-PH inhibited basal currents. We hypothesize that the basal binding sites may be high affinity and saturated in both the whole-cell and inside-out patch experiments. Recent evidence has suggested another region of GIRK4 (Ser209-Arg225) capable of high affinity binding to Gbeta gamma (30). It is possible that such a site accounts for part or all of the basal channel activity.

How does GIRK4*(L339E) impair specifically agonist-induced stimulation? In the simplest model, free Gbeta gamma would be bound to high affinity basal sites. The GIRK4*(IRKL316-Y341) chimera and the GIRK4*(L339E) mutant may impair a low affinity binding of this region to Gbeta gamma subunits. Normally, agonist-induced liberation of Gbeta gamma subunits would increase the local free Gbeta gamma concentration, allowing interaction with a low affinity site, encompassing GIRK4*(Leu339), and leading to stimulation of channel activity. Further work will be required to test this hypothesis.

GIRK1*(L333E) channels displayed similar properties to GIRK4*(L339E). Again, basal currents from this mutant channel were sensitive to Gbeta gamma , but no ACh-induced currents could be detected. Double mutations in heteromultimeric GIRK1(L333E)/GIRK4(L339E) channels expressed in oocytes showed similar properties to the highly active homomultimeric mutants discussed above. Furthermore, mutation of both channels in a heteromultimer was required for the ACh-insensitive phenotype, whereas reduced agonist-induced currents were obtained with one or the other of the two subunits mutated. These results suggest that there is an equivalent contribution of GIRK1 and GIRK4 to Gbeta gamma -mediated ACh-induced activity. Additionally, coexpression of different Gbeta gamma combinations or different receptors such as the beta 2-adrenergic receptor did not alter the unique properties of these mutant channels. This suggests that signaling through different receptors and by different Gbeta gamma combinations activates the channel through conserved interactions.

Biochemical evidence has suggested multiple binding sites in the C- and N-terminal segments of GIRK channels (15, 19, 30). The multiplicity of Gbeta gamma binding sites with effector proteins is in agreement with the finding that the Gbeta gamma -phosducin co-crystals show multiple sites of interaction between the two proteins (31). Our data combine biochemical with functional evidence for more than one Gbeta gamma binding site on the channel. Surprisingly, distinct functional roles could be assigned to multiple binding sites; one designed to interact with Gbeta gamma released from receptor stimulation, whereas additional site(s) may interact with free Gbeta gamma to yield basal activity. Thus, these results suggest that for the K+ channel the multiplicity of interactions may subserve distinct functional roles.

    ACKNOWLEDGEMENTS

We are grateful to Eitan Reuveny for generously sharing with us the modified version of the beta ARK-PH construct and to Dr. Maureen Linder for graciously guiding us with the Gbeta gamma purification. We thank Drs. David Clapham and Robert Margolskee for critical review of the manuscript, Grigory Krapivinsky for advice with the binding studies, and Mariana Max, Eitan Reuveny, and Ming Ming Zhou for helpful discussions.

    FOOTNOTES

* This work was supported by Grants from the National Institutes of Health (HL54185) and American Heart Association (National Center 96011620) (to D. E. L.).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 These authors contributed equally to this work.

§ Associate of the Howard Hughes Medical Institute.

Supported by National Institutes of Health Training Grant (HL07824).

parallel To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Box 1218, Mount Sinai School of Medicine, CUNY, 1 Gustave L. Levy Pl., New York, NY 10029-6574. Tel.: 212-241-6285; Fax: 212-860-3369; E-mail: logothetis{at}msvax.mssm.edu.

    ABBREVIATIONS

The abbreviations used are: ACh, acetylcholine; GIRK, G protein-gated inwardly rectifying K+; PH, pleckstrin homology; GST, glutathione S-transferase; GTPgamma S, guanosine 5'-O-(thiotriphosphate).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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