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REPORT

Scanning Mutagenesis Reveals a Role for Serine 189 of the Heterotrimeric G-Protein Beta 1 Subunit in the Inhibition of N-Type Calcium Channels

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

	ABSTRACT

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Direct interactions between the presynaptic N-type calcium channel and the 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.

	INTRODUCTION

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N-type and P/Q-type calcium channels are inhibited in response to activation of G-protein–coupled receptors (GPCRs), with crucial consequences for synaptic activity, neurotransmitter release, and nociception (Dunlap et al. 1995; 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 Ca_v2 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.

	METHODS

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cDNAs

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 Ca_v2.2 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.

View larger version (25K): [in this window] [in a new window]   FIG. 2. A–C: whole cell current recordings from tsA-201 cells expressing GIRK1/4 channels, as described in METHODS. A: typical set of current traces recorded from a cell coexpressing heterologous WT G1, obtained using steps from a holding potential of –35 mV to test potentials ranging from –120 to +60 mV. B: typical set of current traces, obtained as in A, but from a cell coexpressing G1-S189C. C: typical voltage-ramp traces, plotted as current–voltage relations, obtained from experiments performed with cells coexpressing either no heterologous G, or G1-S189C, or WT G1 (as indicated by labels at the bottom left ends of the traces). D: histogram summarizing analysis of results obtained for the 3 experimental conditions described in C; for all 3 conditions, conductances were calculated for voltage-ramp recording data collected between transmembrane potentials of –100 and –60 mV; columns are shown with SE bars, with corresponding experimental condition indicated by labels below the respective columns; asterisks indicate statistically significant difference from conductances calculated from negative control cells (labeled "–G1"). Numbers in parentheses indicate numbers of cells tested.

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.

	RESULTS

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We previously showed that substitution of residues 140–168 or residues 186–204 of G₁ 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.

View larger version (33K): [in this window] [in a new window]   FIG. 1. A: 3 sets of typical current traces from tsA-201 cells expressing the rat N-type calcium channel, as described in METHODS. Each is a matched set of current traces from sequential test pulses, with the second test pulse preceded by a +150-mV depolarizing prepulse. (In each set the trace with larger current values is from the second test pulse.) Left: traces from a cell expressing no heterologous G. Center: traces from a cell coexpressing the G1-S189C mutant. Right: traces from a cell coexpressing WT G1. B: histogram summarizing the results of paired-pulse facilitation (PPF) experiments performed with all G1 mutants; columns shown with SE bars. Respective positions of mutations in the G1 amino acid sequence are indicated by numbers beneath the corresponding columns (see METHODS for full description of the mutations used). Stars above the S189C ("189") and negative control columns ("Ctrl") indicate that these 2 data sets scored P < 0.05 in an ANOVA on ranks test against positive control data ("WT"). Numbers in parentheses indicate numbers of cells tested.

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.

	DISCUSSION

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Although the bioactive surfaces of peptide toxins and hormones are often studied using comprehensive scanning mutagenesis of all solvent-exposed amino acid residues (Froy et al. 1999; 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., G_1–4) 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.

View larger version (34K): [in this window] [in a new window]   FIG. 3. A: molecular surface rendering of WT G1, calculated using crystal structure data reported by Sondek et al. (1996). Shown in green are surface areas presented by residues 110–112, 140–168, and 186–204, previously identified as key determinants of N-type channel modulation (Doering et al. 2004). Areas presented by the side chains of Tyr111, Asp153, and Ser189 are shown in orange-red. B: ribbon cartoon model representing the WT G1 structure. Areas corresponding to residues 110–112, 140–168, and 186–204 are shown in green; side chains of Tyr111, Asp153, and Ser189 are represented in orange-red. Molecular images were drawn using MOLMOL (Koradi et al. 1996).

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.

	GRANTS

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This work was supported by an operating grant to G. W. Zamponi from the Canadian Institutes of Health Research. G. W. Zamponi is a Canada Research Chair and a senior scholar of the Alberta Heritage Foundation for Medical Research (AHFMR). H. W. Tedford holds postdoctoral fellowships from the AHFMR and the Heart and Stroke Foundation of Canada (HSF-Canada). A. E. Kisilevsky holds studentships from AHFMR and HSF-Canada and J. B. Peloquin is the recipient of a studentship from the Canadian Foundation for Fighting Blindness.

	ACKNOWLEDGMENTS

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We thank C. Doering, C. Bladen, J. McRory, and J. Hamid for assistance with DNA constructs and technical issues of molecular biology and electrophysiology. We also thank L. Chen and W. Barr for assistance with cell culture.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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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