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A Single Gβ Subunit Locus Controls Cross-talk between Protein Kinase C and G Protein Regulation of N-type Calcium Channels

The modulation of N-type calcium channels is a key factor in the control of neurotransmitter release. Whereas N-type channels are inhibited by Gβγ subunits in a G protein β-isoform-dependent manner, channel activity is typically stimulated by activation of protein kinase C (PKC). In addition, there is cross-talk among these pathways, such that PKC-dependent phosphorylation of the Gβγ target site on the N-type channel antagonizes subsequent G protein inhibition, albeit only for Gβ1-mediated responses. The molecular mechanisms that control this G protein β subunit subtype-specific regulation have not been described. Here, we show that G protein inhibition of N-type calcium channels is critically dependent on two separate but adjacent ∼20-amino acid regions of the Gβ subunit, plus a highly conserved Asn-Tyr-Val motif. These regions are distinct from those implicated previously in Gβγ signaling to other effectors such as G protein-coupled inward rectifier potassium channels, phospholipase β2, and adenylyl cyclase, thus raising the possibility that the specificity for G protein signaling to calcium channels might rely on unique G protein structural determinants. In addition, we identify a highly specific locus on the Gβ1 subunit that serves as a molecular detector of PKC-dependent phosphorylation of the G protein target site on the N-type channel α1 subunit, thus providing for a molecular basis for G protein-PKC cross-talk. Overall, our results significantly advance our understanding of the molecular details underlying the integration of G protein and PKC signaling pathways at the level of the N-type calcium channel α1 subunit.

The modulation of calcium channels at presynaptic nerve terminals is a key factor in regulating synaptic efficacy (1,2). It is now well established that the activation of G protein-coupled receptors inhibits presynaptic calcium channel activity and thus neurotransmitter release (3). G protein inhibition of both N-type and P/Q-type calcium channels appears to be exclusively mediated by the G protein ␤␥ subunit (4,5), with the G␤ subunit being the main determinant of calcium channel inhibition. Putative G protein ␤␥ subunit interaction sites have been identified within the intracellular loop linking domains I and II of the calcium channel ␣ 1 subunit (6 -8), as well as in the C-terminal region (9). To date, five different types of G protein ␤ subunits have been identified and shown to mediate varying effects on native and transiently expressed N-type calcium channels (10,11). Moreover, N-type and P/Q-type calcium channels appear to be differentially modulated by different types of G protein ␤ subunits (12), thus providing for a mechanism by which different G proteincoupled receptors may selectively regulate individual presynaptic calcium channel subtypes.
In contrast, activation of protein kinase C (PKC) 1 results in an up-regulation of N-type channel activity (13,14). There is a complex interplay between PKC and G protein pathways such that activation of PKC antagonizes subsequent receptor-mediated G protein inhibition of presynaptic calcium channels (15,16). This effect is mediated via PKC-dependent phosphorylation of a single threonine residue located in the G protein interaction site within the domain I-II linker region of the N-type calcium channel ␣ 1 subunit (17), thus allowing the channel protein to integrate multiple modulatory inputs. Interestingly, this cross-talk between G protein and PKC pathways appears to be a selective feature of the G protein ␤ 1 subunit, thus allowing PKC to selectively antagonize G protein inhibition mediated by a subset of (i.e. predominantly G␤ 1 -coupled) receptors (18). However, although calcium channel structural determinants of G protein regulation and PKC cross-talk have received considerable attention (3), there is relatively scant information that concerns the G protein structural determinants that underlie N-type channel regulation and PKC-G protein cross-talk. Alanine mutagenesis of G␤ residues known to interact with G␣ disrupts G␤ coupling to a series of downstream effectors, including calcium channels, adenylyl cyclase, GIRK channels, and phospholipase ␤ 2 (19,20). This suggests that there may be partial overlap in the G protein ␤ subunit structural determinants that control the functional interactions both within the heterotrimeric G protein complex and with downstream signaling targets. Ford et al. (19) identified six amino acid residues (Lys-78, Met-101, Asn-119, Thr-143, * This work was supported by an operating grant from the Canadian Institutes of Health Research (to G. W. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ These authors contributed equally to this work. § Recipient of a studentship award from the Alberta Heritage Foundation for Medical Research.
¶ Asp-186, and Trp-332) on the G␤ 1 subunit that, when mutated to alanine, reduced the ability of G␤ 1 to inhibit N-type calcium channels. In addition, mutagenesis of two residues (Leu-55 and Ile-80) resulted in an increased ability to regulate N-type channels. However, these residues are completely conserved across all types of G protein ␤ subunit subtypes, which implies that they cannot account for the differential effects of different types of G␤ subunits on calcium channel activity. Hence, additional G␤ structural determinants control G protein modulation of N-type channels.
Here, we utilized chimeric and mutant G protein ␤ subunits to systematically identify G protein structural determinants that control their action on N-type channels. We identify a hot spot of amino acid sequences in the G␤ 1 subunit that is essential for N-type channel modulation. In addition, we localize cross-talk behavior to a single locus on the G␤ 1 subunit, thus identifying a molecular switch that allows the G proteins to detect a phosphorylated N-type calcium channel. In this context, our data close a major gap in our understanding of the complex interplay between G protein and PKC regulation of presynaptic calcium channel activity.

MATERIALS AND METHODS
cDNAs-The cDNAs encoding human G␤ 1 and G␥ 2 , rat G␤ 5 , and EGFP-tagged G␤ 1 and G␤ 5 subunits were described by us previously (12,21). Wild type rat calcium channel subunits were donated by Terry Snutch (University of British Columbia), and the T422E mutant N-type channel was described previously (17).
Chimeric Constructs-Chimeras were created in two steps. For an initial round of chimeras, MluI, ApaLI, and EagI sites were inserted into both G␤ 1 and G␤ 5 encoding cDNAs at exactly complimentary positions, using the QuikChange TM site-directed mutagenesis kit (Stratagene, La Jolla, CA). In each case, the entire coding region was sequenced after mutagenesis. Together with the 5Ј and 3Ј cloning sites (KpnI and XhoI, respectively) and the presence of additional ApaLI and EagI sites in the pMT2 vector sequence, this allowed the swapping of four different regions (i.e. residues 1-47, 48 -168, 168 -280, and 280 -340; numbering according to G␤ 1 sequence; see Fig. 1A) between G␤ 1 and G␤ 5 through cutting and ligating. Successful creation of the chimeras was confirmed via sequencing. Note that positions 47, 168, 280, and 340 in G␤ 1 correspond to positions 54, 179, 294, and 353 in G␤ 5 .
A second round of chimeras was created via PCR, using wild type G ␤ subunits and the initial set of chimeras as templates. Briefly, sense and antisense oligonucleotides, spanning the junctions of the new chimeras, were synthesized. Subsequently, these were used to amplify (with vector upstream and downstream primers) each end of the new chimera, using the appropriate clone as a template. Overlaps included in the junction spanning oligonucleotides allowed the two fragments to anneal at those points. The two fragments were gel-isolated, combined in equimolar concentrations, and subjected to another round of high fidelity PCR, with only the upstream and downstream primers included in the reaction mix. Proofstart DNA polymerase (Qiagen) was used for all PCRs. Purified, full-length PCR products were isolated after restriction digestion and cloned in the appropriate vector. All of the fragments generated by PCR were sequenced after cloning to ensure that no PCR-induced errors were incorporated into the final clones.
Point Mutations in G␤ 1 and G␤ 3 -Point mutations in both G␤ 1 and G␤ 3 were also created using the QuikChange TM kit as described above. The presence of the mutations and the absence of mutagenesis errors were determined via DNA sequencing.
EGFP-tagged Constructs-N-terminal fluorescently tagged chimeric G␤ proteins were created using Clontech (Palo Alto, CA) Living Colors TM C-terminal EGFP vectors. Briefly, chimeric G protein ␤ subunits were excised from the pMT2-XS vector using previously engineered restriction sites (XhoI and KpnI found in the 5Ј and 3Ј regions, respectively) (12) and subcloned in-frame into the EGFP vectors. Correct insertion within the EGFP vector was confirmed with both restriction enzyme digestion and sequencing.
Tissue Culture and Transient Transfection of tsA-201 Cells-Human embryonic kidney tsA-201 cells were grown and transfected with calcium phosphate as described by us previously in detail (21). In each experiment involving calcium channels, wild type or mutant rat Ca v 2.2 calcium channel ␣ 1 subunits were cotransfected with rat ␤ 1b , rat ␣ 2 -␦ 1 , G␥ 2 , and an EGFP expression marker (except in the case where EGFP-tagged G pro-teins were used), plus one of wild type or chimeric G␤ subunits. For experiments involving GIRK channels, GIRK1 and GIRK4 subunits were used instead of calcium channel subunits. To prevent cells from overgrowing, the cells are routinely placed in a 28°C incubator 12 h after transfection. Under these conditions, tsA-201 cells change their morphology such that they appear rounded (see Fig. 1C, inset).
Patch Clamp Recordings and Data Analysis-Glass coverslips carrying transfected cells were transferred to a 3-cm culture dish containing recording solution comprised of 20 mM BaCl 2, 1 mM MgCl 2 , 10 mM HEPES, 40 mM tetraethylammonium hydroxide, 10 mM glucose, 65 mM CsCl (pH 7.2 with tetraethylammonium-hydroxide). Whole cell patch clamp recordings were performed using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) linked to a personal computer equipped with pCLAMP version 6.0, 8.0, or 9.0. Patch pipettes (Sutter borosilicate glass, BF150-86-15) were pulled using a Sutter P-87 microelectrode puller, fire-polished, and showed typical resistances of 3-4 M⍀ . The internal pipette solution contained 108 mM cesium methanesulfonate, 4 mM MgCl 2 , 9 mM EGTA, 9 mM HEPES (pH 7.2). Series resistance was compensated by 80 -85%. Leak currents were negligible. The data were filtered at 1 kHz and recorded directly onto the hard drive of the computer. Unless stated otherwise, the currents were evoked by stepping from Ϫ100 mV to a test potential of ϩ20 mV. G protein inhibition was assessed by application of a strong depolarizing (ϩ150 mV) prepulse (PP) for 50 ms. Typically, only cells with current amplitudes greater than 50 pA 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 ϩPP ) and before (I ϪPP ) the prepulse 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. For experiments involving activation of protein kinase C, PMA (Sigma) was dissolved in dimethyl sulfoxide at a 1 mM stock concentration and diluted into the recording solution at a final concentration of 30 nM. Control solution or solution containing PMA was perfused onto cells via a home-built gravity-driven microperfusion system.
For recordings involving GIRK1 and GIRK4 subunits, whole cell recordings were conducted using an internal solution of 100 mM potassium gluconate, 40 mM KCl, 10 mM HEPES, 5 mM EGTA 5, 1 mM MgCl 2 , and 5 mM NaCl (pH 7.4 with KOH). The external solution contained 25 mM KCl, 10 mM HEPES, 10 mM glucose, and 116 mM NaCl (pH 7.4 with NaOH). Under these conditions, the predicted reversal potential for potassium is approximately Ϫ30 mV. GIRK channel activity was assessed by holding the cells at Ϫ35 mV, followed by an application of a voltage ramp from Ϫ120 to ϩ60 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. Whole cell capacitance ranged from 5 to 40 pF. In this range, there was no correlation between capacitance and whole cell conductance (r 2 ϭ 0.15; not shown), and hence, the data are plotted in Fig. 4 as whole cell conductance rather than current densities. For each batch of transfected cells, we determined the GIRK activity in the absence of G␤ subunits.
All of the data were analyzed using Clampfit (Axon Instruments) and fitted in Sigmaplot 4.0 (Jandel Scientific). Statistical analysis was carried out in SigmaStat via t tests, or as appropriate via ANOVA with a post hoc Tukey test or, for GIRK experiments, ANOVA on the ranks (Dunn method).
Confocal Microscopy-Imaging was carried out at the Seaman Family MR Research Center Confocal Microscopy and Imaging Facility. Briefly, tsA-201 cells were transfected with DNA encoding the N-type calcium channel (Ca v 2.2 ␣ 1 , ␣ 2 -␦ 1 , and ␤ 1b ), G␥ 2 , and EGFP-tagged G protein ␤ subunits. Initially coverslips containing cells of interest were placed in a glass-bottomed Petri dish and visualized with an inverted IX70 Olympus microscope. Confocal images were created using an Olympus Fluoview confocal laser scanning microscope (confocal aperture 2). The cells were stimulated with an argon 488-nm laser. Settings were chosen for laser power, photon multiplier tube gain, and offset so that pixel densities were just below saturation levels. Transmitted image visualization was conducted with Nomarski differential interference contrast microscopy (21). 1 Subunits-We have shown previously that rat N-type calcium channels are potently inhibited by G␤ 1 subunits, whereas G␤ 5 subunits have no significant effect on current activity (Ref. 12; see also Fig. 1C). To determine key G protein ␤ subunit structural determinants that control calcium channel inhibition, the differ-ential G␤ 1 /G␤ 5 effects were used as the basis for a chimeric approach. Initially the G␤ 1 and G␤ 5 subunits were each divided into four complimentary regions that were exchanged in various combinations (see Fig. 1A for example). The chimeric constructs were coexpressed with N-type (Ca v 2.2 ϩ ␤ 1b ϩ ␣ 2 -␦ 1 ) calcium channels, the G␥ 2 subunit, and an EGFP selection marker, and tonic G protein inhibition was assessed by application of strong depolarizing PP as described under "Materials and Methods." As shown in Fig. 1B, G␤ 1 mediates a robust tonic inhibition as reflected by a large increase in current amplitude following the PP. Replacement of the first 47 residues or the last 60 residues of G␤ 1 with G␤ 5 , individually or in combination, preserved the ability of G␤ 1 to inhibit N-type channels, despite the fact that G␤ 1 and G␤ 5 share only 30 and 60% sequence identity in these two regions, respectively. In contrast, as illustrated in Fig. 1C, replacement of either residues 47-168 or residues 168 -280 with corresponding G␤ 5 sequence, alone or in combination with other substitutions, reduced the degree of prepulse relief to that seen in the presence of wild type G␤ 5 (note that prepulse relief observed with G␤ 5 does not differ significantly from conditions where no G proteins are coexpressed). To minimize the possibility that the lack of modulation might be due to the lack of expression or due to inappropriate targeting of the G␤ subunit chimeras, we generated N-terminally EGFP-tagged versions of the wild type and key chimeric G proteins and analyzed their subcellular distribution via fluorescence confocal microscopy. As shown in the inset of Fig. 1C, the wild type and chimeric G protein subunits were detected in the plasma membrane, suggesting that they are indeed properly expressed and targeted in tsA-201 cells. Moreover, the observation that all of the G␤ constructs display robust plasma localization implies that the observed effects are not secondarily due to an inability of certain chimeras to assemble into G␤␥ dimers. In addition, we have shown previously via Western blot and kinetic analyses that transient transfection of wild type G protein results in saturating levels of G␤ which, in turn, leads to a homogenous population of G protein bound N- 1. Effect of chimeric G protein subunits on N-type calcium channel activity. A, schematic representation of a chimeric G protein ␤ subunit adapted from the crystal structure reported by Sondek et al. (31). The G␤ subunit is comprised of an N-terminal helix linked to a rigid seven bladed propeller structure. An initial set of chimeras was constructed by swapping fragments between G␤ 1 (indicated in blue) and G␤ 5 (indicated in orange). For this purpose, the G␤ sequence was divided into four segments (i.e. N terminus-residue 47, residues 47-168, residues 168 -280, and residue 280-C terminus; the numbering corresponds to G␤ 1 sequence, see arrows for approximate positions within the G␤ subunit structure). The nomenclature of the chimeric constructs is based on the origin of the four segments, with the depicted 5151 construct indicating that segments 1 and 3 were derived from G␤ 5 . B, current recordings obtained from N-type (Ca v 2.2 ϩ ␤ 1b ϩ ␣ 2 -␦ 1 ) calcium channels in the presence of wild type G␤ 1 , or chimeric G␤ subunits, before and after application of a strong depolarizing prepulse as outlined under "Materials and Methods." In each case, G␥ 2 and an EGFP marker were coexpressed. Note that each of the chimeric constructs results in robust G protein inhibition. C, summary of the effects of wild type and chimeric G␤ subunits on N-type channel activity, in the form of ratios of current amplitudes obtained after and before application of the prepulse. Error bars denote standard errors, numbers in parentheses reflect numbers of experiments, asterisks indicate statistical significance (p Ͻ 0.05) relative to G␤ 5 (ANOVA). The two arrows in the bar chart highlight chimeras (i.e. 1511 and 1151) in which substitution of a single region abolished the ability of G␤ 1 to regulate N-type channel activity. The dotted line indicates the level of modulation seen with G␤ 5 . Inset, membrane localization of EGFP-tagged wild type and chimeric G protein ␤ subunits, visualized via bight field light microscopy (top row) or fluorescence confocal microscopy (bottom row). Note that the cells appear rounded due to our particular incubation protocol (i.e. 28°C, see "Materials and Methods"). type calcium channels (21). Taken together, these data suggest that the failure to observe G protein inhibition with certain chimeras was not simply due to a lack of expression/targeting.

Determinants of N-type Channel Inhibition by G␤
Overall, our data obtained with the initial set of chimeras suggest that although the N-and C-terminal regions of G␤ are not critical determinants of G protein inhibition of N-type channels, one or more regions between residues 47 and 280 are essential for G protein inhibition. To further elucidate the G protein ␤ subunit structural determinants of N-type channel modulation, we created additional chimeras with sequence substitutions in regions 47-168 and 168 -280. As shown in Fig. 2, replacing G␤ 1 residues 47-116 or 116 -168 with corresponding G␤ 5 sequence resulted in the loss of G protein inhibition. Substitutions of smaller fragments within these two regions revealed that replacement of regions 47-75, 75-100, and 116 -140 did not block the ability of G␤ 1 to inhibit N-type channel activity. On the contrary, the 47-75 construct displayed a dramatically enhanced ability to inhibit N-type channel activity. In contrast, substitution of residues 140 -168 or replacement of an Asn-110, Tyr-111, and Val-112 motif (that is highly conserved in all G protein ␤ subunits with the exception of G␤ 5 ) with G␤ 5 (i.e. Cys-Ala-Ile) sequence eliminated the ability of G␤ 1 to inhibit N-type calcium channels. A similar analysis for residues 168 -280 is shown in Fig. 3. As shown in Fig. 3 (A and  B), substitution of residues 204 -248, 248 -280, and 168 -186 maintained the ability of G␤ 1 to inhibit N-type calcium channels, whereas substitution of residues 186 -204 abolished G␤ 1 modulation. Taken together, there appear to be at least three separate regions that are responsible for the differential effect of G␤ 1 and G␤ 5 on N-type calcium channel activity. It is unlikely that these effects would have arisen from a global disruption of G␤ 1 subunit folding because of the presence of G␤ 5 sequence, because G␤ 1 activity was retained for a majority of the G␤ 5 substitutions that were created (i.e. 1-47, 47-75, 75-100, 116 -140, 168 -186, 204 -248, and 248 -280). To ensure that the effects of the chimeras were specific rather than caused by inappropriate folding, we first attempted to create a gain of function chimera in which regions in G␤ 5 were concomitantly replaced with corresponding G␤ 1 residues 110 -112, 140 -168, and 186 -204. However, the G␤ 5 (110 -112, 140 -168, 186 -204) chimera did not result in significant G protein inhibition (I ϩPP /I ϪPP ϭ 1.11 Ϯ 0.03, n ϭ 22). This suggests that gain of function may require additional residues outside of the three identified regions, which will be subject to further investigation. As an additional approach, we created two point mutations in which residues 111 and 153 of G␤ 1 were replaced with corresponding G␤ 5 residues. These residues are located within the 110 -112 and 140 -168 stretches were chosen based on the surface exposure on the G␤ 1 crystal structure (see Fig.  5B below). Substitution of Tyr-111 with alanine resulted in a dramatic reduction of the degree of prepulse relief (I ϩPP /I -PP ) to 1.48 Ϯ 0.19 (n ϭ 15), and replacement of Asp-153 to asparagine resulted in an even stronger reduction of prepulse relief to 1.37 Ϯ 0.08 (n ϭ 15). These data suggest that even single amino acid substitutions in at least two of the identified regions are sufficient to drastically attenuate the ability of G␤ 1 to inhibit N-type calcium channels, consistent with the idea that the inability of the chimeras to regulate channel activity did not arise secondarily from global structural changes.
To confirm the functionality of those G␤ chimeras that were unable to regulate N-type channel activity, we carried out a series of experiments with GIRK channels. GIRK1 and GIRK4 subunits were coexpressed in tsA-201 cells with either wild type or chimeric/mutant G␤ subunits, and the whole cell GIRK conductance was determined via whole call patch clamp recordings using a voltage ramp protocol. Rather than examining the entire set of chimeras, we focused on the key chimeras and mutants that most narrowly defined the regions involved in N-type channel modulation, i.e. G␤ 1 (100 -112), G␤ 1 (140 -168), G␤ 1 (186 -204), G␤ 1 (Y111A), G␤ 1 (D153N), and the G␤ 5 (110 -112, 140 -168, 186 -204) chimera. As shown in Fig. 4, expression of GIRK1/4 in the absence of exogenous G␤ subunits resulted in some background GIRK activity, consistent with previous work in Xenopus oocytes (22). Cotransfection of G␤ 1 subunits resulted in a significant increase in whole cell conductance by ϳ300%. Interestingly and in contrast with a previous study (22), wild type G␤ 5 subunits also effectively activated GIRK1/4 channels, thus confirming that G␤ 5 subunits are indeed functionally expressed in our system. But more importantly, every single one of the chimeric and mutant G␤ subunits examined mediated a significant increase in GIRK1/4 activity to a level comparable with that seen with wild type G␤ 1 . Hence, we conclude that the lack of effects of these chi-meras/mutants on N-type channel activity did not arise from inadequate protein expression or protein misfolding.
G Protein PKC Cross-talk-We have shown previously that PKC-dependent phosphorylation of Thr-422 in the Ca v 2.2 calcium channel I-II linker selectively antagonizes the G␤ 1 -mediated inhibition of N-type channel activity (18). We also showed that all aspects of this effect could be mimicked by replacing Thr-422 with glutamic acid (17,18), thus eliminating the need for diffuse activation of PKC and consequently the potential for secondary effects/incomplete specificity of PKC activators. Because a chimera containing the first 47 residues of G␤ 1 (Fig.  5A) appeared to behave like wild type G␤ 1 with regard to N-type channel regulation (recall Fig. 1), we examined whether the PKC cross-talk could still be observed with this construct. Although the T422E mutation reduced the ability of G␤ 1 to inhibit N-type channel activity, the 5111 chimera failed to recognize the presence of the T422E substitution (Fig. 5B), thus indicating that a key G␤ 1 structural determinant is located in the N-terminal region. Because the PKC cross-talk is only observed with G␤ 1 , we examined the N-terminal 47-amino acid stretch for residues unique to this subunit (Fig. 5C, bold letters). As shown in Fig. 5D, replacement of Asp-5 with glutamic acid had no effect on cross-talk. In contrast, replacement of Asn-35 and Asn-36 with corresponding sequences in G␤ 3 abolished the effects of the T422E I-II linker mutation, suggesting that one or both of these asparagine residues sense the presence of a negative charge within the I-II linker G proteinbinding domain (7). If so, then one should be able to confer cross-talk onto other G protein subunits by substituting aspar- agine residues in complimentary positions. To test this hypothesis, we carried out site-directed mutagenesis in the G␤ 3 subunit (which modulates N-type channels effectively but whose action is not antagonized by PKC) (18) and compared the abilities of the mutant G␤ 3 (S35N,G36N) construct to inhibit wild type and T422E mutant N-type channels. Consistent with our hypothesis, the mutant G␤ 3 subunit inhibited the T422E channel significantly (p Ͻ 0.05) less effectively than the wild type Ca v 2.2 channel (Fig. 5E).
To confirm that the observations were not an artifact of the T422E calcium channel mutant, we repeated the experiments shown in Fig. 5 (D and E) using proper activation of PKC via 30 nM of the phorbol ester PMA. For each cell, the degree of prepulse relief was measured before application of PMA and 3 min after PMA application. As seen in Fig. 5F, activation of PKC did not affect the degree of prepulse relief observed with the G␤ 1 (N35S,N36G) mutant. In contrast, mutagenesis of G␤ 3 residues 35 and 36 to corresponding G ␤1 sequence (i.e. G␤ 3 (S35N,G36N)) conferred the cross-talk behavior, such that the degree of prepulse was significantly reduced (p Ͻ 0.01, paired t test) following the application of PMA.
Taken together, these data suggest that the G␤ 1 subunit contains a precise locus of two amino acids that allows this subunit to sense the presence of a negative charge (such as a phosphate group) on the N-type calcium channel ␣ 1 subunit.

DISCUSSION
Comparison with Previous Work-Our work constitutes the first systematic approach toward delineating key G␤ subunit regions that are essential for inhibiting N-type calcium channel activity. Unlike previous studies that were based on alanine mutagenesis of G␤ subunit residues known to be involved in G␣ subunit binding (i.e. Refs. 19 and 20), we employed a chimeric approach that was based on the differential abilities of G␤ 1 and G␤ 5 to inhibit N-type calcium channels. We show that mutagenesis of residues Asn-110, Tyr-111, and Val-112, a motif that is highly conserved in all G␤ subunits other than G␤ 5 , virtually abolished all G protein regulation of the N-type channel. This region is located outside of the known G␣ interaction domain (Refs. 23 and 24 and Fig. 6) and has not been previously identified as an important functional domain on the G␤ 1 subunit. G protein inhibition was also lost upon substitution of G␤ 1 residues 140 -168 and 186 -204. Hence, the G protein ␤ subunit structural determinants that control signaling to N-type calcium channels appear to be different from those implicated in coupling to other effector systems such as adenylyl cyclase, GIRK channels, or phospholipase ␤ 2 (i. e. residues 72-105, 115-135, and 143,  186, 228, and 332) (19, 22, 25-31). However, in lieu of specific examination of the action of our chimeras on effectors other than GIRK channels, it is difficult to gauge whether the FIG. 4. Activation of GIRK1/4 channels by wild type and selected chimeric and mutant G protein ␤ subunits. A, raw current traces of GIRK1/4 channels in the presence of G␤ 1 subunits and elicited by stepping from a holding potential of Ϫ35 mV to various test potentials ranging from Ϫ130 mV to ϩ60 mV. B, representative GIRK1/4 currents in the absence or the presence of G protein ␤ subunits acquired via a ramp protocol as outlined under "Materials and Methods." C, summary of GIRK1/4 activity induced by wild type and chimeric G protein ␤ subunits in comparison with the activity observed in the absence of G␤. The bars indicate mean whole cell GIRK conductance determined via ramp protocols as described under "Materials and Methods." The error bars denote the standard errors, the numbers in parentheses reflect numbers of experiments, and the asterisks denote statistical significance relative to ϪG␤.
regions identified in our study are exclusively involved in signaling to N-type channels.
Within regions 140 -168 and 186 -204, respectively, 12 and 9 amino acid residues are identical in G␤ 1 and G␤ 5 and can therefore not account for the observed effects (the conserved residues include Thr-143 and Asp-186, which were implicated previously as being important for N-type channel modulation) (19). Examination of the localization of the nonconserved resi- FIG. 5. A, schematic representation of the 5111 chimera. B, degree of prepulse relief observed with wild type (WT) Ca v 2.2 or mutant T422E Ca v 2.2 calcium channels in the presence of G␤ 1 or the 5111 chimera. Note that the replacement of Thr-422 with glutamic acid antagonizes G␤ 1 action but has no effect on modulation by the 5111 chimera. C, sequence alignment of the N-terminal helix regions of G␤ 1 through G␤ 5 . The residues shown as bold letters are unique to G␤ 1 . D, effect of mutagenesis of unique G␤ 1 residues in PKC-G protein cross-talk. Note that the antagonistic effect of the T422E mutation on G protein inhibition is abolished upon mutagenesis of asparagine residues 35 and 36. E, induction of G protein-PKC cross-talk into the G␤ 3 subunit after insertion of asparagines residues into positions 35 and 36. All of the error bars are the standard errors, and the asterisks denote statistical significance (p Ͻ 0.05, t test) between the degree of inhibition observed with the wild type and T422E mutant N-type channel. F, effect of protein kinase C activation on the degree of prepulse relief observed following the coexpression of G␤ 1 (N35S,N35G) and G␤ 3 (S35N,G36N). In each case, the degree of prepulse relief was measured prior to and 3 min after application of 30 nM PMA. Note that PMA significantly reduces the effect of G␤ 3 (S35N,G35N) but not that of the G␤ 1 point mutant. The error bars are the standard errors, and the asterisk reflects the statistical significance at the 0.05 level (paired t test ϩPMA versus ϪPMA).
FIG. 6. Localization of structural determinants of G␤ 1 inhibition of Ntype calcium channels on the threedimensional structure of the G␤ subunit. Protein structure data were obtained from crystal structure coordinates (32) and visualized via Viewer Pro software (Accelerys Inc.). A, residues 35 and 36 are visualized in red, and the Asn-110, Tyr-111, and Val-112 motif is indicated in yellow (note that residues 110 and 112 are buried in the protein, with only Tyr-111 being exposed). The residues shown in blue and green, respectively, reflect the amino acids in regions 140 -168 and 186 -204. B, same as A but depicting only those residues within the identified regions that are not conserved between G␤ 1 and G␤ 5 . The residues depicted in pink are known to be involved in interactions with the G␣ subunit (23,24). dues in regions 110 -112, 140 -168, and 186 -204 reveals a pattern in which all of the exposed residues appear to be arranged in a ribbon-like fashion at the protein surface opposite to that containing the G␣ interaction domain (Fig. 6B). Among these exposed residues, Tyr-111 and Asp-153 were individually found to be critical for G protein inhibition. The lack of overlap with residues known to be involved in G␣ binding contrasts with what is observed with a number of other G␤␥ effectors that interact with the G␣ interaction region following dissociation of the heterotrimeric G protein complex (19). It is important to note that our data do not necessarily disagree with previous findings that mutagenesis of conserved residues within the G␣-binding region can affect G␤ 1 inhibition of Ntype calcium channels (19,31), because our chimeric approach was designed to elucidate the molecular basis of the differences between G␤ 1 and G␤ 5 regulation of N-type channels rather than identification of all residues that contribute to N-type channels inhibition. The notion that our intended gain of function chimera was not sufficient to mediate significant G protein inhibition of the channel is also consistent with the existence of additional structural determinants.
Substitution of residues 45-75 of G␤ 1 with G␤ 5 sequence resulted in a curious enhancement of the degree of N-type channels inhibition. This stretch of amino acids contains a leucine residue in position 55, which when mutated to alanine has been reported to increase N-type channel inhibition (19), in agreement with present data. This region also contains Ser-67, which, when mutated to the corresponding G␤ 5 lysine residue, has been reported to abolish G protein inhibition altogether (31). Our observation that the 45-75 chimera mediated robust G protein inhibition appears at odds with this finding; however, it is conceivable that the effects of the Ser-67 substitution are masked by the enhancing effect of Leu-55. Additional mutagenesis of individual residues will be required to elucidate the precise roles of residues 55 and 67 in calcium channel inhibition. Taken together, our data indicate that a ribbon-like structure comprised of residue Tyr-111 and residues in regions 140 -168 and 186 -204 of G␤ 1 is essential for N-type channel inhibition, with residues 111 and 153 being of particular importance.
Molecular Mechanism of G protein-PKC Cross-talk-The Ntype calcium channel contains at least three separate physical binding sites for G␤␥; two of these sites are formed by ϳ20amino acid stretches within the domain I-II linker (6, 7) plus an additional site in the C-terminal region (9). The observation that multiple regions within the G␤␥ subunit were found to be critical for G protein inhibition is consistent with the existence of multiple G protein microbinding sites on the N-type channel ␣ 1 subunit. The second of the two I-II linker G␤␥ regions contains the PKC phosphorylation site formed by residue Thr-422 (7,17). In vitro phosphorylation of this site blocks G␤␥ binding to this region (7), and replacement of Thr-422 with glutamic acid mimics all aspects of phosphorylation (17,18). Thus, the T422E mutation provides a convenient means of investigating G protein ␤ subunit determinants that contribute to the cross-talk phenomenon without having to resort to activation of PKC via pharmacological means.
Our findings indicate that residues Asn-35 and/or Asn-36 near the N-terminal helix of the G␤ 1 subunit were necessary and sufficient for sensing the presence of this glutamic acid residue or the true phosphorylation of the native threonine residue. The location of this "phosphorylation sensor" on the G␤ 1 subunit is somewhat surprising, because this region is not typically associated with coupling to any of the known G␤␥ effectors (see above). Yet the role of these two residues in supporting G protein-PKC cross-talk appears to be highly specific, because substitution of these residues into the G␤ 3 subunit that is normally incapable of recognizing phosphorylation of Thr-422 (18) conferred the cross-talk behavior. We can, at this point, only speculate about the actual mechanism by which G␤ residues 35 and 36 functionally couple to the N-type channel I-II linker containing the Thr-422 site. We have shown previously that disruption of G␤␥ binding to this region results in a complete loss of G protein inhibition (7). Hence, if there is a direct interaction between G␤ subunit residues Asn-35/Asn-36 and Thr-422, the phosphorylation event would only serve to partially disrupt G␤␥ binding because G protein inhibition is merely reduced but not eliminated following activation of PKC (17,18). On the other hand, the data obtained with the 5111 chimera together with the low (ϳ30%) degree of sequence conservation in residues 1-47 suggests that this region of G␤ is not a major determinant of G protein action on N-type channels. Yet between residues 44 and 53, the amino acid sequence of among all G␤ subunits is highly conserved (Fig. 5C). This raises the possibility that this region could interact with the N-type channel I-II linker but that phosphorylation of residues Thr-422 might weaken binding via an interaction with G␤ 1 residues Asn-35 and Asn-36, thus accounting for the reduced G protein inhibition following activation of PKC. Further mutagenesis of residues 44 -53 will, however, be required to test this hypothesis.
Assuming that a site near/at residues 35 and 36 does indeed interact with the calcium channel I-II linker region flanking Thr-422, then this begs the question as to which region of the N-type channel interacts with the ribbon-like structure depicted in Fig. 6B (i.e. residues 111, 140 -168, and 186 -204). As stated above, the N-type channel contains two additional binding domains for G␤␥: a region in the C-terminal (9) and a second I-II linker site ϳ30 residues upstream of Thr-422 (6,7). Disruption of the G␤␥ binding to this region completely eliminates G protein inhibition (7), whereas deletion of the C-terminal site has only a minor effect (17). Considering that replacement of these G␤ 1 subunit domains with G␤ 5 sequence completely abolished G protein action, we thus favor the I-II linker site over the C terminus as a potential interacting partner. We therefore envision a model in which G␤␥ is held in place through interactions of two distinct sites on the G␤ protein with two spatially separate regions within the calcium channel I-II linker. In this model, the functional interaction between the phosphorylated Thr-422 I-II linker residue and residues 35/36 on the G␤ 1 subunit would destabilize the overall binding interaction, thus reducing the extent of G protein inhibition of the channel. The C terminus of the N-type channel might contribute toward stabilizing overall G protein binding (perhaps by interacting with previously identified residues in the G␣ interaction region).
Taken together, our data close a major gap in our understanding of the molecular basis underlying cross-talk between G protein and PKC regulation of N-type calcium channels. The presence of a specific site on the G␤ subunit that serves as a molecular detector of PKC-dependent phosphorylation of the N-type calcium channel provides a unique means of integrating multiple signaling pathways at the level of a proteinprotein interaction. This may allow for precise regulation of N-type calcium channel activity and, consequently, synaptic transmission.