Critical Determinants of the G Protein γ Subunits in the Gβγ Stimulation of G Protein-activated Inwardly Rectifying Potassium (GIRK) Channel Activity*

The βγ subunits of G proteins modulate inwardly rectifying potassium (GIRK) channels through direct interactions. Although GIRK currents are stimulated by mammalian Gβγ subunits, we show that they were inhibited by the yeast Gβγ (Ste4/Ste18) subunits. A chimera between the yeast and the mammalian Gβ1 subunits (ymβ) stimulated or inhibited GIRK currents, depending on whether it was co-expressed with mammalian or yeast Gγ subunits, respectively. This result underscores the critical functional influence of the Gγ subunits on the effectiveness of the Gβγ complex. A series of chimeras between Gγ2 and the yeast Gγ revealed that the C-terminal half of the Gγ2 subunit is required for channel activation by the Gβγ complex. Point mutations of Gγ2 to the corresponding yeast Gγ residues identified several amino acids that reduced significantly the ability of Gβγ to stimulate channel activity, an effect that was not due to improper association with Gβ. Most of the identified critical Gγ residues clustered together, forming an intricate network of interactions with the Gβ subunit, defining an interaction surface of the Gβγ complex with GIRK channels. These results show for the first time a functional role for Gγ in the effector role of Gβγ.

The ␤␥ subunits of G proteins modulate inwardly rectifying potassium (GIRK) channels through direct interactions. Although GIRK currents are stimulated by mammalian G␤␥ subunits, we show that they were inhibited by the yeast G␤␥ (Ste4/Ste18) subunits. A chimera between the yeast and the mammalian G␤ 1 subunits (ym␤) stimulated or inhibited GIRK currents, depending on whether it was co-expressed with mammalian or yeast G␥ subunits, respectively. This result underscores the critical functional influence of the G␥ subunits on the effectiveness of the G␤␥ complex. A series of chimeras between G␥ 2 and the yeast G␥ revealed that the C-terminal half of the G␥ 2 subunit is required for channel activation by the G␤␥ complex. Point mutations of G␥ 2 to the corresponding yeast G␥ residues identified several amino acids that reduced significantly the ability of G␤␥ to stimulate channel activity, an effect that was not due to improper association with G␤. Most of the identified critical G␥ residues clustered together, forming an intricate network of interactions with the G␤ subunit, defining an interaction surface of the G␤␥ complex with GIRK channels. These results show for the first time a functional role for G␥ in the effector role of G␤␥.
Heterotrimeric guanine nucleotide GTP-binding (G) proteins are composed of ␣, ␤, and ␥ subunits at a ratio of 1:1:1 (1). G proteins play important roles in a variety of transmembrane signaling pathways that are activated by extracellular hormones, neurotransmitters, chemokines, local mediators, and sensory stimuli (2)(3)(4). An agonist-bound G protein-coupled receptor activates the G proteins by generating GTP-bound ␣ subunits and triggering the release of the ␤␥ subunits from the heterotrimer. Both the ␣ and ␤␥ subunits are then able to regulate downstream effectors through direct interactions. Once the G␣-GTP subunit is inactivated through GTP hydrol-ysis, the G␣-GDP subunit reassociates with the G␤␥ subunits to form the inactive heterotrimer, which can interact with the G protein-coupled receptor and lead to a new signaling cycle.
G protein ␥ subunits are a large family of proteins with distinctly different primary structures, with Ͻ20% identity among 13 isoforms. In contrast, the ␤ subunits display Ͼ90% identity among the G␤ 1 -␤ 4 isoforms (5,6). Under physiological conditions, the G␤␥ complex acts as a functional monomer where the ␤ and ␥ subunits never dissociate from each other. Multiple isoforms, including 5 G␤ and 13 G␥ subunits, have been identified in mammals so far (5,7). The diversity seen in the number of subunit isoforms and the degree of sequence identity among them implies that G␥ subunits may confer specificity to particular G␤␥ complexes in modulating effector function. Post-translational modifications of the ␥ subunits have been shown to be involved in controlling the interaction of ␤␥ dimers with their ␣ subunits (8), in membrane anchoring (9 -11); and in coupling to specific receptors (12,13) or effectors, e.g. phospholipase C (14), phosphoinositide kinase (15), ␤-adrenergic receptor kinase (16), ion channels (10), and microtubules (17). However, specific effects on downstream effectors result mainly from the diversity conferred by the combination of the various ␤ and ␥ isoforms. The exact role of ␥ subunits in G protein signaling to particular effectors such as the G protein-activated inwardly rectifying potassium (GIRK) 1 channels remains poorly understood. One important strategy for probing the role of G␥ subunits in GIRK function would be to identify structural determinants of G␥ subunits that might be essential for G␤␥ effects on GIRK channels.
GIRK channels were the first example of an effector found to be directly modulated by the G␤␥ dimers (18). Four mammalian GIRK channels identified so far (19 -22) are functionally expressed as homo-or heterotetramers in multiple tissues including heart (GIRK1/GIRK4) and brain (predominantly GIRK1/GIRK2) (23), where these channels have been best studied. GIRK channels belong to the inwardly rectifying K ϩ (Kir) channel family that is structurally characterized by a pore helix that controls a loop that forms the K ϩ selectivity filter, flanked by two putative transmembrane helices, M1 and M2, that extend to intracellular N and C termini. Both the N and C termini of GIRK channels harbor the sites of interaction with the G␤␥ subunits (24 -33). Expression of either GIRK1 or GIRK4 alone in heterologous cell systems, Xenopus oocytes, or mammalian cell lines, reveals moderate to no K ϩ currents (23). Introduction of a mutation within the pore helix region of * This work was supported by National Institutes of Health Grants HL54185 (to D. E. L.) and GM61119 (to J. P. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 1. Yeast G␤␥ inhibits GIRK4* channel activity. In A, the upper panels show representative barium-sensitive current versus voltage relationships constructed by averaging data from oocytes expressing GIRK4* (left panel), GIRK4* ϩ yeast ␤␥ (middle panel), and GIRK4* ϩ ␤ 1 ␥ 2 (right panel). The GIRK4* channel currents were inhibited by yeast G␤␥ but were activated by mammalian ␤ 1 ␥ 2 subunits. The lower panels show IRK1 currents (left panel) IRK1 ϩ yeast ␤␥ and summary data for all experiments shown (right panel). In the summary plot, basal currents at Ϫ80 mV were averaged for a representative batch of oocytes tested (right, *, p Ͻ 0.01, unpaired Student's t test, n ϭ 7-9). In B, the yeast ␤␥ inhibited, whereas the mammalian ␤␥ stimulated, GIRK4* currents. Currents measured at Ϫ80 mV are shown. Yeast or mammalian G␤ and G␥ expressed alone or co-expressed with the complimentary subunit of the other species did not show any significant effect on GIRK4* currents (*, p Ͻ 0.01, n ϭ 6 -9). GIRK1 (F137S) or GIRK4 (S143T, GIRK4*) channels results in highly active G protein-sensitive K ϩ currents (34). Use of the modified homomeric channel simplifies the experimental design for structure-function studies and provides an efficient approach to addressing the contribution of each of the subunits of the G␤␥ dimer in signaling to the channel. Co-expression of G␣ subunits with GIRK channels inhibits basal current activity, presumably through binding free G␤␥ subunits that are responsible for the basal activity (25).
In the present study, we have found that the G protein subunits most distantly related to the mammalian ones, the yeast G␤␥ (Ste4/Ste18) (5), inhibit GIRK4* currents. Combining biochemical and electrophysiological approaches, we have screened a series of chimeras between G␤ 1 or G␥ 2 and their yeast counterparts. A minimal region localized in the middle of the primary amino acid sequence of ␥ 2 was identified to be necessary for activation of GIRK4* channels. Ten residue differences between G␥ 2 and its yeast counterpart, most of which are clustered together in the G␥ 2 three-dimensional structure, proved critical for channel activation. This cluster of residues is located close to G␤ residues that have been previously identified to be functionally important (35). These studies define a molecular surface that is critical for G␤␥ effects on GIRK channels, suggesting that multiple residues of ␥ 2 play a role in regulating GIRK4* currents.

EXPERIMENTAL PROCEDURES
Construction of Various Recombinant Plasmids-Yeast genomic DNA was isolated from DY150 cells (Clontech) according to standard protocol. cDNAs encoding Ste4 and Ste18 (GenBank TM accession numbers: M23982 and M23983) were amplified using PCR with Pfu DNA polymerase (Stratagene) and subcloned into the Xenopus expression vector pGEMHE (36). Similarly, cRNAs for GIRK4*, G␤ 1 , and G␥ 2 were also subcloned into pGEMHE. Gene splicing by overlap extension was used to generate the chimeric cDNAs between G␤ 1 and STE4 or G␥ 2 and STE18. Site-specific mutations were introduced by the QuikChange site-directed mutagenesis kit (Strategene). FLAG-G␤ 1 was constructed by attaching the FLAG epitope (DYKDDDDK) to the N terminus of the cDNA. The correct sequence of each construct was verified by DNA sequencing (DNA Sequencing Facility, Cornell University, or Invitrogen).
All constructs were linearized with NheI or SphI (New England Biolabs), and in vitro transcription of cRNA was performed using Ambion's mMessage mMachine kit (Ambion) with T7 polymerase. cRNAs were electrophoresed on 1% formaldehyde gels, and concentrations were estimated from two dilutions compared with the RNA marker (Invitrogen). cRNAs were dissolved in nuclease-free water to a concentration of 1 mg/ml. One ng of each cRNA was generally injected into each oocyte, unless otherwise indicated. The cRNAs listed below were injected in the following approximate quantities: GIRK channel subunits, 1.0 ng/species; IRK1 channel, 1.0 ng; m2 receptor, 1.5 ng; G␤ subunit, 1.0 ng; G␥ 2 subunit, 1.0 ng; STE4, 1.0 ng; STE18, 1.0 ng; chimeras and mutants, 1.0 ng.

FIG. 2. A chimera between the mammalian and the yeast G␤ subunits
gives the mammalian or yeast phenotype on GIRK4* currents upon co-expression with the corresponding G␥ subunit. A, schematic of mammalianyeast G␤ chimeras used in this study. Mammalian ␤ 1 (m␤) and yeast ␤ (y␤) subunits are depicted in open and black rectangles, respectively. The chimera ym␤ contains the N-terminal 35 residues of yeast preceding the mammalian ␤ subunit. The numbers on the top of each segment represent the amino acid region taken from the specified subunit. In B, basal currents measured at Ϫ80 mV come from oocytes expressing each chimera with GIRK4* except control groups. Currents have been normalized relative to the GIRK4* control. Chimera ym␤ stimulated GIRK4* currents when expressed with mammalian G␥ 2 , but inhibited currents when expressed with the yeast G␥ (*, p Ͻ 0.01, n ϭ 8 -15). m␤␥, mammalian G␤ 1 ␥ 2 ; y␤␥, yeast G␤␥. In C, the inhibited basal current of GIRK4* channels mediated by chimera ym␤ with yeast G␥ can be overcome by co-expressing increasing concentrations of the mammalian G␥ cRNA. m␥/y␥(1:1), m␥/y␥ (3:1), and m␥/y␥ (6: 1)represent the injected m␥ cRNA of 1, 3, and 6 ng/oocyte as compared with 1 ng of y␥ cRNA, respectively (*, p Ͻ 0.01, n ϭ 7-12). sium reversal potential (E K ), and currents were constantly monitored by 500-ms pulses to a command potential of Ϫ80 mV for 200 ms followed by a step to ϩ80 mV for another 200 ms, and the cycle was repeated every 2 s. Periodically, a protocol was applied with a command potential from Ϫ100 with steps to ϩ100 mV in 10-mV increments. Current amplitudes were measured at the end of the 200-ms pulse at each potential. Control currents were evaluated 2-5 min after impaling the oocytes, whereas Ba 2ϩ -insensitive currents were evaluated once steadystate inhibition was achieved, 1-3 min after application of 3 mM Ba 2ϩ . Basal current represents the difference between the control and the Ba 2ϩ -insensitive currents. Error bars in the figures represent mean Ϯ 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 was tested for each experiment shown.
Preparation of Crude Membranes-The injected or uninjected oocytes were lysed with a glass homogenizer in lysis buffer (150 mM NaCl, 50 mM Tris, 1 mM EDTA, pH 7.5, supplemented with a protease inhibitor mixture (Sigma). Next, they were incubated on ice for 1 h. Following two rounds of low speed centrifugation (900 ϫ g for 5 min at 4°C), the insoluble debris was removed, and the clear supernatant was subjected to high speed centrifugation (100,000 ϫ g for 45 min at 4°C). The pellet was dissolved in lysis buffer with 1% Nonidet P-40 in a volume of 2 l/oocyte. Immunoprecipitation, Trypsin Assay, and Immunoblotting-Crude membranes were mixed with the FLAG (M2) agarose affinity gel (Sigma) at 4°C overnight. Immune complexes with beads were washed three times and mixed with the lysis buffer. A limited trypsin digestion assay of the FLAG-␤ 1 ␥ 2 complexes was performed as described previously (38) with small modifications. Briefly, the protein bound to the beads was directly subjected to trypsin digestion (L-1-tosylamido-2phenylethyl chloromethylketone-treated trypsin, Sigma T-8642) at 37°C for 30 min. The reaction was terminated by the addition of equal volume of sample buffer (100 mM Tris-HCl (pH 6.8) 4% SDS, 5% 2-mercapthethanol, 0.2% bromphenol blue, and 20% glycerol) and was boiled for 5 min. After removal of the beads by a transient spin, the same amount of protein was loaded on a 12% SDS-polyacrylamide gel. Following electrophoresis, the samples were transferred to nitrocellulose membranes (Millipore) using a semidry electroblotter (120 mV for 45 min at room temperature). The membranes were blocked with blocking buffer (50 mM Tris-HCl (pH 7.5), 80 mM NaCl, 0.1% Tween 20, 5% non-fat dry milk, and 0.02% NaN 3 ), and the protein was immunoblotted with anti-G␤ polyclonal antibody (T-20, Santa Cruz Biotechnology). The bound antibody was detected by horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology), and chemiluminescence was used to visualize the bands (Amersham Biosciences).

RESULTS
Yeast G␤␥ Inhibits GIRK4* Basal Currents-We set out to test the effect of yeast G␤␥ subunits on GIRK4* channels. Oocytes were injected with cRNA for GIRK4*. We measured basal K ϩ currents using a two-electrode voltage clamp (Fig.  1A). As expected, co-expression of the channel with G␤ 1 ␥ 2 subunits resulted in stimulated inwardly rectifying currents, as FIG. 3. A small region on mammalian G␥ 2 that is important for GIRK4* current activation mediated by the G␤␥ dimer. A, alignment of the yeast G␥ against the full-length G␥ 2 . Among the 71 amino acids, 11 (in the black background) were identical to the corresponding residues in the yeast G␥, and 13 (in the gray background) showed similarity of residue features. The comparison was based on the published structural motifs of G␥ 2 (44). B, schematic illustration of chimeric constructs between G␥ 2 and yeast G␥. G␥ 2 is depicted in the open rectangle, whereas the yeast G␥ is depicted in the black rectangle. The chimeras were named as GS and identified by the positions of residues that they contain from each of the G␥ subunits. The numbers shown are always referring to G␥ 2 positions. In C, basal currents are shown measured at Ϫ80 mV from oocytes injected with equal amounts of cRNA for each ␥ chimera in the same background of GIRK4* and wild-type G␤ 1 cRNAs. The chimera (GS-6) harboring the residues 35-63 of G␥ 2 significantly stimulated GIRK4* currents as compared with oocytes expressing the channel alone, but the reverse chimera (GS-9) containing the yeast ␥ region corresponding to the residues 35-63 of G␥ 2 failed to activate the GIRK4* channel. The further narrowed chimera (GS-11) containing the minimal region 35-50 of G␥ 2 caused significant channel stimulation of activity. As expected, the chimera (GS-2) harboring the G␥ 2 -(35-71) gave currents similar to wild-type G␥ 2 control (*, p Ͻ 0.01, n ϭ 8 -12). compared with control (34). Expression of yeast G␤␥ subunits significantly inhibited basal (Fig. 1A) but not agonist-dependent GIRK4*currents (data not shown). The effects of yeast G␤␥ subunits on GIRK currents appeared similar to those of the mammalian G␤ 5 ␥ 2 subunits (35,39). In contrast, the inwardly rectifying K ϩ channel IRK1, which is insensitive to G proteins (25,40), was not affected by the presence of yeast G␤␥ subunits (Fig. 1A). We next mixed and matched mammalian and yeast G␤ and G␥ subunits and tested their effects on GIRK4* currents. Only G␤ and G␥ dimers of the same species showed either stimulatory (mammalian) or inhibitory (yeast) effects on GIRK4* currents (Fig. 1B).
Yeast G␥ and Mammalian G␥ 2 Exert Opposite Effects on the Function of GIRK4* Channels by Associating with a Yeast/ Mammalian G␤ Chimera-To determine the molecular determinants of the functional difference between the yeast G␥ and the mammalian G␥ 2 on the G␤␥-mediated stimulation of GIRK4* channels, we constructed chimeras between the yeast G␤ and the mammalian G␤ and screened for stimulatory or inhibitory effects on GIRK4* currents. One chimera, where the first 35 amino acids of the yeast G␤ were added to the intact mammalian G␤ 1 subunit (ym␤), could yield the expected effect on GIRK channel activity upon association with either the mammalian or the yeast G␥ subunit. cRNAs of the ym␤ chimera, GIRK4* channels, the mammalian G␥ 2 (m␥), or the yeast G␥ (y␥) were co-injected into oocytes (Fig. 2A). The controls, mammalian G␤ 1 ␥ 2 (m␤␥) or yeast G␤␥ (y␤␥), were tested each time on the same batch of oocytes. Fig. 2B shows the agonistindependent (basal) current measured by expression of GIRK4* alone or together with mammalian and or yeast G␤␥ subunits. The ym␤ chimera stimulated GIRK4* currents upon co-expression with the mammalian G␥ and inhibited currents upon co-expression with the yeast G␥. This result strongly suggested that the N-terminal first 35 amino acids of the yeast G␤ is sufficient to confer the inhibitory phenotype of the yeast G␤␥ to GIRK4* currents, without affecting the stimulatory effect of the mammalian G␥ 2 . Moreover, this result underscores the importance of the G␥ subunit in the G␤␥ complex as the same G␤ subunit (ym␤) was stimulatory, when complexed with the mammalian G␥ subunit, or inhibitory, when complexed with the yeast G␥ subunit. When stoichiometric amounts of both the mammalian and yeast G␥ were co-expressed with the ym␤ chimera, no significant effect on GIRK4* currents was observed. However, when progressively greater amounts of FIG. 4. Ten residues on G␥ 2 reduced the ability of G␤␥ to stimulate GIRK4* activity. In A, 33 mutants of G␥ 2 (spanning the region from residue 35 to 71 of the subunit) in which residues were replaced with the corresponding yeast G␥ residues were screened by measuring basal currents at Ϫ80 mV from oocytes co-expressing GIRK4* and wild-type G␤ 1 . Ten mutants shown in boldface (A35L, E42S, D48N, P49H, P53G, V54L, P55K, P60S, F61S, R62N) significantly reduced the ability of G␤ 1 to stimulate GIRK4* currents as compared with oocytes expressing GIRK4* alone and the channel with wild-type G␤ 1 ␥ 2 , respectively. The remaining 23 mutants activated the channel by forming dimer with G␤ 1 in a manner similar to control. Data were expressed as normalized basal current of the control GIRK4* (*, p Ͻ 0.01, n ϭ 8 -25). B, limited trypsin protection assay for the identified G␥ 2 mutants, which were co-expressed with FLAG-G␤ 1 (fG␤1) in oocytes. The crude membrane proteins of oocytes were subjected to precipitation by anti-FLAG antibody and incubated either with or without trypsin for 30 min at 37°C, as indicated. Digested proteins were subsequently analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotted with G␤ C-terminal antibody as described under ''Experimental Procedures.'' Molecular masses of the separated fragments were estimated using the relative mobility of markers run alongside (not shown). The sample of uninjected oocytes showed that no G␤␥ complexes were precipitated (lanes 1 and 2). Expressed FLAG-␤ 1 alone showed a correct band (lane 3) but was completely digested in the presence of trypsin (lane 4). The G␥ 2 mutants, like wild-type G␥ 2 , showed protection of a typical ϳ26-kDa fragment of the C terminus of G␤ 1 in the digestion with trypsin (lanes . This experiment was repeated three times, yielding similar results. mammalian G␥ were expressed relative to the yeast G␥, then a correspondingly greater stimulatory effect was obtained. These data suggest that the competition of the appropriate G␤␥ for the channel sites yields either a stimulatory effect (the mammalian phenotype) or an inhibitory effect (the yeast phenotype).
The C-terminal Half of the G␥ 2 Subunit Is a Crucial Contributor to the GIRK4* Current Stimulation by G␤ 1 ␥ 2 -To identify a minimal region of G␥ 2 responsible for activating GIRK4* channel currents, chimeric constructs between the mammalian G␥ 2 and the yeast G␥ were made based on a sequence comparison between the two subunits (showing a 15% identity between the G␥ 2 and the yeast G␥ proteins) (Fig. 3A). Each of these G␥ chimeras were co-injected with G␤ 1 , and their effects on GIRK4* currents were compared with control. G␥ chimeras GS-2, GS-6, and GS-11 showed significant stimulation of GIRK4* currents upon co-expression with G␤ 1 . All these chimeras had in common the G␥ 2 amino acid region between residues 35 and 50, suggesting that this region contains important determinants for the stimulatory effects of G␥ 2 on GIRK4* currents. Since the GS-11 chimera did not produce as much GIRK4* current as the GS-2 chimera or the wild-type G␥ 2 , it is likely that the residue 51-71 region contains determinants in addition to those found in the residue 35-50 region that are important for maximal G␤␥ stimulation of GIRK4* currents. Another chimera, GS-10 incorporating the unique 13-residue insert Lys-68 -Val-80 in the yeast G␥ sequence onto the mammalian G␥ (m1-44y68 -80m45-71), did not interfere with the ability of the G␤/GS-10 complex to stimulate GIRK currents (data not shown). Thus, the 13-residue insert did not appear to be responsible for the inhibitory effect of the yeast G␥ on the G␤␥ complex with the mammalian G␤ subunit.
Ten Amino Acids of G␥ 2 Reduce the Ability of G␤␥ to Stimulate GIRK4* Currents-We proceeded to identify the specific amino acids within the residue 35-71 region of G␥ 2 responsible for the G␥ contribution to the stimulatory effects of the G␤␥ complex on GIRK4* currents. Each residue of G␥ 2 was mutated to its counterpart in the yeast G␥ and was co-expressed with GIRK4* channels in oocytes. Whole-cell currents were recorded, allowing us to screen for the ability of each mutant to increase channel currents relative to the wild-type G␤ 1 (Fig.  4A). Of the 33 mutants of G␥ 2 tested, 23 mutants showed the ability to enhance GIRK4* currents to levels that were not significantly different from those of the wild-type G␤ 1 ␥ 2 . The remaining 10 single-point mutants (A35L, E42S, D48N, P49H,  P53G, V54L, P55K, P60S, F61S, and R62N) of G␥ 2 , however, significantly decreased the ability to activate the channel current but did not completely abolish stimulation of channel activity, implying that negation of the contribution from multiple residues of G␥ 2 might be required for disrupting full stimulation of the GIRK channel by G␤ 1 ␥ 2 subunits. The inability of the mutants to cause full GIRK stimulation raises the question of whether the impairment of the channel function could be due to a change in the native conformation of G␤ 1 ␥ 2 .
To test for such a possibility, we linked a FLAG epitope at the N terminus of G␤ 1 to distinguish it from endogenous G␤ and identified the expressed G␤ 1 in oocytes by immunoprecipitation with an anti-FLAG antibody conjugated to agarose beads (Sigma). The FLAG-tagged G␤ 1 showed similar stimulation of K ϩ currents as compared with the non-tagged G␤ 1 co-expressed with G␥ 2 (data not shown). For the pulled-down proteins, limited tryptic digestion was performed (9,38). Fig. 4B shows that a typical band of 26 kDa was protected from trypsin digestion. In contrast, expression of G␤ 1 alone failed to confer trypsin resistance, which is accomplished by proper association of the G␤ with a G␥ subunit. (Fig. 4B, lanes 3 and 4). These results confirm that the G␥ 2 mutants that show significant functional impairment still assemble properly with wild-type G␤ 1 to produce G␤␥-stimulated currents. The weaker enhancement of the channel currents by the G␥ mutants is likely to be caused by a functional impairment rather than a global structural defect.
G␥ Residues Co-localize with G␤ Residues, All of Which Are Critical for Mediating the G␤␥ Effects on GIRK Channel Activity-To better understand the nature of interactions between G␥ 2 and GIRK channels, we mapped the identified critical residues onto the known crystal structure of the G␤ 1 ␥ 2 complex. Fig. 5A (left panel) shows a ribbon diagram of the G␤ 1 ␥ 2 complex adapted from the published coordinates (5). G␤ 1 is shown in white, and G␥ 2 is shown in yellow. Identified residues on G␥ 2 are colored in cyan. Except for Ala-35 and Glu-42, the remaining 8 identified residues cluster in the same region of the complex. The G␥ 2 structure is composed of two ␣-helical domains and two loops. Eight clustered residues are in a loop in close proximity to the lipid modification that is used to anchor the protein to the membrane. All 7 of the critical residues are located around a groove that is formed from the interaction of G␤ and G␥. Ser-67 in G␤, which was previously identified to play an important role in controlling GIRK channel activity (35), resides on the side of this groove defined by the majority of the identified mutations. Several residues in this region maintain interactions between G␤ and G␥. Mapping the 7 FIG. 5. Localization on the protein structure of G␥ residues critical for GIRK activation. Left panel, a ribbon model of the G␤ 1 ␥ 2 complex rendered from published coordinates (5). G␥ is shown in yellow, and G␤ is shown in white. G␥ residues identified in this study are shown in cyan. Except Ala-35, all other critical residues cluster in a region close to the C terminus of G␥ 2 . These residues are in close proximity to a region in G␤ 1 that has been shown to play a critical role in GIRK channel activation (e.g. G␤-Ser-67) (35). Right panel, the molecular surface of the G␤␥ complex shows overall shape and distribution of the residues identified. Again, G␥ is shown in yellow, G␤ is shown in white, and the functionally important residues are shown in cyan.
functionally important residues onto the G␤ 1 ␥ 2 structure clearly indicates that these residues are surface-accessible (Fig. 5, right panel). Furthermore, these residues form a continuous surface with Ser-67 on G␤. Overall, the positioning of these residues defines a surface on the G␤␥ structure that is critical in the activation of GIRK channels.
The D36T and D48N Combination Mutant of G␥ 2 Abolishes G␤␥-mediated K ϩ Currents-Based on the above evidence that no single-point mutant of G␥ 2 could totally abolish activation of the GIRK channel, we next sought to address whether multiple residues were required for the G␤␥-mediated stimulation of channel currents. It has been shown that three consecutive amino acids in G␥ 2 , Ala-Asp-Leu (residues [35][36][37], conferred the ability to assemble with G␤ 2 (38). Does the triple-point mutant also impair the ability of the G␤ 1 ␥ 2 to assemble into a dimer and thus in turn retard channel activation? Fig. 6A shows that both the double (A35L/D36T) and triple G␥ 2 mutant (A35L/D36T/L37I) stimulated channel function, suggesting that mutation of these G␥ 2 residues did not interfere with G␤ 1 assembly. Interestingly, in screening 23 multiple-point mu-  Fig. 4 and normalized relative to the GIRK4* control. Every combination that included mutations D36T and D48N (underlined) abolished G␤␥ activation of the GIRK channels. This suggests that the co-existence of Asp-36 and Asp-48 in G␥ 2 was critical for G␤␥-stimulated activity. Other combination mutants containing either D36T or D48N significantly enhanced K ϩ currents relative to control (*, p Ͻ 0.01, n ϭ 8 -13). In B, the double-point mutant of G␥ 2 (D36T/D48N) together with wild-type G␤ 1 failed to stimulate K ϩ currents relative to control (*, p Ͻ 0.01, n ϭ 6 -10). C, trypsin protection assays of uninjected oocytes or oocytes injected with cRNA for FLAG-␤ 1 alone, FLAG-␤ 1 ␥ 2 , and FLAG-␤ 1 ␥ 2 (D36T/D48N). Crude membranes incubated with anti-FLAG beads and with or without trypsin. Lanes 1 and 2 from uninjected oocytes showed no protein bands. Oocytes injected with the cRNA of FLAG-␤ 1 (lanes 3 and 4) showed a 38-kDa band that was completely digested by trypsin. The typical 26-kDa C-terminal G␤ 1 fragment was protected in both the FLAG-tagged G␤ 1 ␥ 2 and the FLAG-tagged G␤ 1 ␥ 2 (D36T/D48N) (lanes 6 and 8). The data shown are representative of three similar experiments. tants, we found that only the mutants which involved both the D36T and D48N residues abolished G␤␥ stimulation of K ϩ currents (e.g. A35L/D36T/L37I/D48N, A35L/D36T/L37I/D48N/ F61S, and A35L/D36T/D48N but not A35L/D48N, L37I/D48N, etc.) (Fig. 6A). Residue D36T alone did not impair stimulation of GIRK4* currents significantly, whereas residue Asp-48 did significantly reduce but did not abolish the ability of G␤␥ to stimulate GIRK4 currents. Thus, we proceeded to test the double-point mutant (D36T/D48N) of G␥ 2 , which displayed current levels similar to those of the unstimulated control channel (Fig. 6B). These results suggest that the combination of the G␥ 2 mutations D36T and D48N impair the ability of the G␤␥ subunits to stimulate GIRK4* activity. The trypsin protection assay revealed a normal pattern by the G␤ 1 ␥ 2 complex, indicating that the defect of the double mutant did not result from a gross structural impairment. DISCUSSION In mammals, 20 G␣, 5 G␤, and 13 G␥ subunits have been identified so far (4,7). The possible diversity for G protein hetrotrimeric complex formation in mammals is therefore great. Yet in simpler organisms such as yeast (e.g. Saccharomyces cerevisiae), only a single G␤ (STE4), a single G␥ (STE18), and two G␣ (GPA1 and GPA2) gene copies are found (41)(42)(43), and thus, the possibility or need for diversity is limited. Sequence comparison of mammalian versus yeast G protein subunits reveals lower levels of sequence conservation than those encountered among mammalian subunits (44). It has been shown previously that K ϩ currents are directly stimulated by the mammalian G␤ 1 ␥ 2 complex (18, 45) by binding both the N and C termini of GIRK channels (24 -33). In the present study, we have demonstrated that yeast G␤␥, unlike the mammalian G␤ 1 ␥ 2 , displayed inhibition on basal GIRK currents. The inhibitory effect of the yeast G␤␥ is analogous to that seen with G␤ 5 ␥ 2 , which when overexpressed binds the channel and prevents stimulatory G␤␥ from activating GIRK currents (35,39).
Recent work aiming to identify the interaction sites between the G␤␥ subunits and the channel has focused primarily on the interacting surfaces of the G␤ subunits with the channels or of G␤ subunits with G␣ (35, 46 -48). G␤ 1 subunits alone were shown to bind to GIRK channels, but they failed to enhance channel currents without forming a dimer with the G␥ 2 subunits (49), suggesting the importance of the G␥ 2 subunits on channel activation. Here, we added the 35 N-terminal end amino acids of the yeast G␤ onto the mammalian G␤ 1 protein and produced a chimeric G␤ protein. This chimera stimulated the GIRK current upon association with the mammalian G␥ subunit, whereas it inhibited this current upon association with the yeast G␥ subunit. This result strongly suggested that the turn-on (activation) or turn-off (inhibition) of channel activity depended on the conformations of different G␤␥ complexes influenced by different G␥ subunits. This finding prompted us to utilize a chimeric strategy to identify the region of the mammalian G␥ responsible for the stimulation of GIRK currents.
Our chimeric approach revealed that the C-terminal half of G␥ 2 was critical for the stimulatory contributions of the mammalian G␥ subunit on the G␤␥ complex. Through site-directed mutagenesis, we identified 10 mutants that significantly reduced stimulation of K ϩ currents without affecting proper interactions with the G␤ subunit, as assessed by a trypsin protection assay. In a recent study, we identified three functionally important G␤ 1 residues that interact with GIRK channels (35). One of those, Ser-67, which does not interact with G␣ subunits, affected basal K ϩ currents when mutated. Mutation of the neighboring G␥ 2 (Arg-62) to amino acids with distinct chemical features, such as Ala, Glu, Lys, or Phe, did not produce a phenotype significantly different from that of the wild-type G␥ 2 (data not shown). Thus, the changes obtained with mutation of the 7 G␥ 2 residues seem rather specific and may affect interactions of the G␤ 1 (Ser-67) and other associated G␤ residues with the GIRK channel. These results suggest that more than one residue of G␥ 2 may be involved in the activation of the channel by the G␤ 1 ␥ 2 complex. Some attention has recently been paid to the role of different G␥ isoforms in the interactions of the G␤␥ complex with effectors (50). Yet it remains unclear how the G␥ subunits influence effectors through their interactions with G␤ subunits. In the present study, we identified the G␥ 2 double-point mutant (D36T/D48N), which totally abolished G␤␥ stimulation of GIRK currents, without affecting proper interactions with the G␤ subunit. This finding suggests not only that G␥ is required for the G␤␥ activation of GIRK channels (49) but also that it modulates effector function by fine-tuning the effectiveness of the G␤␥ dimer. Structural evidence has shown that residues Asp-36 and Asp-48 of G␥ 2 could form putative salt bridge and hydrogen bonds with G␤ 1 (5). This may mean that the strict stereochemical requirements in the hydrogen bond-driven interactions between G␤ and G␥ had not been reached in the conformation of the G␤ 1 ␥ 2 (D48N) mutant, despite the fact that there were 7 other residues of G␥ 2 forming hydrogen bonds with G␤ 1 (5). This change may explain why the mutation attenuated stimulation of the channel activity by the G␤␥ complex.
In summary, yeast G␤␥ specifically inhibited GIRK4* basal currents in Xenopus oocytes, but not the G␤␥-insensitive IRK1 currents. The region (residues 35-71) of G␥ is required for full stimulation of GIRK4* currents by the G␤ 1 ␥ 2 subunits. Ten amino acid residues within this region of the G␥ subunit form an intricate network of interactions with G␤, specifying a critical region of the G␤␥ heterodimer that determines the stimulatory effects on the GIRK4 channel. Two amino acids of G␥ 2 (Asp-36 and Asp-48), which interact with the G␤ 1 subunit by putative salt bridge and hydrogen-bonding interactions, maintain the fine conformation of the G␤ 1 ␥ 2 subunits required for full stimulation of GIRK4* activity. The precise interactions of these functionally critical residues that have been identified in the G␤ and G␥ subunits will have to await structural determination of the GIRK channel with the G␤␥ complex.