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REPORT
Department of Physiology and Biophysics, Faculty of Medicine, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada
Submitted 1 March 2006; accepted in final form 3 April 2006
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ABSTRACT |
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subunit of the heterotrimeric G-protein complex cause voltage-dependent inhibition of N-type channel activity, crucially influencing neurotransmitter release and contributing to analgesia caused by opioid drugs. Previous work using chimeras of the G-protein subtypes G1 and G5 identified two 20–amino acid stretches of structurally contiguous residues on the G1 subunit as critical for inhibition of the N-type channel. To identify key modulation determinants within these two structural regions, we performed scanning mutagenesis in which individual residues of the G1 subunit were replaced by corresponding G5 residues. Our results show that G1 residue Ser189 is critical for N-type calcium channel modulation, whereas none of the other G1 mutations caused statistically significant effects on the ability of G1 to inhibit N-type channels. Structural modeling shows residue 189 is surface exposed, consistent with the idea that it may form a direct contact with the N-type calcium channel 
1 subunit during binding interactions. |
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INTRODUCTION |
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; Wheeler et al. 1994; Zamponi 2001). It is now known that heterodimers of the G-protein and 
subunits interact directly with N-type and P/Q-type channels, causing steady-state inhibition of the channels and increasing their time constant of activation. Both aspects of this modulation depend on membrane potential and can be relieved by either application of strong depolarizations or trains of action potentials (Dolphin 2003; Page et al. 1997). The extent of inhibition is dependent on both calcium channel subtype and on the G-protein subunit isoform (Arnot et al. 2000; Garcia et al. 1998; Ruiz-Velasco and Ikeda 2000), with G5 subunits mediating little or no detectable voltage-dependent inhibition of N-type currents, whereas G1 results in robust inhibition of these channels (Doering et al. 2004).
Efforts to map the inhibitory binding interactions of the N-type and P/Q-type channels with the G-protein subunit have yielded evidence suggesting that three intracellular regions of the pore-forming Cav2 subunits of the channels—the amino terminus, the loop-linking domains I and II, and the carboxy terminal region—all contribute to enable binding of the G-protein complex (Agler et al. 2005; Canti et al. 1999; De Waard et al. 1997; Page et al. 1997; Qin et al. 1997; Zamponi et al. 1997). Complementary efforts to map regions of the G-protein subunit that are involved in N-type and P/Q-type channel modulation have resulted in the identification of several amino acid residues on the G1 subunit, many of which overlapped with the G interaction region (Agler et al. 2003, 2005; Ford et al. 1998; Mirshahi et al. 2002a). By using series of chimeras between G1 and G5 we recently identified a structurally contiguous region of the G1 molecule that, when replaced by the corresponding G5 sequence, resulted in complete loss of inhibitory action against the N-type channel (Doering et al. 2004). This structurally contiguous region of G1 constitutes residues 110–112, 140–168, and 186–204 of the primary G1 structure. Within these regions, mutations of G1 residues Tyr111 and Asp153 resulted in loss of voltage-dependant inhibition, suggesting their importance for direct binding interactions with the N-type channel (Doering et al. 2004).
Here we further characterize the molecular determinants that underlie the inhibitory action of G1 on the N-type channel. We describe construction of a series of G1 mutations within regions 140–168 and 186–204, in which individual residues were replaced with those of G5, and examination of their effects on N-type calcium channel inhibition by paired-pulse facilitation assays. Of all of the mutants examined, only Ser189 of G1 emerged as a crucial residue for voltage-dependent inhibition of the N-type channel, suggesting that modulation of N-type channels involves highly localized G subunit structural determinants.
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METHODS |
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cDNAs encoding human G1 and G2, rat G5 were previously described (Arnot et al. 2000; Feng et al. 2001). Wild-type (WT) rat calcium channel subunit cDNAs were donated by T. Snutch (University of British Columbia). Constructs encoding GIRK1 and GIRK4 subunits were provided by Dr. Hubert van Tol (Center for Addiction and Mental Health, Toronto, Ontario).
G1 mutants
Using overlap extension PCR methodologies (Ho et al. 1989), the following mutants of human G1 were constructed: G141M, G144N, C148A, R150S, L152T, V158L, S160A, T164G, M188L, S189C, S191D, D195S, L198T, and A203G. In each case, this constitutes a change to the residue at the corresponding position of the primary structure of the rat G5 molecule. Mutagenic PCRs were performed using Pfu ultra polymerase (Stratagene), according to the manufacturer’s suggestions. Full-length mutant G1 cDNAs were digested with Xho I and Kpn I (these restriction sites were included in the 5' ends of the nonmutagenic flanking primers designed to anneal to the 5' and 3' ends of the human G1 open reading frame), then subcloned into Xho I-, Kpn I–double-digested pMT2-XS expression vector. All inserts were sequenced to confirm the presence of the mutations and to rule out PCR errors.
Tissue culture and transient transfection
Human embryonic kidney tsA-201 cells were grown and transfected with calcium phosphate as previously described in detail (Doering et al. 2004). In each experiment involving calcium channels, wild-type or mutant rat Cav2.2 calcium channel 1 subunits were cotransfected with rat 1b, rat 2-
1, human G2, and an enhanced green fluorescent protein (EGFP) expression marker, plus one of wild-type or mutant G subunits. For experiments involving GIRK channels, GIRK1 and GIRK4 subunits were used instead of calcium channel subunits. To prevent overgrowth of cells, culture dishes were placed in a 28°C incubator 3–9 h after the washing step performed to remove DNA precipitate from the cultures.
Voltage-clamp recordings of N-type channel currents
Glass coverslips carrying transfected cells were transferred to a 3-cm culture dish containing recording solution consisting of (in mM) 20 BaCl2, 1 MgCl2, 10 HEPES, 40 tetraethylammonium hydroxide, 10 glucose, and 65 CsCl (pH 7.2 with tetraethylammonium-hydroxide). Whole cell patch-clamp recordings were performed as described previously (Doering et al. 2004), using internal pipette solution consisting of (in mM) 108 cesium methane-sulfonate, 4 MgCl2, 9 EGTA, and 9 HEPES (pH 7.2). Currents were evoked by stepping from –100 mV to a test potential of +20 mV. G-protein inhibition was assessed by application of a +150-mV prepulse (PP) for 50 ms. Only cells with current amplitudes >50 pA and <1.2 nA were used for analysis. The degree of prepulse relief of tonic G-protein inhibition was determined as the ratio of peak current amplitudes seen after (I+PP) and before (I–PP) the prepulse—referred to hereinafter as the paired-pulse facilitation (PPF) ratio and reflects the ability of a given G-protein subunit to inhibit N-type current activity. The PP paradigms were programmed using the "train" and "user list" functions in pCLAMP (Axon Instruments).
Voltage-clamp recordings of GIRK channel currents
Whole cell recordings were performed using an internal solution of (in mM) 100 potassium gluconate, 40 KCl, 10 HEPES, 5 EGTA, 1 MgCl2, and 5 NaCl (pH 7.4 with KOH). External solution consisted of (in mM) 25 KCl, 10 HEPES, 10 glucose, and 116 NaCl (pH 7.4 with NaOH). Under these conditions, the predicted reversal potential of potassium is about –30 mV. GIRK channel activity was tested by holding the cells at –35 mV, followed by application of a voltage ramp from –120 to +60 mV over 525 ms. Only cells displaying inward rectification were used for analysis, and whole cell GIRK conductance was obtained by a linear fit to the inward current observed between the potentials of –100 and –60 mV during the voltage ramp. Whole cell capacitance ranged from 10 to 186 pF. In this range, there was no significant correlation between capacitance and whole cell conductance (Pearson correlation coefficients for matched arrays of capacitance and conductance from cells expressing GIRK1/4 channels were 0.233, 0.247, and 0.609 for data from cells coexpressing either no heterologous G, heterologous G1-S189C, or heterologous WT G1, respectively). Thus data are plotted in Fig. 2B as whole cell conductance rather than current densities. GIRK channel data presented herein were all recorded from one batch of tsA-201 cells of the same passage and culture, cotransfected simultaneously in separate plates, under identical conditions with the exception of the G-protein–expression vectors used (or omitted) to obtain the desired coexpression pattern in the individual transfections.
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All electrophysiological data were analyzed using Clampfit (Axon Instruments) and fitted in Sigmaplot 2000 (SPSS) or Microsoft Excel. Statistical analysis was carried out in SigmaStat 2.03 (SPSS). A Kruskal–Wallis one-way ANOVA on ranks (Dunn’s method) was performed on N-type calcium channel data; assuming normal distribution (based on passed normality test) a one-way ANOVA was performed using a post hoc Tukey test on GIRK channel data.
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RESULTS |
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1 with the corresponding residues of G5 yields a chimera that cannot induce voltage-dependent inhibition of the N-type channel (Doering et al. 2004). Alignment of the human G1 sequences for residues 140–168 and 186–204 with the corresponding rat G5 sequences revealed 14 nonconserved residues. To identify which of these individual amino acid residues were critical for G-protein modulation of the N-type channel, responsible for this effect, we systematically replaced G1 residues with those corresponding to the G5 sequence. The resulting mutant G1 constructs were coexpressed in HEK (human embryonic kidney) cells with N-type channel subunits and an EGFP expression marker. Voltage-dependent G-protein inhibition of N-type currents was assessed by using a PPF paradigm (see METHODS), wherein alternating test potentials were recorded after application of +150-mV depolarizing prepulses intended to disrupt inhibitory interaction of G subunits and N-type channels (Fig. 1). As controls and to establish a baseline, N-type channels were also coexpressed either with heterologous WT G1 or without any heterologous G, and PPF was tested under both conditions. As illustrated by current traces shown in Fig. 1A, application of prepulses to cells coexpressing heterologous WT G1 resulted in substantially larger and more rapidly activating N-type currents (Fig. 1A, right traces), but prepulses applied to cells not expressing heterologous G typically resulted in only negligible changes (Fig. 1A, left traces), as expected from the absence of G-protein modulation.
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1 mutants are summarized in Fig. 1B. Among the 14 mutants, 11 yielded PPF ratios that were similar to those obtained with WT channels. Two additional mutants, G144N and M188L, appeared to result in a somewhat reduced degree of prepulse facilitation; however, although a Student’s t-test indicated significant differences for these two residues (P values of 0.003 and 0.004, respectively), the respective distributions of PPF values failed the normality test and analysis by ANOVA on ranks indicated that this reduction did not reach statistical significance in either case. In contrast, the S189C mutant yielded an average PPF ratio slightly lower than that of negative control assays (1.13 and 1.16, respectively), which was significantly different from that observed with WT G1 (difference of ranks score, 141.9; P < 0.05).
To eliminate the possibility that the S189C mutant might not express, or not fold correctly, we examined the abilities of both G1-S189C and WT G1 to activate GIRK1/4 channels expressed in tsA-201 cells. As illustrated by raw current trace ensembles in Fig. 2, A and B, large inward K+ currents were observed at negative test potentials in cells coexpressing GIRK1/4 channels and either G1-S189C or heterologous WT G1, with significant rectification at more positive test potentials. Inward K+ currents recorded as negative controls from cells with no heterologous G were substantially smaller (see Fig. 2C). ANOVA analysis of whole cell conductances (calculated for ramp potentials between –100 and –60 mV) confirmed a statistically significant difference between K+ conductances of cells with no heterologous G compared with cells expressing either G1-S189C or WT G1 (Fig. 2D), but with no statistically significant difference between the latter two conditions. These results thus confirm the expected functionality of the G1-S189C mutant in the activation of GIRK1/4 channels, consistent with the view that Ser189 is required for direct modulatory interactions with the N-type calcium channel, but not for proper G folding and expression.
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DISCUSSION |
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; Kristensen et al. 1997; Maggio and King 2002; Nadasdi et al. 1995; Tedford et al. 2001, 2004), the G subunit is 10- to 50-fold larger than the typical subjects of these studies—a poor prospect for comprehensive scanning mutagenesis, in view of the resources that would be required. Structure–activity relationship (SAR) studies of the G subunit have thus avoided comprehensive scanning approaches, instead imposing limits on the structural variants tested by using chimeras of G subtypes (Mirshahi et al. 2002b), or in other cases using targeted mutations of structural regions suspected to be significant based on interactions observed in cocrystal structures (Agler et al. 2003).
Here, we used a scanning mutagenesis approach, but limited it to examination of 14 G1 residues in sequence regions 140–168 and 186–204, areas previously identified in our tests of a panel of chimeras of G1 and G5 (Doering et al. 2004). Our data show that only one of these 14 residues (Ser189) significantly contributed to voltage-dependent channel modulation. Taken together with our previous results, there appear to be three residues of G1—Tyr111, Asp153, and Ser189—that are required for voltage-dependent inhibition of N-type channels (Doering et al. 2004). Consistent with their critical role in modulating N-type channels, these residues are all highly conserved in G1 orthologs expressed in human, rat, and mouse. Moreover, sequence alignment indicates that they are also conserved in any of the known human G subtypes (i.e., G1–4) that are capable of modulating the N-type channel (Arnot et al. 2000). As illustrated in Fig. 3, the three residues are noncontiguous in the three-dimensional structure of G1—a situation with many precedents because SAR studies of conotoxins, spider toxins, and also G modulation of effectors other than N-type calcium channels have all identified noncontiguous residues presumed to participate directly in functional contacts with their respective targets (Agler et al. 2003; Lew et al. 1997; Maggio and King 2002; Mirshahi et al. 2002b). The side chains of Tyr111 and Asp153 each protrude outward from the rest of the structure and would presumably be quickly accessible for interactions with intracellular loops of the 1B subunit of the N-type channel. By contrast, Ser189 is situated in a concavity in the center of the structure, with only the -OH functionality at the distal end of its side chain exposed to the aqueous solution at the structural surface. Thus the kinetics of its binding interactions with the N-type channel would be slow relative to Tyr111 and Asp153, although this would have to be confirmed by biochemical means. However, precedents exist in the literature both for binding epitopes that protrude from protein structures and also for less-accessible binding epitopes situated in concavities of protein surfaces (Bogan and Thorn 1998; Kim et al. 1995; Tedford et al. 2004). We should also note that Ser189 does not appear to form a serine kinase consensus site (as evaluated by PROSITE analysis), and thus the loss of N-type channel inhibition is not attributable to altered phosphorylation of G1.
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5 indicate that this subunit is unable to modulate N-type channel activity (Arnot et al. 2000). Interestingly, human G52 has been shown to inhibit heterologously expressed human N-type calcium channels (Zhou et al. 2000), and mouse G52 can inhibit native N-type currents in rat superior cervical ganglion neurons (Garcia et al. 1998; Ruiz-Velasco and Ikeda 2000). Considering the propensity of certain G5 subunits to regulate N-type channels, why then would replacement of a G1 residue with corresponding G5 residue result in a loss of N-type channel regulation, in particular when this G5 residue is conserved in rat, mouse, and human? One possible explanation may lie in the G5 subunit isoforms used in the latter two studies. The N-terminus region of mouse (GenBank accession number P62881) G5 contains about 40 additional amino acid residues compared with the rat G5 subunit used in our previous work (GenBank accession number AAS59141), and this extended N-terminus is not seen with G1. Considering that the remainder of the mouse G5 sequences are >99% identical to the rat isoform, this implies an important role of the extended N-terminus in N-type channel modulation. Because the G1 and G5 subunits used in our experiments do not contain extended N-termini and our G5 subunit is incapable of inhibiting N-type channel activity, the loss of G1 subunit regulation of the channel that occurs on substitution of certain key residues with G5 sequence is thus expected. The human G5 subunit used by Zhou and coworkers (2000) is virtually identical to shorter rat isoform used in our study, although the former study involved human rather than rat Cav2.2 channels, thus making firm comparisons difficult.
Substitution of two additional residues, Gly144 and Met188 (which are conserved in G1 through G4), was found to reduce the degree of prepulse relief, although the statistical significance of this effect depended on the statistical analysis used (i.e., ANOVA vs. t-test). These two residues are situated on the opposite face of the G subunit compared with Tyr111, Asp153, and Ser189 (not shown), and thus they are unlikely to mediate their effects by altering channel interactions with these three residues. However, they are located in close proximity to other G1 residues (i.e., Thr142, Asp119, Met101) previously implicated in N-type channel modulation by Ford and colleagues (1998), thus perhaps accounting for a possible partial effect. It is unlikely that the substitution of these two residues would result in gross alterations in the overall G structure because substitution of larger regions of G1 flanking these residues with G5 sequence preserves the ability of such chimeric constructs to activate GIRK channels (Doering et al. 2004).
Particularly remarkable is the presence of several unique ensembles of modulatory epitopes in the structure of G, all of which contribute to G modulation of a different effector molecule—e.g., N-type and P/Q-type calcium channels, GIRK channels, adenylyl cyclase, and phospholipase 2 (Agler et al. 2003; Ford et al. 1998; Mirshahi et al. 2002a). All of these effector interaction sites are localized to the G interaction domain of G1 (Agler et al. 2003; Ford et al. 1998; Mirshahi et al. 2002a). However, the N-type channel may be unique among these effectors because the critical modulatory residues identified by our present study are situated on the G surface opposite to that involved in G binding (see Fig. 3). The presence of multiple, widely dispersed N-type channel interaction sites on the G subunit is consistent with the notion that several G binding regions (i.e., N-terminus, I–II linker region) have been identified on the channel (Agler et al. 2005; Zamponi et al. 1997). The alignment of the three critical residues highlighted in Fig. 3 in a ribbonlike fashion may be indicative of specific contact points with a single one of these channel regions. Ultimately, mutation complementation experiments may be needed to identify which channel residues participate in such mutual interactions. Nonetheless, our data point to highly specific sequence differences between G5 and the four other known G subunit subtypes that underlie the G subtype specificity of N-type channel modulation.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. W. Zamponi, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr. NW, Calgary, T2N 4N1, Canada (E-mail. zamponi{at}ucalgary.ca)
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ABSTRACT
INTRODUCTION
