Structural Basis for Modulation of Gating Property of G Protein-gated Inwardly Rectifying Potassium Ion Channel (GIRK) by i/o-family G Protein α Subunit (Gα_i/o)

Background: Although Gβγ is known to activate GIRK, Gα_i/o also modulates GIRK gating.

Results: The α2/α3 helices of Gα_i3 in the GTP-bound state directly bind to the αA helix of GIRK.

Conclusion: The complex model explains how Gα_i/o sequesters Gβγ efficiently from GIRK upon GTP hydrolysis.

Significance: The structural basis for the rapid closure of GIRK by Gα_i/o is provided.

Abstract

G protein-gated inwardly rectifying potassium channel (GIRK) plays a crucial role in regulating heart rate and neuronal excitability. The gating of GIRK is regulated by the association and dissociation of G protein βγ subunits (Gβγ), which are released from pertussis toxin-sensitive G protein α subunit (Gα_i/o) upon GPCR activation in vivo. Several lines of evidence indicate that Gα_i/o also interacts directly with GIRK, playing functional roles in the signaling efficiency and the modulation of the channel activity. However, the underlying mechanism for GIRK regulation by Gα_i/o remains to be elucidated. Here, we performed NMR analyses of the interaction between the cytoplasmic region of GIRK1 and Gα_i3 in the GTP-bound state. The NMR spectral changes of Gα upon the addition of GIRK as well as the transferred cross-saturation (TCS) results indicated their direct binding mode, where the K_d value was estimated as ∼1 mm. The TCS experiments identified the direct binding sites on Gα and GIRK as the α2/α3 helices on the GTPase domain of Gα and the αA helix of GIRK. In addition, the TCS and paramagnetic relaxation enhancement results suggested that the helical domain of Gα transiently interacts with the αA helix of GIRK. Based on these results, we built a docking model of Gα and GIRK, suggesting the molecular basis for efficient GIRK deactivation by Gα_i/o.

Introduction

G protein-gated inwardly rectifying potassium ion channel (GIRK)³ (1) is a member of the inwardly rectifying potassium ion channel (Kir) family, regulating heart rate, neuronal excitability, and other physiological events (2, 3). GIRK functions as a tetramer in which a long, ∼88 Å K⁺ permeation pathway is formed by the transmembrane (TM) and cytoplasmic pore (CP) regions (4, 5). The crystal structure of GIRK revealed two K⁺-ion gates (5): the helix bundle crossing on the cytoplasmic side of the TM region and the loops on the membrane side of the CP region, referred to as G-loops. Although the gating of Kir family proteins is generally regulated by phosphatidylinositol 4,5-bisphosphate, the opening of the GIRK gate is also triggered by the direct binding of the G protein βγ subunits (Gβγ). The stimulation of G protein-coupled receptors (GPCRs) causes the exchange of GDP with GTP on the α subunit (Gα), which decreases the affinity of Gα for Gβγ. Gβγ then dissociates from Gα in the GTP bound state (Gα(GTP)) and associates with GIRK, resulting in the opening of the GIRK gate (2, 3, 6–11). Recently, our NMR analyses revealed that Gβγ binds to the border region of the two neighboring subunits of the GIRK tetramer. This Gβγ binding causes the reorientation of the tetramer subunits and the conformational change of the gate in the CP region (G-loops), which probably leads to the opening of the TM gate (12). When the GPCR stimulation ends, the GTP on Gα is hydrolyzed to GDP, and then Gα in the GDP-bound state (Gα(GDP)) removes Gβγ from GIRK, resulting in the closure of the GIRK gate.

Although Gβγ directly binds to and activates GIRK, Gα also plays important roles in regulating GIRK. In native tissues, GIRK is activated only through the stimulation of GPCRs that couple to the G proteins belonging to the i/o-family (G_i/o) (13). The activation and the deactivation of GIRK are also accelerated when the channels are co-expressed with Gα_i/o (14–16).

Efficient GIRK activation seems to be attributed to the preassociation of the heterotrimeric G protein in the i/o-family (Gα_i/o(GDP)βγ) with GIRK, in which Gα_i/o is directly associated with both GPCR and GIRK before GPCR activation (17, 18). Furthermore, the direct association of Gα_i/o(GDP)βγ with GIRK in the absence of GPCR-stimulation reportedly suppresses the basal activity of GIRK while maintaining the maximal evoked current by Gβγ in the presence of GPCR stimulation (14, 15, 19–22). This regulation mechanism is called “priming,” by which Gα_i/o reportedly plays an important role by facilitating a better response to GPCR activation.

On the other hand, the deactivation is accelerated by the direct interaction of Gα_i/o(GTP) with GIRK (18). The interaction enables Gα_i/o to rapidly bind to Gβγ upon GTP hydrolysis, which is presumably assisted by the regulator of G protein signaling (RGS) (23), resulting in the closure of the GIRK gate. Because the re-association of Gα_i/o(GDP) and Gβγ allows Gα_i/o(GDP)βγ to enter the next cycle of GIRK activation, the rapid reassociation would also accelerate the activation of GIRK (15). Thus, the direct interaction between GIRK and Gα_i/o(GTP) could contribute to the efficient activation and deactivation of the GIRK channel. In a FRET study investigating the conformational changes of the GIRK1/2 channel in Xenopus oocytes, the coexpression of Gα_i/o(GTP) with Gβγ conferred different FRET patterns, and the maximal GIRK current, as compared with those observed when Gβγ and Gα_i/o, were separately expressed (19).

Altogether, it is now evident that Gα_i/o not only functions as the donor and acceptor of Gβγ but also modulates the gating property of GIRK. To reveal the underlying molecular mechanism by which the Gα_i/o modulates GIRK, several studies have proved the interactions between Gα_i/o and the cytoplasmic region of GIRK. Gα_i/o binding to the N- and C-terminal fragments of GIRK was examined by pulldown assays in vitro (14, 15, 19, 20, 22, 24, 25), indicating that the C-terminal region of GIRK is essential for the i/o-family-specific binding to GIRK. On the other hand, little is known about the GIRK binding site on Gα_i/o. Gα consists of a GTPase domain and a helical domain (26, 27) in which GDP or GTP is sandwiched by both domains and GPCRs bind to the GTPase domain (28). An electrophysiological study using a Gα_i1/Gα_q chimera, in which the helical domain was replaced, revealed that the helical domain of Gα is responsible for the specific activation of the GIRK channel (25). Thus, it remains unclear how Gα_i/o modulates the GIRK gating property, and therefore, the elucidation of the molecular recognition mode of Gα_i/o and GIRK is required.

In this study we performed the NMR analyses of the interaction between the cytoplasmic region of GIRK1 and Gα_i3(GTP), which accelerates the deactivation of GIRK. The NMR spectral changes of Gα upon the addition of GIRK as well as the transferred cross-saturation (TCS) results indicated that Gα and the cytoplasmic region of GIRK1 directly bind to each other, with an estimated K_d value of ∼1 mm. We identified the binding sites on Gα and GIRK, respectively, and examined the binding mode by paramagnetic relaxation enhancement (PRE) experiments. Based on these results, we built a docking model of Gα and the cytoplasmic region of GIRK, suggesting the molecular basis for the efficient deactivation of the channel by Gα_i/o.

EXPERIMENTAL PROCEDURES

Expression and Purification of Cytoplasmic Regions of Mouse GIRK1

The N- and C-terminal cytoplasmic regions of mouse GIRK1 (residues 41–63 and 190–386) were fused into a single polypeptide, which is hereafter referred to as GIRK_CP-L. The C-terminal region of GIRK_CP-L is 15 residues longer than that of GIRK_CP (residues 41–63 and 190–371). We previously analyzed the interaction of GIRK_CP with Gβγ and confirmed its validity by the crystal structure (29). Preliminary TCS experiments using GIRK_CP and Gα_i3 indicated that the C-terminal region of GIRK_CP was involved in the interaction with Gα_i3, suggesting that some interacting residues are missing in GIRK_CP. Therefore, we extended the GIRK_CP construct for 15 amino acids to obtain GIRK_CP-L, which was used for the further analyses.

GIRK_CP-L was expressed in Escherichia coli cells. The uniformly ²H,¹⁵N-labeled GIRK_CP-L samples for the NMR analyses were prepared by growing E. coli in M9 minimal medium containing ¹⁵NH₄Cl, [²H]glucose, and [²H/¹⁵N]Celtone® base powder in 99% ²H₂O. GIRK_CP-L was purified according to the same procedure as for GIRK_CP (30). GIRK_CP-L mainly eluted as a tetramer from the size exclusion chromatography column. The uniformly ²H,¹⁵N-labeled and uniformly ²H-labeled GIRK_CP-L samples for the TCS experiments were prepared without the denaturing and refolding procedure to preserve the amide hydrogen atoms as ²H in the core of the protein. The GIRK_CP-L samples were incubated at 303 K for >48 h before the TCS experiments to prevent ²H/¹H exchange during the TCS measurements.

Assignments of NMR Signals of GIRK_CP-L

The ¹H,¹⁵N transverse relaxation-optimized spectroscopy (TROSY) signals of GIRK_CP-L were well dispersed and mostly overlapped with those of GIRK_CP. The backbone assignments of residues 41–63 and 190–366 of GIRK_CP-L were transferred from those of GIRK_CP (BioMagResBank accession number 11067) (30). The assignments of residues 367–377 were established by triple resonance experiments (HNCACB and HNCA) (31, 32) using uniformly ²H,¹³C,¹⁵N-labeled GIRK_CP-L as shown in supplemental Table 1. The backbone amide resonances for Asn-378–Val-386 could not be assigned because their signals were very weak or not observed, probably due to either the fast exchange of their amide hydrogen atoms with those of water molecules or the line-broadening due to conformational exchange.

Expression and Purification of Gα_i3

The Gα_i3 protein, with an N-terminal decahistidine tag followed by an HRV3C protease cleavage site, was expressed in E. coli cells. The uniformly ²H,¹⁵N-labeled Gα_i3 samples for NMR analyses were prepared by growing E. coli in M9 minimal medium containing ¹⁵NH₄Cl, [²H]glucose, and [²H/¹⁵N]Celtone® base powder in 99% ²H₂O. Gα_i3 was purified as described (12, 33). Briefly, Gα_i3 was purified to homogeneity by chromatography on a HIS-Select® Nickel Affinity Gel (Sigma) column, His-tag cleavage by PreScission^TM Protease (GE Healthcare), and removal of the cleaved His tags and the PreScission^TM Protease (GE Healthcare) by HIS-Select® Nickel Affinity Gel (Sigma).

Spin Labeling of Gα_i3

For PRE experiments, we first prepared the Gα_i3 mutant referred to as Hexa III, in which all six exposed Cys residues were substituted (Gα_i3 (C3S-C66A-C214S-C305S-C325A-C351I)), according to the previous report (34). Using this construct as a template, cysteine substitutions were separately introduced to Ile-82 and Ser-153 by the QuikChange® system (Stratagene). All mutations were confirmed by DNA sequencing.

Spin-labeling was performed in a buffer containing 10 mm Hepes-NaOH (pH 7.0), 50 mm KCl, 10 mm MgCl₂, and 0.60 mm GTPγS. The Gα_i3 mutants were incubated with S-(1-oxy-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate (MTSL) at a molar ratio of 1:3 for each protein:MTSL at room temperature for 4 h. Under these conditions we confirmed that only the most reactive cysteine residue was modified, whereas the remaining buried native cysteine residues were not modified. The excess MTSL was removed by extensive washes with the buffer by ultrafiltration using an Amicon® Ultra filter unit (Millipore). For the diamagnetic state experiments, ascorbate was added to the MTSL-labeled Gα_i3 mutants at a molar ratio of 1:3 protein:ascorbate, and the solution was incubated at 4 °C for 12 h. The ascorbate was then removed by ultrafiltration. Thus, we prepared the MTSL-labeled Hexa III in the diamagnetic state.

NMR Analyses

All NMR experiments were performed on a Bruker Avance 600 spectrometer equipped with a cryogenic probe. The ¹H,¹⁵N TROSY spectra of Gα_i3 in the presence of various amounts of GIRK_CP-L were observed at 308K. TCS and PRE experiments were performed at 303 K. All spectra were processed by the Bruker TopSpin 2.1 software, and the data were analyzed by Sparky (T. D. Goddard and D. G. Kneller, Sparky 3, University of California, San Francisco, CA). The error bars are based on the signal-to-noise ratio calculated by the Sparky software. The backbone NMR signal assignments of Gα_i3 were reported previously (35).

To examine the spectral changes of Gα_i3 induced by the presence of GIRK_CP-L, we prepared five samples, each containing the uniformly ²H,¹⁵N-labeled Gα_i3 (0.28 mm) mixed with GIRK_CP-L at molar ratios (Gα_i3:GIRK_CP-L tetramer) of 1:0, 1:0.9, 1:1.8, 1:2.7, and 1:3.6 in the NMR sample buffer (10 mm HEPES-NaOH (pH 7.0), 50 mm KCl, 10 mm MgCl₂, 0.6 mm GTPγS, 5 mm DTT), 1 mm sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), 10% ²H₂O, 90% ¹H₂O). We then observed the ¹H,¹⁵N TROSY spectrum of each sample. The residues with overlapping resonances were omitted from the analyses.

The TCS experiments were performed as described with minor modifications (12, 36). In the TCS experiments observing Gα_i3, a solution containing uniformly ²H,¹⁵N-labeled Gα_i3 (0.3 mm) and unlabeled GIRK_CP-L (0.25 mm as a tetramer) was prepared in buffer (10 mm HEPES-NaOH (pH 6.5), 50 mm KCl, 5 mm DTT, 1 mm DSS, 0.8 mm GTPγS, 20% ¹H₂O, 80% ²H₂O). The saturation frequency was set at 0.83 ppm, and the maximum radiofrequency amplitude was 0.17 kHz for WURST-2 (adiabatic factor Q₀ = 1). The saturation duration and the relaxation delay were set at 1.5 and 2.5 s, respectively. To evaluate the effect of the residual aliphatic protons within Gα_i3, TCS experiments were also performed under the same conditions as those mentioned above, with the sample containing uniformly ²H,¹⁵N-labeled Gα_i3 (0.3 mm) and uniformly ²H-labeled GIRK_CP-L (0.25 mm as a tetramer). It should be noted that the residues with signal overlapping and/or signal-to-noise ratios less than 10 were excluded from the analyses.

In the TCS experiments observing GIRK_CP-L, a solution containing uniformly ²H,¹⁵N-labeled GIRK_CP-L (0.25 mm as a tetramer) and unlabeled Gα_i3 (0.4 mm) was prepared in buffer (10 mm HEPES-NaOH (pH 6.5), 50 mm KCl, 5 mm DTT, 1 mm DSS, 0.8 mm GTPγS, 20% ¹H₂O, 80% ²H₂O). The saturation frequency was set at 0.83 ppm, and the maximum radiofrequency amplitude was 0.17 kHz for WURST-2 (adiabatic factor Q₀ = 1). The saturation duration and the relaxation delay were set at 3.0 and 2.0 s, respectively. To evaluate the effect of the residual aliphatic protons within Gα_i3, TCS experiments were also performed under the same conditions as those mentioned above, with the sample containing uniformly ²H,¹⁵N-labeled GIRK_CP-L (0.25 mm as a tetramer) and uniformly ²H-labeled Gα_i3 (0.4 mm). It should be noted that the residues with signal overlapping and/or signal-to-noise ratios less than 10 were excluded from the analyses.

In the PRE experiments of the paramagnetic state, samples containing uniformly ²H,¹⁵N-labeled GIRK_CP-L (0.075 mm as a tetramer) mixed with 0.2 mm oxidized Hexa III-Cys-MTSL(ox) were prepared. In the experiments of diamagnetic state, samples containing uniformly ²H,¹⁵N-labeled GIRK_CP-L (0.075 mm as a tetramer) mixed with reduced Hexa III-Cys-MTSL(red) in buffer (10 mm HEPES-NaOH (pH 7.0), 50 mm KCl, 1 mm DSS, 10% ²H₂O, 90% ¹H₂O) were prepared. The ¹H,¹⁵N TROSY spectrum of each sample was recorded. The residues with overlapping resonances were omitted from the analyses. PRE was calculated as paramagnetic to diamagnetic signal intensity ratios (I_para/I_dia) (37).

Construction of Complex Models

The complex models of Gα-GIRK and Gα-GIRK-Gβγ were obtained with the HADDOCK software (38).

First, we built a homology model of Gα_i3(GTP) by the MODELLER software (39) using the crystal structure of Gα_i1(GTPγS) (PDB code 1GIA) as a template, whose amino acid sequence is 94% identical to that of Gα_i3. The crystal structure of GIRK_CP (PDB code 1N9P) and the structure of Gβγ in the crystal structure of Gα_i1(GDP)β₁γ₂ (PDB code 1GP2) were used, respectively, for dockings. The active residues used in the definition of the ambiguous interaction restraints for docking are listed in supplemental Table 2.

The Gα-GIRK-Gβγ ternary complex model was built as follows. First, we built a docking model of the Gβγ-GIRK complex with parameters listed in supplemental Table 2. Then, the Gα-GIRK and Gβγ-GIRK complex models were superimposed by the GIRK structure.

RESULTS

NMR Spectral Changes of Gα_i3 upon Binding to GIRK_CP-L

To investigate the direct binding between Gα_i3 and GIRK_CP-L, we observed a series of ¹H,¹⁵N TROSY spectra of 0.28 mm uniformly ²H,¹⁵N-labeled Gα_i3 in the absence or presence of 0.25, 0.50, 0.75, and 1.0 mm GIRK_CP-L as a tetramer. As the concentration of GIRK_CP-L increased, most signals exhibited decreased intensity due to line-broadening without changing their chemical shifts, whereas a number of signals exhibited further intensity reductions and eventually disappeared in the presence of 0.75 mm GIRK_CP-L. In addition, several signals exhibited small but significant chemical shift changes (Fig. 1, A and B). Although the overall intensity reductions are caused by the slowing of the overall tumbling motion upon binding to GIRK_CP-L, the further intensity reductions and apparent chemical shift changes reflect the direct binding of GIRK_CP-L to Gα_i3. The weighted averages of the chemical shift differences (Δδ) were calculated using the equation (Δδ = [(Δδ_HN)² + (Δδ_N/6.5)²]^1/2). The titrations curves of Δδ were fit to the following theoretical formula to obtain the value of the dissociation constant (K_d).

where Δδ_sat is the Δδ value when a saturating amount of GIRK_CP-L is added (ppm), and [Gα_i3]_tot = 0.28 mm. The fitting of the titration curves of the chemical shift changes for the signals from Lys-209 and Trp-258 resulted in dissociation constant (K_d) values of 0.6 and 1.1 mm, respectively (Fig. 1C).

FIGURE 1.

NMR spectral change of Gα_i3 upon the addition of GIRK_CP-L. A, shown is an overlay of the ¹H,¹⁵N TROSY spectra of the uniformly ²H,¹⁵N-labeled Gα_i3 (0.28 mm) in the absence (black) and presence (red) of 2.7 eq of GIRK_CP-L (0.75 mm as a tetramer). The signals with chemical shift differences larger than 0.008 ppm are labeled. The signal from Gly-45, which is one of the residues showing significant intensity reduction without a significant chemical shift change, is also labeled in parentheses. B, overlays of the spectra in the presence of 0–2.7 eq of GIRK_CP-L (as a tetramer) are displayed for Gly-45 (left) and Trp-258 (right) as typical examples of the signals with significant intensity reductions and chemical shift changes, respectively. C, titration curves of the chemical shift differences for Lys-209 (left) and Trp-258 (right) are shown. D, shown are plots of the chemical shift differences of Gα_i3 in the absence and presence of 2.7 eq of GIRK_CP-L (as a tetramer). The error bars were calculated based on the digital resolutions of the spectra. The minimum values of Δδ within the error ranges (Δδ_min) were utilized for the evaluation. Bars corresponding to the residues with significant chemical shift differences, which have the minimum values of Δδ within the error ranges (Δδ_min) larger than 0.008 ppm, are colored red. Asterisks in D and E indicate the residues with no data due to signal overlapping, lack of assignments, or insufficient signal-to-noise ratio. The secondary structure elements of Gα_i3 are depicted in gray below the sequence, and α2, α3, α4, and β6 are colored green. E, shown are plots of the normalized intensity ratios (R) of uniformly ²H,¹⁵N-labeled Gα_i3 upon the addition of 1.8 eq of the GIRK_CP-L tetramer to Gα_i3. The intensities of the free Gα_i3 were divided by a scaling factor of 1.78 (see supplemental Methods) and then were used for the calculation of the intensity ratios. The error bars were calculated based on the signal-to-noise ratios. Bars corresponding to the signals with the maximum value of R, within the error ranges (R_max) lower than 0.42 are colored cyan. F, mapping of the affected residues on the Gα_i1 structure in which 333 of 354 residues (94%) are identical to those of Gα_i3 (PDB code 1GIA) is shown. The amide nitrogen atoms of the residues with apparent chemical shift differences and intensity reductions are shown as balls colored red and cyan, respectively. Proline residues and residues with no data are colored black. The α2, α3, α4, and β6 are colored green, and GTPγS is depicted by teal sticks.

We evaluated the apparent chemical shift differences (Δδ) of Gα_i3 in the absence of GIRK_CP-L and the presence of 0.75 mm GIRK_CP-L, in which 37–50% of Gα_i3 was in the GIRK_CP-L-bound state, as estimated by the K_d value of 0.6–1.1 mm (Fig. 1D). The residues with significant chemical shift changes are Glu-207, Lys-209, Trp-211, His-213, Phe-215, Glu-216, Ser-246, Trp-258, Arg-312, and Thr-316, for which the minimum values of Δδ within the error ranges (Δδ_min = Δδ − error) are larger than 0.008 ppm. It should be noted that the threshold, 0.008 ppm, corresponds to 0.016–0.022 ppm between the free and GIRK_CP-L-bound states.

We also evaluated the signal intensity reductions, as the accelerated intensity reductions are caused by the differential line broadening, i.e. the chemical shift changes in the slow to intermediate exchange regime, upon binding to GIRK_CP-L (Fig. 1B) (40). Fig. 1E shows the intensity ratios (R) in the presence and absence of GIRK_CP-L, which were corrected by multiplying by the scaling factor of 1.78, for the increase in the molecular weight upon binding (see supplemental Methods for details). The scaling factor of 1.78 is obviously too large for the N- and C-terminal residues (residues 1–31 and 349–354), because these residues tumble faster than those in the other regions of Gα_i3, as evidenced by the small line widths and the small values of the chemical shift indices (35, 41), resulting in the R values larger than 1.0. Except for these signals, most signals showed the R values ranging from 0.40 to 0.80, suggesting that the tumbling motion is slowed in the presence of GIRK_CP-L, presumably due to the increase in sample viscosity. The residues with significantly larger signal intensity reductions, which exhibited the maximum R value within the error range (R_max = R + error) lower than 0.42, are Ala-41, Ser-44, Gly-45, Lys-46, Ser-47, Thr-177, Gly-203, and Ile-253.

Fig. 1F shows the mapping of these affected residues on the crystal structure of Gα (PDB code 1GIA), where the residues with significantly large Δδ_min and small R_max are colored red and blue, respectively. Most of the residues with chemical shift changes are located in two regions; that is, the region from the α2 helix and the following loop (Glu-207, Lys-209, Trp-211, His-213, Phe-215, and Glu-216) and the region from the α3 helix and the following loop (Ser-246 and Trp-258). The other residues, Arg-312 and Thr-316, are located on the loop between the α4 helix and the β6 strand. On the other hand, the residues with significant intensity reductions are located around the GTP binding site, in which Ser-44, Gly-45, Lys-46, Ser-47, and Gly-203 directly interact with the phosphate group of GTPγS.

As shown in Fig. 1F, the affected residues exhibited significant chemical shift changes, indicating that they are involved in the direct GIRK_CP-L binding site and/or in the site(s) exhibiting a conformational change upon binding.

GIRK_CP-L Binding Site on Gα_i3 Revealed by Transferred Cross-saturation Experiments

To identify the GIRK_CP-L binding site on Gα_i3, TCS experiments were performed. The saturation of the GIRK_CP-L resonances by the irradiation with radio frequency pulses caused the signal intensity reductions of the ¹H,¹⁵N TROSY signals of the Gα_i3 residues, which should be located in the GIRK_CP-L binding site (Fig. 2A) (42, 43).

FIGURE 2.

TCS from GIRK_CP-L to Gα_i3. A, shown is a selected portion of the ¹H,¹⁵N TROSY spectra of the uniformly ²H,¹⁵N-labeled Gα_i3 (0.30 mm) in the presence of GIRK_CP-L (0.25 mm as a tetramer), which was recorded without (left) and with (right) radio frequency irradiation. Cross-sections are also shown for the signals from Ala-99 and Trp-258. B, procedures were the same as A, except that ²H-labeled GIRK_CP-L was used instead of unlabeled (¹H-labeled) GIRK_CP-L, as a negative control. C, shown is a plot of the difference in the reduction ratios (ΔRR) originating from the backbone amide groups with and without irradiation in the presence of unlabeled GIRK_CP-L and ²H-labeled GIRK_CP-L (see also supplemental Fig. 1). The residues indicated by asterisks are those with no data mostly due to overlapping of the resonances or insufficient signal-to-noise ratio. The error bars were calculated based on the signal-to-noise ratios. Bars corresponding to the residues with significant intensity reductions (minimum values of ΔRR (ΔRR_min) > 0.08) are colored red. The secondary structure elements of Gα_i3 are depicted in gray below the sequence, and αA, αB, α2, and α3 are colored green. D, mapping of the affected residues in the TCS experiment on the Gα structure (PDB code 1GIA) is shown. The backbone nitrogen atoms of the affected residues are shown as red balls in the ribbon diagram of the Gα structure (left). The affected residues are colored red on the surface representations of the Gα structure, whereas the residues with no data, including proline residues, are colored black (center and right). The αA, αB, α2, and α3 are colored green.

In the case of a larger protein system, such as Gα_i3 and GIRK_CP-L, with molecular masses of 41 and 103 kDa, respectively, the enhanced ¹H homonuclear dipolar-dipolar interactions might cause intramolecular saturation transfer from the residual protons in Gα_i3 (the exchangeable hydrogen atoms in the NH, OH, and SH groups and/or the hydrogen atoms, due to the incomplete ²H-labeling of ²H,¹⁵N-labeled Gα_i3). To exclude these effects, we also performed a control experiment by using ²H-labeled GIRK_CP-L instead of the unlabeled (i.e. ¹H-labeled) protein to reflect only the artificial effects described above (Fig. 2B, supplemental Fig. 1, gray) and subtracted the intensity reduction ratios of this control experiment from those obtained by using unlabeled GIRK_CP-L (supplemental Fig. 1, orange). The differences in the reduction ratios, ΔRR, are shown in Fig. 2C, with the error bars calculated based on the signal-to-noise ratios. The minimum values of ΔRR within the error ranges (ΔRR_min = ΔRR − error) were utilized for the evaluation.

The residues with large intensity reductions (ΔRR_min > 0.08, Fig. 2C) are located on the helical domain (Gly-89 in the αA helix; Gly-112 and Ala-114 in the αB helix) and the GTPase domain (Arg-208, Lys-209, Trp-211, Ile-212, His-213, Glu-216, and Gly-217 in the α2 helix and the following loop; Met-240, Lys-248, Leu-249, Ile-253, Asn-256, and Trp-258 in the α3 helix and the flanking loops; Glu-186 in the β2 strand). The mapping of these residues on the structure of Gα revealed that the residues identified on the GTPase domain of Gα_i3 are clustered, indicating that this site (hereafter, referred to as the “α2/α3 site”) mainly contributes to the GIRK_CP-L binding (Fig. 2D). This is also supported by the two alanine mutants of the identified residues (I212A and W258A) that exhibited impaired binding affinity for GIRK_CP-L (supplemental Fig. 2). It should be noted that Glu-186 seems separated from the cluster by the intervening residue, Phe-199 (supplemental Fig. 3). The amide group of Phe-199 is buried in the protein and is more than 6 Å away from the contiguous surface for GIRK_CP-L binding, resulting in the lack of cross-saturation for Phe-199. Therefore, we conclude that the α2/α3 site is the major GIRK_CP-L binding site.

The residues identified on the helical domain (Gly-89, Gly-112, and Ala-114), which are distant from the α2/α3 site, do not form a contiguous surface but are located on the same side of the protein surface as the α2/α3 site, suggesting that these residues transiently contact GIRK_CP-L. We further investigated the interactions of the helical domain with GIRK_CP-L (see below).

Gα_i3 Binding Site on GIRK_CP-L Revealed by TCS Experiments

Conversely, to identify the Gα_i3 binding site on GIRK_CP-L, TCS experiments observing GIRK_CP-L were performed. In the same manner as the TCS experiments to identify the binding site on Gα_i3, we evaluated the cross-saturation from unlabeled Gα_i3 (supplemental Fig. 4, orange) by subtracting the intensity reduction ratios of the control experiment in the presence of ²H-labeled Gα_i3 (supplemental Fig. 4, gray). The differences in the reduction ratios, ΔRR, are shown in Fig. 3A, with the error bars calculated based on the signal-to-noise ratios. The minimum values of ΔRR within the error ranges (ΔRR_min) were utilized for the evaluation.

FIGURE 3.

TCS from Gα_i3 to GIRK_CP-L. A, shown is a plot of the difference in the reduction ratios (ΔRR) originating from the backbone amide groups with and without radio frequency irradiation in the presence of unlabeled Gα_i3 and ²H-labeled Gα_i3 (see also supplemental Fig. 3). The residues indicated by asterisks are those with no data, mostly due to overlapping resonances or insufficient signal-to-noise ratio. The error bars were calculated based on the signal-to-noise ratios. Bars corresponding to the residues with significant intensity reductions, which have the minimum values of ΔRR within the error ranges (ΔRR_min) larger than 0.02, are colored red. The primary sequence of GIRK_CP-L (residues 41–63 and 190–386) is displayed in the single-letter amino acid code followed by the residue number. The secondary structure elements of GIRK_CP-L are depicted in gray below the sequence, based on the crystal structure (PDB code 1N9P), and the αA helix is colored green. B, mapping of the affected residues in the TCS experiment on a single subunit of the GIRK_CP tetramer (PDB code 1N9P) is shown. Residues 371–386 are depicted by gray tubes. Side views of the GIRK_CP-L structures parallel to the membrane plane, where the membrane side is above the molecules, are depicted by ribbon diagrams with balls for the amide nitrogen atoms of the affected residues (left). The affected residues are colored red on the surface representations of the GIRK_CP-L structure, whereas the residues with no data, including proline residues, are colored black (center and right). The αA helix is colored green in the ribbon diagrams. Views from the outside (left and center) and the inside of the tetramer (right) are shown. The four subunits of the GIRK_CP tetramer are shown in a surface representation for reference. C, mapping of the affected residues (colored red) in the TCS experiment on both of the two adjacent subunits of a GIRK_CP tetramer (white and gray), viewed from the outside (left) and the inside (right) of the tetramer. Schematic drawings of the GIRK_CP tetramer (subunits A, B, C, and D), viewed from the membrane side, are included to indicate the view of the surface models. The dashed blue line indicates the boundary between the two neighboring subunits.

The residues with large intensity reductions (ΔRR_min > 0.02) are Glu-242, Val-358, Leu-365, Leu-366, Met-367, Ser-368, Ser-369, Leu-371, Ile-372, and Ala-373 as well as one of the C-terminal unassigned residues (hereafter, referred to as CT1). These residues were mapped on a single subunit (Fig. 3B) and two adjacent subunits (Fig. 3C) of GIRK_CP (PDB code 1N9P). All of these residues, except for Glu-242 are located on the C-terminal region around the αA helix of GIRK_CP-L (Fig. 3). Therefore, we concluded that these residues are the Gα_i3 binding residues. Glu-242 would be involved in the direct binding to the Gα_i3, as explained under “Discussion.”

It should be noted that the first two residues, Ile-372 and Ala-373, and CT1 were detected among the extended C-terminal 15 residues from GIRK_CP. The other C-terminal residues showed no significant intensity reductions. Thus, we concluded that the extension for 15 residues is sufficient for the interaction with Gα_i3.

Contribution of Helical Domain of Gα_i3 to Interaction with GIRK_CP-L, as Investigated by PRE Experiments

The interaction of the helical domain of Gα_i3 with GIRK_CP-L was investigated by PRE experiments. PRE arises from magnetic dipolar interactions between the nuclear spin and the unpaired electron spin of the paramagnetic center, which enhances the relaxation of the nuclear spins, leading to the line-broadening and thus the intensity reduction of the NMR signals of the residues within about 20 Å of the spin label (37, 44). In addition, PRE can detect a transient interaction, because the PRE effect occurs within 250–500 μs (45), which is a much shorter duration than that of the cross-saturation (the effective saturation time of 200–300 ms for the TCS experiments in this study) (43).

The spin-labeling reagent, MTSL, which can be chemically introduced to cysteine side chains, was used to label Gα_i3. First, we prepared a mutant of Gα_i3 (C3S-C66A-C214S-C305S-C325A-C351I) in which the six natively existing cysteine residues that are exposed and reactive are substituted with other residues (hereafter, referred to as Hexa III). It should be noted that the corresponding mutant of Gα_i1 (C3S-C66A-C214S-C305S-C325A-C351I), named Hexa I, was folded properly and functional (34). We also confirmed the proper folding of Hexa III by the ¹H,¹⁵N TROSY spectrum of ²H,¹⁵N-labeled Hexa III (data not shown). Then, either Ile-82 or Ser-153 was substituted by Cys and chemically modified by MTSL (Hexa III-I82C-MTSL and Hexa III-S153C-MTSL). The Ile-82 residue is located at the center of the three residues identified by TCS (Gly-89, Gly-112, and Ala-114), and Ser-153 exists on the opposite side from I82. Because the paramagnetism of MTSL disappears upon reduction by the addition of ascorbate, the PRE effects were evaluated by the signal intensity ratios of the ¹H,¹⁵N TROSY spectra of ²H,¹⁵N-labeled GIRK_CP-L between the oxidized and reduced states in the presence of Hexa III-I82C-MTSL or Hexa III-S153C-MTSL.

Fig. 4A shows that the PRE effects from Hexa III-I82C-MTSL were observed for 13 signals of GIRK_CP-L: Thr-353, Tyr-356, Ser-357, Leu-365, Leu-366, Met-367, Ser-368, Ser-369, Leu-371, Ile-372, Ala-373, A375, and CT1, which reside in the C-terminal region of GIRK_CP-L. It should be noted that most of the other residues of the C-terminal region could not be analyzed due to signal overlapping, and thus these residues might also be affected by MTSL. On the other hand, only Cys-53 was detected for Hexa III-S153C-MTSL (Fig. 4B), which might be caused by the partial MTSL modification of Cys-53. Because the spin label was introduced at I82C, which is at the center of the three residues identified by TCS, we conclude that the Ile-82 side of the helical domain can approach within 20 Å of the C-terminal region of GIRK_CP-L.

FIGURE 4.

PRE results of the interaction between GIRK_CP-L and Gα_i3. A and B, left, shown is a plot of the signal intensity RRs of paramagnetic to diamagnetic for Hexa III-I82C-MTSL (A) and Hexa III-S153C-MTSL (B). The residues indicated by asterisks are those with no data due to overlapping resonances or insufficient signal-to-noise ratio. The error bars were calculated based on the signal-to-noise ratios. Bars corresponding to the residues with significant intensity reductions, which have the minimum values of RR within the error ranges (RR_min) larger than 0.20, are colored red. The primary sequence of GIRK_CP-L (residues 41–63 and 190–386) is displayed in the single-letter amino acid code followed by the residue number. Center, mapping of the affected residues in the PRE experiment on a single subunit of the GIRK_CP structure (PDB code 1N9P) is shown. Residues 371–386 are depicted by gray tubes. Side views of the GIRK_CP structures parallel to the membrane plane are depicted by surface representations viewed from the outside of the tetramer, where the membrane side is above the molecules. The affected residues are colored red. The proline residues and the residues with no data are colored black. Right, shown is mapping of the spin-labeled site of Gα_i3 on the structure of Gα (PDB code 1GIA).

DISCUSSION

Direct Interaction between Gα_i3 and Cytoplasmic Region of GIRK

NMR analyses were performed to probe the interaction between GIRK_CP-L and Gα_i3. Because cross-saturation is a phenomenon depending on the intermolecular ¹H-¹H distances, the TCS results from GIRK_CP-L to Gα_i3 (Fig. 2) and vice versa (Fig. 3) indicated that the α2/α3 site of Gα_i3 and the αA helix of GIRK_CP-L directly interact with each other. Our relaxation matrix calculations (43, 46) in which the on and off rates of the GIRK_CP-L-Gα_i3 interaction are also considered, suggested that under the current experimental conditions the cross-saturation effect should be observed for the amide hydrogen atoms of GIRK_CP-L within 6 Å from Gα_i3 and for those of Gα_i3 within 5 Å from GIRK_CP-L. Furthermore, the direct interaction was verified by the mutations of the interacting residues on Gα_i3, which impaired the affinity for GIRK_CP-L (I212A and W258A, supplemental Fig. 2).

Most of the residues with apparent chemical shift changes are located in the GIRK_CP-L binding site consisting of the α2 and α3 helices of Gα_i3, whereas the other residues, Arg-312 and Thr-316, exist on the α4/β6-loop that is adjacent to the α3 helix, reflecting the conformational changes of these residues upon GIRK_CP-L binding (Fig. 1). The fitting of the titration curves of the chemical shift changes of Lys-209 and Trp-258, the GIRK_CP-L binding residues, resulted in the K_d values of 0.6 and 1.1 mm, respectively, which are within the range of the fitting error (Fig. 1C). Thus, the K_d value for the binding of Gα_i3 and GIRK_CP-L was estimated as 1 mm. Although this K_d value is quite large as a value for a protein-protein interaction, it would fall in the nanomolar to micromolar range on the cell membrane, considering the reduced dimensionality effects (12, 47, 48). The K_d value on the order of 1 mm is 4 times of the K_d value of 0.25 mm that we previously reported for the GIRK_CP-Gβγ interaction (12), which is consistent with the report that the affinity of the cytoplasmic region of GIRK1 for Gβγ was 4–5-fold stronger than that for Gα_i3 (15).

As shown in Fig. 1, E and F, accelerated signal intensity reductions were observed for the Gα_i3 residues at the GTP binding region, which reflect the larger chemical shift changes for the residues in the intermediate exchange regime. These residues are distant from the α2/α3 site, which is the direct GIRK_CP-L binding interface, suggesting that the conformational change around the GTP binding site is induced by GIRK_CP-L binding to the α2/α3 site. In particular, Ser-44, Gly-45, Lys-46, Ser-47, and Gly-203 are the residues directly interacting with the β- or γ-phosphate groups of GTP. This might be related to the report that the GTPase activity of Gα_s is enhanced upon binding to its effector, adenylate cyclase (49), which also binds to the α2/α3 site of Gα_s (50). However, the significance of the conformational change at the GTP binding site in terms of GIRK regulation remains unclear and is beyond the scope of this paper.

Binding Mode of Gα and Cytoplasmic Region of GIRK

Although the TCS results revealed that the αA helix of GIRK_CP-L directly binds to the α2/α3 site of Gα_i3, the PRE results indicated the αA helix of GIRK_CP-L is within 20 Å of Ile-82 of Gα_i3. However, the αA helix of GIRK_CP-L, which binds to the α2/α3 site, should be more than 20 Å away from the spin-labeled site, suggesting that another αA helix in a neighboring subunit of the GIRK_CP-L tetramer must come close to the spin-labeled site (Fig. 5).

FIGURE 5.

The proposed model of Gα(GTP) and the cytoplasmic region of the GIRK complex. Left, two adjacent subunits of a GIRK_CP tetramer (PDB code 1N9P), viewed from the inside of the tetramer, are depicted by a ribbon diagram, and a homology model of Gα_i3 built from the crystal structure of Gα_i1(GTPγS) (PDB code 1GIA) by the MODELLER software (39) is shown in a surface representation. Residues 371–386 of GIRK1 are depicted schematically. The nitrogen atoms of the GIRK_CP-L residues identified in the TCS experiments and the residues with PRE effects from MTSL, modified to I82C on the helical domain of Gα_i3 (green), are shown as red and magenta balls, respectively. The α2/α3 residues of Gα_i3 identified in the TCS experiments are colored blue. Center, shown is the binding model of Gα_i3 in the GTP-bound state and the cytoplasmic region of GIRK1 (PDB code 1N9P), obtained with HADDOCK software (38). The residues on GIRK_CP-L and the α2/α3 residues on Gα_i3 identified by TCS were defined as the active residues in the program so as to form the interface of the complex. The residues 358–370 on GIRK were defined as “semi-flexible segments” to allow them to move during the simulated annealing. Only the two adjacent subunits of the GIRK tetramer are shown from the inside of the tetramer. Right, the model displayed at the center is rotated by 180 degrees, and all of the subunits of the GIRK tetramer are shown.

To build a model of the Gα_i3-GIRK_CP-L complex satisfying the NMR-derived structural information, rigid body docking was performed by using the HADDOCK program (38). Because the structure of Gα_i3(GTP) is not available, we built a homology model by the MODELLER software of Gα_i3(GTP) from the crystal structure of Gα_i1(GTPγS) (PDB code 1GIA) in which 94% of the 354 residues are identical to Gα_i3 (39) and used it for the construction of the complex model. The residues in the α2/α3 site of Gα and in the αA helix of GIRK_CP-L, identified by the TCS experiments, were specified as the “active residues” in the program so that they formed the interface of the complex. Although the information about the residues on the helical domain derived from the TCS and PRE experiments was not used for the calculation, the helical domain of Gα was calculated to be proximal to the αA helix in a neighboring subunit of GIRK_CP-L, probably due to the restraint of Glu-242 on the subunit interface of GIRK_CP-L, as determined from the TCS experiments (Fig. 5). In this binding mode, Glu-242 of GIRK_CP-L at a distance from the αA helix, approaches the side chain of Glu-186 of Gα_i3, which accounts for its intensity reduction in the TCS experiment.

The three residues that TCS identified on the helical domain of Gα_i3 (Gly-89, Gly-112, and Ala-114) showed relatively weak cross-saturation, and they do not form a continuous binding surface, presumably because the αA helix of GIRK_CP-L transiently accesses the Gα_i3 helical domain. This can be accounted for by the conformational flexibility of the αA helix. In the crystal structure, the αA helix of GIRK_CP is stabilized by the crystal contacts with the αA helix of another tetramer. In addition, the C-terminal part of the helix (residues 367–368) exhibited very weak or no NOEs that are typical for an α helix (data not shown), and the chemical shift index calculated by the ¹³C chemical shifts indicated that the C-terminal residues after Met-367 are unstructured. Altogether, we concluded that the α2/α3 site of Gα_i3 mainly recognizes the αA helix of GIRK_CP-L in one subunit of the tetramer, whereas the helical domain of Gα_i3 transiently approaches the αA helix in another neighboring subunit.

The N terminus of Gα is modified by a lipid moiety in vivo and anchored to the cell membrane (51), which can be accounted for by this binding mode. Although the most N-terminal Gα residue in the crystal structure, Val-34, resides at about 35 Å away from the possible intracellular cell membrane in the current binding mode (supplemental Fig. 5), the N-terminal residues 1–33 of Gα, which would form a 40 Å-long α helix in the complex with Gβγ, are presumably unstructured and adopt a longer length than 40 Å in the GTP-bound state.

Molecular Recognition Mode between Gα_i3 and GIRK_CP-L

Fig. 6 shows the surface properties of the α2/α3 site of Gα_i3 and the αA helix of GIRK_CP-L. The α2/α3 site possesses a hydrophobic cleft at its center, which is surrounded by polar residues, whereas the αA helix also contains a cluster of hydrophobic residues that are exposed to the outside. This suggests that the complementarity of the shape at the direct binding sites, formed by these hydrophobic residues, is important for the binding. Indeed, the mutations of the hydrophobic residues of Gα_i3, I212A and W258A, resulted in the impaired affinity for GIRK_CP-L (supplemental Fig. 2).

FIGURE 6.

Properties of the binding interfaces on Gα_i3 and GIRK1. An “open-book” display of the Gα-GIRK complex model (Fig. 5) is shown in surface representations. Residues 371–386 of GIRK_CP-L are depicted schematically. The residues on GIRK_CP-L and the α2/α3 residues on Gα_i3 identified by TCS (labeled by bold letters) and their surrounding residues with Cα atoms within 6.0 Å, are colored according to their side-chain properties: acidic, basic, hydrophobic, and hydrophilic residues are colored red, blue, green, and yellow, respectively.

The α2/α3 site on Gα, which is highly conserved among the Gα family members, is known to be the effector binding surface of Gα in the GTP-bound form, as revealed by the crystal structures of Gα in complex with its effectors such as Gα_s-adenylate cyclase (50) and Gα_q-G protein receptor kinase (52), and with peptides (53). In these complexes, the hydrophobic and acidic side chains from the effectors and peptides are inserted into the hydrophobic cleft of the α2/α3 site on Gα. GIRK is also recognized in a similar manner to those of the effectors and the peptides. It should be noted that the conformational flexibility of the αA helix might play a role in its interaction with the hydrophobic cleft of α2/α3 on Gα.

The helical domain of Gα is reportedly important for the i/o-family-specific activation of the GIRK channel, as determined with a Gα_i1/Gα_q chimera (25). However, the main GIRK binding site for the Gα_i3(GTP) revealed in this study was the α2/α3 site on the GTPase domain, not on the helical domain. The activation specificity should be accomplished by the binding of Gα_i/o(GDP)βγ to GIRK, which seems to occur at a different site from that for Gα_i/o(GTP).

Previously, the Gα_i/o binding regions in GIRK1 were investigated by pulldown assays using the cytoplasmic fragments of GIRK (14, 15, 20, 22, 24). The cytoplasmic C-terminal residues 320–369 of GIRK1 were indicated to be important for strong binding to Gα_i3 (14), whereas the N-terminal fragment of GIRK1 (residues 1–84) also bound to Gα_i/o(GTP) (15, 20). The Gα_i3 binding residues of GIRK1 revealed in this study are involved in the previously defined C-terminal region, and we identified three additional residues, Leu-371, Ile-372, and Ala-373. These residues vary among the GIRK subtypes, and GIRK1 possesses more hydrophobic and fewer charged residues than the other subtypes (supplemental Fig. 6), suggesting that the Gα_i/o binding affinity might differ among the subtypes. Unfortunately, the N-terminal binding region was not identified here, as the construct used in our study lacks residues 1–40 and 64–84.

Modulation of GIRK by G_i/o

GIRK is activated by the Gβγ binding, and Gα_i/o modulates the gating property of GIRK. Gα_i/o(GDP)βγ is assumed to be precoupled with GPCRs and GIRK, which facilitates the efficient gate opening of GIRK, with the high specificity of GPCR signaling to GIRK (17, 18). Gα_i/o(GDP)βγ binding to GIRK suppresses the basal K⁺ current of GIRK while maintaining the maximal current evoked by the GPCR stimulation, which is referred to as priming (14, 15, 19–22). On the other hand, Gα_i/o(GTP) binding to GIRK accelerates its deactivation (15).

Recently, a two-site model was proposed for the G protein binding sites on GIRK (“anchoring site” and “activation site”) (19, 22). The anchoring site is relevant to precoupling and priming for GIRK when the site accommodates Gα_i/o(GDP)βγ and to the binding of Gα_i/o(GTP) when Gβγ shifts to the activation site, which was revealed by our NMR analyses as the border of the two neighboring subunits of the GIRK tetramer (12). In this study we have identified the anchoring site for Gα_i/o(GTP) on GIRK (Figs. 5 and 7). Notably, it is unknown whether the site is identical to the one for Gα_i/o(GDP)βγ. Upon ligand stimulation of the precoupled GPCR, the GDP-GTP exchange on Gα_i/o in the complex with Gβγ at the anchoring site for Gα_i/o(GDP)βγ decreases the affinity of Gα_i/o for Gβγ, allowing Gβγ to activate GIRK by binding to the activation site. When the GPCR stimulation ends, GTP on Gα_i/o at the anchoring site for Gα_i/o(GTP) is rapidly hydrolyzed to GDP with assistance from RGS (23). Thus, Gα_i/o, at the anchoring site for Gα_i/o(GTP) immediately re-associates with Gβγ, leading to the closure of the GIRK gate.

FIGURE 7.

A model of the Gα_i/o-GIRK-Gβγ ternary complex. Left, the nitrogen atoms of the Gβγ binding residues on GIRK1 (12) are mapped as magenta balls on the complex model of Gα and the GIRK tetramer (Fig. 5). Right, shown is a ternary complex model of Gα-GIRK-Gβγ. First, the binding model of Gβγ-GIRK was built by using HADDOCK with parameters listed in supplemental Table 2. The Gβγ binding residues on GIRK_CP-L identified in our previous study (12) and the residues on Gβγ reported to be important for GIRK binding (54, 55) were defined as the active residues in the software. Then the Gα-GIRK complex model shown in Fig. 5 and the Gβγ-GIRK complex model were superimposed by the GIRK structure, rendering the Gα-GIRK-Gβγ ternary complex model. Gβ and Gγ are colored magenta and violet, respectively. The residues involved in the intermolecular interactions between Gα and Gβγ, within a distance of 4 Å in the Gα_i1(GDP)β₁γ₂ (PDB code 1GP2) structure, are colored orange.

In this study we revealed that the cytoplasmic region of GIRK1 and the GTP-bound Gα_i3 directly bind to each other, and the αA helix of GIRK1 corresponds to the anchoring site for Gα_i/o(GTP). The mapping of the Gβγ binding residues (12) on the structure of the Gα-GIRK complex indicated that the binding sites of Gα_i3(GTP) and Gβγ on GIRK do not overlap with each other, and therefore, Gα_i3(GTP) and Gβγ can simultaneously bind to GIRK (Fig. 7). Furthermore, the mapping of the RGS binding site on the structure of Gα suggested that RGS can bind to Gα_i3 in the complex with GIRK (supplemental Fig. 7). Therefore, the Gα_i/o-GIRK-Gβγ ternary complex model obtained here provides the structural basis for the rapid closure of the GIRK channel upon signal termination through the efficient removal of the proximal Gβγ from GIRK.

It should be noted that the α2 helix of Gα_i3 in the GIRK binding site (the α2/α3 site) is located in the switch II region, which is known to alter its conformation upon GDP-GTP exchange and is included in the Gβγ binding site. Thus, the anchoring site of Gα_i/o(GDP)βγ is different from that of Gα_i/o(GTP) revealed here. Structural analyses of the interaction between Gα_i/o(GDP)βγ and GIRK will provide a complete understanding of the modulation mechanism of the GIRK-gating by G_i/o proteins.