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Subunit with Inward Rectifier K+ Channel Kir3
Departments of Pharmacology (Q.Z., H.N, S.N., T.Ko.) and Anatomy and Cell Biology, College of Medicine, University of Illinois at Chicago, Illinois 60612 (T.Ka., Y.N.)
Received May 12, 2003; accepted August 8, 2003
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Abstract |
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subunits bind and activate G protein-coupled inward rectifier K+ (GIRK) channels. This protein-protein interaction is crucial for slow hyperpolarizations of cardiac myocytes and neurons. The crystal structure of G shows a seven-bladed propeller with four strands in each blade. The G/G
interacting surface contains sites for activating GIRK channels. Furthermore, our recent investigation using chimeras between G1 and yeast (STE4) suggested that the outer strands of blades 1 and 2 of G1 could be an interaction area between G1 and GIRK. In this study, we made point mutations on suspected residues on these outer strands and investigated their ability to activate GIRK1/GIRK2 channels. Mutations at Thr-86, Thr-87, and Gly-131, all located on the loops between -strands, substantially reduced GIRK channel activation, suggesting that these residues are G/GIRK interaction sites. These mutations did not affect the expression of G1 or its ability to stimulate PLC2. These residues are surface-accessible and located outside G/G interaction sites. These results suggest that the residues on the outer surface of blades 1 and 2 are involved in the interaction of G with GIRK channels. Our study suggests a mechanism by which different effectors use different blades to achieve divergence of signaling. We also observed that substitution of alanine for Trp-332 of G1 impaired the functional interaction of G1 with GIRK, in agreement with the data on native neuronal GIRK channels. Trp-332 plays a critical role in the interaction of G1 with G as well as all effectors so far tested.
; Dascal et al., 1993; Kubo et al., 1993; Wickman and Clapham, 1995; Stanfield et al., 2002). The activity of GIRK channels, in turn, is regulated by G proteins (Breitwieser and Szabo, 1985; Pfaffinger et al., 1985; Kurachi et al., 1986). Furthermore, the subunits (G) but not the subunit (G) of G proteins were shown to activate the GIRK channels (Logothetis et al., 1987; Ito et al., 1992, Reuveny et al., 1994; Huang et al., 1995; Krapivinsky et al., 1995). Thus, the interaction between G and GIRK is a crucial process for the physiological functions of the brain and the heart and, as such, is likely to be one of the sites for pharmacological interventions. Despite its importance, the molecular mechanism of this protein-protein interaction is poorly understood. One approach to clarify this interaction is to investigate the events that take place on the side of the K+ channel. Another is to elucidate the processes occurring on the G molecules.
The aim of this study was to further clarify the residues of the G molecule that interact with GIRK. X-ray crystallography has revealed that G is a seven-bladed propeller structure with four antiparallel strands for each blade. G interacts with G through the top surface of the propeller. The outer -strands of each blade form the outer surface of the torus-like structure (Wall et al., 1995; Lambright et al., 1996; Sondek et al., 1996). Because G is capable of interacting with downstream effectors only if G is detached from G, it has been assumed that the interaction sites of G with G may also be the sites that interact with various effectors. Indeed, mutagenesis studies have identified various G-contacting residues of G that are crucial for the interaction between G and various effectors, such as phospholipase Cs (PLCs), adenylyl cyclases, and GIRK channels (Ford et al., 1998; Li et al., 1998).
The G-interacting surface of G may not be the only region to bind the effectors. Albsoul-Younes et al. (2001) constructed a set of G1 chimera proteins by replacing a sequence on or near the outermost strand of each of the blades with the corresponding sequence in yeast G (STE4). These proteins were tested for their ability to activate native GIRKs in brain neurons. The results indicated that the chimeras on the outer segments of blades 1 and 2 (called D1 and CD2) significantly impaired the GIRK activation (Albsoul-Younes et al., 2001), suggesting that these locations are important for the interaction between G and GIRKs. These locations do not belong to the interaction sites between G and G.
Because we know that the outer segments of blades 1 and 2 are crucial for the G/GIRK interaction, the next step is to precisely identify the amino acid residues in these regions that are crucial for the GIRK activation. In the present experiments, we introduced point mutations on each of the nonconserved (between G1 and STE4) residues on D1 and CD2 to its corresponding residue in STE4 (Albsoul-Younes et al., 2001). Each of these mutant G1 cDNAs was expressed in human embryonic kidney (HEK) 293 cells and was tested for the ability to produce inwardly rectifying K+ currents. In this way, we have identified three G1 residues located on the outer strands of blade1 and blade 2; mutations of these residues impaired the GIRK activation and therefore may play a crucial role in GIRK activation. The results suggest that in addition to the G-interacting surface, these residues on the outer surface of blades 1 and 2 are specifically involved in the interaction of G with GIRK channels. Recently, Mirshahi et al. (2002) also identified 2 residues on blades 1 and 2 that could be the interaction sites with GIRK but not with G. Altogether, therefore, 5 residues are putative sites at which G/GIRK interaction would take place outside the G/G interface. An abstract of the present work has appeared (Zhao et al., 2002).
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Materials and Methods |
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1 cDNA was from the mouse and G2 cDNAs was from the bovine. All cDNAs used were subcloned into pCMV5 vector (Andersson et al., 1989). Point mutations were introduced by using the QuikChange site-directed mutagenesis kit (Stratagene; La Jolla, CA). The mutations were verified by the sequence facility at the University of Chicago. Cell Culture and Transfection. HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) at 37°C with 10% CO2. Transient transfections of HEK293 cells were performed with Effectene (QIAGEN, Valencia, CA) according to the manufacturer's protocol. The total amount of cDNAs for the same experiment was kept constant by adding empty vectors.
Electrophysiology. HEK293 cells in 60-mm dishes were transfected with cDNAs of wild-type or mutant G1, G2, GIRK1, GIRK2, and GFP. The amount of each cDNA was 0.3 µg, except 0.1 µg for GFP. Twenty-four hours later, the cells were replated onto 35-mm dishes. The dish has a small well (about 1.2 cm in diameter) that was coated with rat-tail collagen (Roche Molecular Biochemicals, Indianapolis, IN). Forty-eight to 56 h after transfection, the whole-cell patch clamp was performed on the stage of a fluorescence inverted microscope. We did experiments on isolated cells with GFP fluorescence. The bathing solution contained 146 mM sodium gluconate, 10 mM potassium gluconate, 2.4 mM CaCl2, 1.3 mM MgCl2, 5 mM HEPES-NaOH, and 0.5 µM tetrodotoxin, pH 7.4. The pipette solution contained 151 mM K gluconate, 5 mM HEPES-KOH, 0.5 mM EGTA-KOH, 0.1 mM CaCl2, 4 mM MgCl2, 3 mM Na2-ATP, and 0.2 mM GDP, pH 7.2. Membrane potentials were corrected for the liquid junction potential between the pipette solution and the bathing solution (the bathing solution side was 7 mV positive). Cell capacitance was determined by cancellation of the transient responses to square-wave voltage inputs as directed in the user's manual for the EPC-7 patch clamp (List Electronic, Darmstadt, Germany). Series resistance was compensated electronically, usually by about 60%, and the remaining resistance was compensated mathematically by a linear approximation. The pCLAMP programs (version 6.03; Axon Instruments, Inc.) were used for the acquisition and analysis of the data. Recordings were performed at room temperature. Statistical results were expressed as mean ± S.E.M.
Western Blot Analysis. HEK293 cells in 35-mm dishes were transfected with 0.75 µg of G1 and 0.75 µg of FLAG-G2. Forty-eight h after transfection, the cells were harvested and lysed on ice for 20 min in 200 µl of lysis buffer (20 mM HEPES-NaOH at pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 25 mM -glycerophosphate, 1 mM Na3VO4, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 0.5% Triton X-100, and 10% glycerol). Lysates were centrifuged at 15,000g for 20 min at 4°C. The supernatants were heated at 100°C for 5 min in a sample buffer (50 mM Tris-HCl, pH 6.8, 8% glycerol, 1% SDS, 2% 2-mercaptoethanol, and 0.008% bromphenol blue). Aliquots (20 µl) of the samples were loaded on 5% stacking, 15% separating SDS-PAGE gel. After electrophoresis, the proteins were transferred to a BA81 nitrocellulose membrane and blocked with blocking buffer (50 mM Tris-HCl, pH 8.0, 2 mM CaCl2, 80 mM NaCl, 5% skim milk, 0.2% Nonidet P-40, and 0.02% NaN3). The proteins were detected by G (T-20) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-FLAG M2 monoclonal antibody (Sigma, Saint Louis, MO). The bound antibodies were visualized by an enhanced chemiluminescence detection system, using horseradish peroxidase-conjugated anti-rabbit or anti-mouse Ig secondary antibodies (Amersham Bioscience, Arlington Heights, IL).
PLC Assay in HEK293 Cells. HEK293 cells at 60 to 70% confluence in 24-well plates were transfected with PLC2, G1 (wild type or mutant), G2, and Go. The total amount of cDNA was 0.8 µg per well. One day after transfection, the cells were labeled with 2 µCi/ml [3H]inositol in 0.5 ml of inositol-free DMEM with 1% fetal bovine serum for 24 h. The next day, the cells were washed for 10 min and then stimulated for 50 min at 37°C with 0.5 ml of inositol-free DMEM supplemented with 25 mM HEPES, pH 7.4, and 10 mM LiCl. The reactions were terminated by 20 mM formic acid (0.75 ml), and the cells were incubated at 4°C for 30 min. The samples were neutralized to pH 7 to 8 by 100 µl of 3% ammonium hydroxide, collected, and centrifuged at 15,000 rpm for 5 min. The supernatants were loaded on 1 ml of Dowex AG 1-X8 columns pre-equilibrated with 10 ml of 4 M ammonium formate and 0.2 M formic acid followed by 5 ml of 0.18% ammonium hydroxide solutions. The flow-through of the sample and of the 1 ml of 0.18% ammonium hydroxide solution loaded after the sample was collected as the inositol fraction. The columns were washed with 4 ml of 40 mM ammonium formate and 0.1 M formic acid. The inositol phosphates were eluted with 1 ml of 4 M ammonium formate and 0.2 M formic acid. The inositol fraction and the inositol phosphate fraction were measured by liquid scintillation counting.
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Results |
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12 Cotransfection in HEK293 Cells. We used HEK293 cells as a heterologous expression system to test the ability of wild-type and mutant Gs to activate GIRK channels. GIRK1/GIRK2 channel subunits alone or GIRK1/GIRK2 together with G12 were expressed. Whole-cell currents were evoked by a series of voltage steps from -127 to -27 mV at a 10-mV increment. Holding potential was -77 mV. Figure 1, A and B, shows that cells transfected with GIRK1/GIRK2 and G12 cDNAs produced large currents. The currents showed inward rectification and the reversal potential (-65.4 ± 0.8 mV; n = 28) corresponded to the K+ equilibrium potential (EK) (-68.4 mV).
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The current amplitudes at -97 mV were chosen to represent the capability of various G1 subunits to activate the GIRK current (Fig. 1C). Transfection with wild-type G12 plus GIRK1/GIRK2 evoked large currents, whereas cells transfected with GIRK1/GIRK2 (without G12) produced smaller currents. These small currents would have been derived from the transfected GIRK1/GIRK2 activated by the endogenous G. If neither GIRK1/GIRK2 nor G was transfected, the current was very small (about one third of the current with GIRK1/GIRK2 only) (Kawano et al., 1999). This small current would have originated from unknown channels endogenous in HEK293 cells (Kawano et al., 1999).
In addition, Fig. 1C shows GIRK1/GIRK2 currents induced by two G1 mutants, the chimera D1 and a point mutation W332A. The D1 mutant, the same as used by Albsoul-Younes et al. (2001), was generated by replacing the G1 sequence on and near the outer strand of blade 1 (S84 to P94) with the corresponding yeast (STE4) sequences (Fig. 2). W332A is a point mutant on the G-contacting surface of G1 (Albsoul-Younes et al., 2001). Both the chimera and W332A produced substantially smaller GIRK current than the wild-type G12. These data on the genetically identified GIRK1/GIRK2 channels coincide well with the previous data on native GIRK channels in locus ceruleus neurons (Albsoul-Younes et al., 2001).
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Identifying the Critical Residues in the D1 Region for G-GIRK Interaction. We mutated each nonconservative G1 residue over the D1 mutation region (i.e., from Ser84 to Pro94) to its corresponding STE4 residue (Fig. 2A); these are Y85A, T86S, T87G, N88L, V90Q, and H91N. We noticed that two residues, Tyr85 and Val90, are nonconservative not only between 1 and STE4 but also between 1 and 5. The subunit G5 is a distinct member of the G family. Instead of activating GIRK channels, G5 inhibits GIRK currents (Lei et al., 2000). We therefore made two additional mutants, Y85F and V90E, by replacing the G1 residue with the corresponding residue of G5.
Each of these mutant G1 cDNAs was cotransfected with GIRK1/GIRK2 and G2. Figure 2, B, C, and D, summarizes the GIRK current stimulation by these G1 mutants in D1 region. Compared with wild-type G12, mutants T86S and T87G were much less effective in activating GIRK currents (p < 0.01 and p < 0.05, respectively) (Fig. 2B). No other mutants showed any significant differences from the wild-type 1 in their ability to enhance the GIRK current (Fig. 2, C and D).
Identifying the Critical Residues in the CD2 Region for G-GIRK Interaction. The result obtained by Albsoul-Younes et al. (2001) indicates that the G1 mutant CD2 is less capable of activating GIRK channels in locus ceruleus neurons. In the CD2 mutant, the G1 sequence (Leu126-Asn132) on the loop connecting the two outer strands of blade 2 was replaced with its counterpart in STE4 (Fig. 3A). We generated five G1 mutants, L126V, T128S, R129K, G131V, and N132A, by mutating each of the nonconservative G1 residues over the CD2 region to its corresponding STE4 residue. We also made one deletion mutation, K127
(Fig. 3A). These mutants were tested for their ability to stimulate GIRK channels coexpressed in HEK293 cells. As shown in Fig. 3B, G1 mutant G131V showed a substantially lower ability to activate the GIRK current compared with wild type. All other mutants in the CD2 region were able to enhance GIRK current in a manner not significantly different from that of the wild type (Fig. 3, B and C).
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Basal Currents or Agonist-Induced Currents. Mirshahi et al. (2002), by using the oocyte heterologous system, identified two G residues, Ser67 and Thr128. Both of these residues are located outside the G/G contact region and are likely to be interaction sites between G and GIRK channels. They observed that mutations of these residues inhibited the GIRK "basal currents", which are presumably activated by the endogenous G. One might ask whether our mutants impair the generation of the GIRK basal current.
In HEK293 cells, unlike the oocyte system, the "basal GIRK current" is relatively small compared with the current induced by exogenous G12 (compare the first column with the fourth column in Fig. 1C). This makes it difficult to determine whether our mutations (T86S, T87G, and G131V) impaired the "basal GIRK current". From our results, therefore, we can conclude only that our mutants impaired the GIRK current stimulated by exogenous G12.
Expression of G1 Mutants in HEK293 Cells. To examine the expression levels of the G1 mutants, HEK293 cells expressing G1 and FLAG-G2 were subjected to the Western blot analysis. An antibody raised against a peptide at the carboxyl terminus of G1 (T-20, Santa Cruz Biotechnology) was used for immunoblotting. Figure 4 shows representative Western blots from cells expressing the G1 mutants. Low levels of G (endogenous G) (the leftmost lane) could be detected in cells transfected with vectors alone (Fig. 4, A and B). All three mutant G1s that had a low ability to activate GIRK channels-T86S, T87G, and G131V-were expressed at a level comparable with that of the wild-type G1 (Fig. 4A); thus, the functional impairment is not caused by the differences in the expression level. All other G1 mutants that can activate the channels as effectively as wild type were also expressed at a level similar to that of the wild-type 1 (Fig. 4B).
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It is noted that expression of G2 in the Western blot is fairly variable in the example of Fig. 4. However, inspection of all Western blots obtained in this experiment (five runs for Fig. 4A and six runs for Fig. 4B) indicated that there seemed to be no correlation between the Western blot expression of G2 and the functional ability of various mutants and the wild type. It is thus unlikely that the functional defect of the mutants is caused by differences in the G2 expression.
Activation of PLC2 by G1 Mutants. G regulates not only GIRKs but also several other effectors including PLCs. To test whether the G1 mutants retain their ability to activate PLC, we transfected HEK293 cells with PLC2, G1, and G2 in either the absence or the presence of Go. For each sample, the amount of inositol phosphates produced by the lithium treatment was normalized to the total amount of free inositol and inositol phosphates. As shown in Fig. 5, transfection of PLC2 alone resulted in the production of a small amount of inositol phosphates. The wild-type G12 caused a 7-fold increase in inositol phosphate production, which could be completely suppressed to the basal level by cotransfection of Go. The bottom panel shows immunoblots of the samples from the same experiment. Without G12 sequestration by Go, the expression level of G1 was more or less proportional to the amount of inositol phosphate produced. Taken together, the elevation of the inositol phosphate production represented the activation of PLC2 by G12 coexpressed in the cells. In conclusion, this experiment shows that despite their inefficiency at activating the K+ channels, the G1 mutants T86S, T87G, and G131V were as effective as wild type in stimulating PLC2 activity. In addition, the inhibitory effect of coexpression of Go on PLC2 activation by these mutants suggests that they can interact with Go in a way similar to wild-type G1.
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Albsoul-Younes et al. (2001) observed that the CD2 mutant, in which the G1 segment near the outer strand of blade 2 was replaced with the corresponding STE4 sequence, was less potent in activating locus ceruleus GIRK channels and in stimulating the PLC2 activity. However, the present study showed that a CD2 region mutant, G131V, which was less effective in activating GIRK channels (Fig. 3B), was able to activate PLC2 as effectively as the wild-type G1. This may mean that residues in the CD2 region other than Gly131 is involved with PLC2 activation.
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Discussion |
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, G, and GIRK. The signals initiated by G can be transmitted to downstream effectors (including GIRK channels) only if G is dissociated from G. This feature suggested that the interaction domain between G and G is shared with the interaction domain between G and effectors (Ford et al., 1998; Li et al., 1998). As to the interaction between G and GIRK1/GIRK4, the results by Ford et al. (1998) indicate that six G1 residues located on the G/G interface could be the interaction sites between G and GIRK (Fig. 6A, 
). Furthermore, Albsoul-Younes et al. (2001), using GIRK channel in brain neurons (presumably GIRK1/GIRK2), found that two more residues on G1, Trp332, and Asp246, belong to this group (Fig. 6A, 
).
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G5 is a particular type of G subunit. G5 inhibits the GIRK channel, whereas all other Gs activate it (Lei et al., 2000). Mirshahi et al. (2002) have recently determined the G residues that are responsible for this difference; these residues may be the interaction sites between G and GIRK. Their results indicate that Ser67 and Thr128 of G (Fig. 6A, 
) may be the location of G/GIRK interaction. These sites are located outside the G/G interaction domain.
Albsoul-Younes et al. (2001) used STE4 to search for the G/GIRK interaction sites. STE4, being the member of the G family that is most distantly related to mammalian Gs, does not activate GIRK (Peng et al., 2000). Albsoul-Younes et al. (2001) constructed a set of chimera DNAs, in which the sequence at or near the outer strand of each blade was replaced by the corresponding sequence of STE4. The mutant G12 proteins were purified and were tested on native GIRK channels in locus ceruleus neurons. Substitution of STE4 outer strands of blades 1 and 2 for the wild type resulted in impairment of GIRK channel activation, suggesting that some residues on the outermost sheets of blades 1 and blade 2 are the G/GIRK interaction sites.
In the present study, we used genetically identified GIRK channels (GIRK1/GIRK2) instead of native GIRK channels in locus ceruleus neurons. By creating point mutations on the regions identified previously (Albsoul-Younes et al., 2001), we have localized the G residues that are responsible for the functional impairment of the chimera between STE4 and G1. Our results indicate that three residues on G1 (Thr86, Thr87, and Gly131; Fig. 6A, 
) are putative interaction sites between G1 and GIRK. It is noted that all three residues are located outside the G/G contact points and they are all located at sites accessible from outside (Fig. 6B). Together with the work by Mirshahi et al. (2002), there are now five residues of this category (Fig. 6A, filled symbols), suggesting that the functional roles of these residues are not trivial. Interestingly, all five residues are located on the loops connecting the strands; this arrangement is consistent with the notion that active sites of protein-protein interactions are often located on loops between -strands.
In summary (Ford et al., 1998; Albsoul-Younes et al., 2001; Mirshahi et al., 2002; present study), there are three domains that are involved in the interaction among G, G, and GIRK: 1) a G/GIRK interaction area outside the G/G interaction site (Fig. 6A, filled symbols), 2) an area belonging both to the G/G interaction sites and to the G/GIRK interaction sites (Fig. 6A, open symbols), and 3) an area belonging to the G/G interaction region but outside the G/GIRK interaction sites (not shown). In other words, the G/G interaction area and the G/GIRK interaction area are not identical, but the two areas intersect.
Protein-Protein Interactions Involving G. Various signaling pathways from many receptors converge on G proteins. Convergence is caused by the simple fact that the same G proteins couple to many different receptors. The signals then diverge from the G proteins to multiple effectors such as GIRK, calcium channels, PLCs, and adenylyl cyclases. The results from this study and others have revealed that these effectors (or any interacting proteins) associate with G through both the G-interacting surface and part of the outer-surface of the blade structure. It has also become clear that each of these proteins is using a specific part of the outer surface of G. Certain mutations of the outer strands of blades 2, 6, and 7 impair PLC2 activation but not the regulation of adenylyl cyclases (Panchenko et al., 1998; Albsoul-Younes et al., 2001). Mutations of certain residues on the outer surface of blades 1 and 2 affected GIRK activation but not PLC2 or adenylyl cyclase type II activation (Albsoul-Younes et al., 2001; present study). Phosducin binds G not only on the G/G interaction site but also on the outer strands of blades 1 and 7. However, phosducin does not bind to the three putative G/GIRK interaction sites described in this study (Thr86, Thr87, and Gly131; Fig. 6A) (Gaudet et al., 1996). It seems that G is using its blade structure for the regulation of different nonhomologous effector molecules. This could be one of the mechanisms for achieving divergence of signaling.
On the other hand, certain residues on G seem to play a more universal role for the protein-protein interaction. One of them is Trp332. Alanine mutation of the Trp332 residue in G1 impaired the subunit's ability to activate GIRK1/GIRK2 (Fig. 1C) as well as the native GIRK channel in locus ceruleus (Albsoul-Younes et al., 2001). Recently, Nishida and MacKinnon (2002), based on their crystallographic study of N- and C-termini of GIRK and in analogy with the interaction sites between phosducin and G (Gaudet et al., 1996), proposed the idea that the -helix near the end of the GIRK C terminus may interact with G at Trp332. It is known that not only is Trp332 an interaction site between G and GIRK and between G and phosducin but also between G and G (Lambright et al., 1996). In addition, Trp332 interacts with several effectors such as PLC2, PLC3, and adenylyl cyclase 2 (Ford et al., 1998; Li et al., 1998). In this respect, Trp332 seems to be a universally key residue for the protein-protein interactions involving G.
One of the marked differences between G/G association and G/phosducin association is that the former association does not entail conformational changes of G (Lambright et al., 1996), whereas the latter does (Gaudet et al., 1996; Loew et al., 1998). Because of certain similarities between the G/phosducin association and the G/GIRK association, the question arises of whether conformational changes of G occur accompanying the G/GIRK interaction. Crystallographic studies will answer this.
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Acknowledgements |
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complex and John M Collins for suggestions in the writing. |
Footnotes |
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ABBREVIATIONS: GIRK, G protein-coupled inward rectifier K+ channels; PLC, phospholipase C; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium.
Address correspondence to: Tohru Kozasa, Department of Pharmacology, College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612. E-mail: tkozas{at}uic.edu
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