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The Journal of Neuroscience, November 15, 1998, 18(22):9163-9170

G-Protein -Subunit Specificity in the Fast Membrane-Delimited Inhibition of Ca²⁺ Channels

David E. García^{1, 5}, Bin Li³, Rafael E. García-Ferreiro⁵, Erick O. Hernández-Ochoa⁵, Kang Yan⁴, Narasimhan Gautam⁴, William A. Catterall³, Ken Mackie^{1, 2}, and Bertil Hille¹

Departments of ¹ Physiology and Biophysics, ² Anesthesiology, and ³ Pharmacology, University of Washington, Seattle, Washington 98195, ⁴  Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110, and ⁵ Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Apartado Postal 70250, C.P. 04510, México, Distrito Federal México

	ABSTRACT

Top Abstract Introduction Materials & Methods Results Discussion References

We investigated which subtypes of G-protein  subunits participate in voltage-dependent modulation of N-type calcium channels. Calcium currents were recorded from cultured rat superior cervical ganglion neurons injected intranuclearly with DNA encoding five different G-protein  subunits. G₁ and G₂ strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors. The G₅ subunit produced much weaker effects than G₁ and G₂, whereas G₃ and G₄ were nearly inactive in these electrophysiological studies. The specificity implied by these results was confirmed and extended using the yeast two-hybrid system to test for protein-protein interactions. Here, G₁ or G₂ coupled to the GAL4-activation domain interacted strongly with a channel sequence corresponding to the intracellular loop connecting domains I and II of a ₁ subunit of the class B calcium channel fused to the GAL4 DNA-binding domain. In this assay, the G₅ subunit interacted weakly, and G₃ and G₄ failed to interact. Together, these results suggest that G₁ and/or G₂ subunits account for most of the voltage-dependent inhibition of N-type calcium channels and that the linker between domains I and II of the calcium channel ₁ subunit is a principal receptor for this inhibition.

Key words: G-proteins; calcium channel; norepinephrine; yeast 2-hybrid; sympathetic neurons; ion channel modulation

	INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Inhibition of neuronal calcium currents by G-protein-coupled receptors probably occurs in every type of neuron. It is a ubiquitous mode of modulation of electrical activity and synaptic function by neighboring neurons. A common form uses a signaling pathway whose components appear to be restricted to the cell membrane (membrane-delimited), develops in <1 sec, and is voltage-dependent, i.e., the inhibition can be partially relieved by a strong depolarizing pulse (for review, see Hille, 1994). A well studied example is the inhibition of N-type calcium channels by norepinephrine (NE) acting on ₂ adrenergic receptors in superior cervical ganglion (SCG) neurons. This type of inhibition is mediated by -subunits of G-proteins, apparently acting directly on the channel (Herlitze et al., 1996; Ikeda, 1996).

The specificity for G and G subunits has not been investigated, and the target site for the interaction of G-protein subunits with calcium channels is the subject of some debate. By analogy with other G-effector proteins, several investigators have proposed that the target site on voltage-gated calcium channels includes a QXXER motif in the intracellular loop connecting domains I and II (L_I-II) of the ₁ subunits of classes A, B, and E (the P/Q-, N-, and R-type) calcium channels (De Waard et al., 1997; Herlitze et al., 1997; Page et al., 1997; Zamponi et al., 1997). This QXXER sequence overlaps with the region required for interaction with the calcium channel  subunit. In contrast, some investigators argue that an interaction of G subunits with the C terminus of calcium channel ₁ subunit is the functionally important one (Zhang et al., 1996a; Qin et al., 1997). Here, we inject DNA for various G-protein  subunits into the nucleus of adult rat SCG neurons and find that only certain G subunits will induce inhibition of the endogenous N-type Ca²⁺ currents. Then, we used the yeast two-hybrid assay to look for interactions between G subunits and a domain of the channel. We find that exactly the same G subunits that inhibit current also interact well with the linker between domains I and II of the ₁ subunit of the class B (N-type) calcium channel. Together, these results identify the G subunits that can mediate fast, membrane-delimited, and voltage-dependent inhibition of N-type calcium channels, and they identify a target domain of the channel that has the appropriate binding specificity.

	MATERIALS AND METHODS

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Cell culture and intranuclear microinjection. Single SCG neurons were enzymatically dissociated from 5-week-old male rats (Sprague Dawley rats were used for the experiments in Figs. 1-3 in Seattle, and Wistar rats were used in the experiments in Figs. 4 and 5 in Mexico) as described previously (Beech et al., 1991; Bernheim et al., 1991). After a 4 hr wait for attachment to the substrate, the neurons were intranuclearly microinjected using an Eppendorf 5242 pressure microinjector and 5171 micromanipulator system (Eppendorf, Madison, WI). The injection solution contained varying amounts of G-protein expression plasmids mixed with two injection markers, 1 mg/ml 10,000 kDa dextran-fluorescein (Molecular Probes, Eugene, OR), and green-fluorescent protein (GFP) plasmid as an expression reporter. Injection at pressures of 10-20 kPa for 0.5-0.8 sec resulted in no obvious increase in cell volume. After 12-16 hr, successfully injected neurons were identified by their characteristic greenish-blue GFP fluorescence using an inverted microscope equipped with epifluorescence and fluorescein optics.

Electrophysiological recording. Currents were recorded using the whole-cell voltage-clamp technique (Hamill et al., 1981) with a patch-clamp amplifier at room temperature. Pipettes (0.85-2 M) were pulled from microhematocrit glass and fire polished. During recording, neurons were constantly perfused locally (1-2 ml/min) with the appropriate external solution. Solution reservoirs were selected by means of solenoid valves, and solution changes were accomplished in <10 sec. Voltage protocols were generated, and data were digitized, recorded, and analyzed using BASIC-FASTLAB (Indec Systems, Capitola, CA).

To measure calcium currents, we used two sets of solutions. In the experiments of Figures 1-3, the pipette solution contained (in mM): 125 N-methyl-D-glucamine, 20 TEA-Cl, 10 HEPES, 0.1 tetracesium-BAPTA, 4 MgCl₂, 0.1 leupeptin, 4 Na₂ATP, and 0.3 Na₂GTP, pH adjusted to 7.2 with methanesulfonic acid. The external solution contained (in mM): 140 TEA-OH, 10 HEPES, 2 CaCl₂, 15 glucose, 0.0001 TTX, and 0.002 nifedipine, pH adjusted to 7.3 with methanesulfonic acid. In the experiments of Figures 4 and 5, the pipette solution contained (in mM): 125 methanesulfonic acid, 20 TEA-Cl, 10 HEPES, 0.1 tetracesium-BAPTA, 4 MgCl₂, 5 MgATP, 0.3 Na₂GTP, and 0.1 leupeptin, pH adjusted to 7.2 with CsOH. The external solution contained (in mM): 162.5 TEA-Cl, 10 HEPES, 8 glucose, 2 CaCl₂, 1 MgCl₂, 0.0002 TTX, and 0.002 nifedipine, pH adjusted to 7.3 with TEA-OH.

The Ca²⁺ current of rat SCG neurons is carried ~85-90% in N-type channels and the remainder in L-type channels, with no detectable P/Q component (Mintz et al., 1992). Therefore, the N-type Ca²⁺ current could be defined as the component of the current sensitive to 100 µM Cd²⁺ in the presence of 2 µM nifedipine. Currents were sampled at 10 kHz. To emphasize the effects of kinetic slowing of Ca²⁺ current activation, current amplitudes were always taken as the mean value of recorded points between 5 and 6 msec after the start of the depolarizing test pulse. This time is before the current reaches a peak. Because the magnitude of the Ca²⁺ current was dependent on cell size, aggregate current data are presented as current densities normalized to cell capacitance. To avoid one source of systematic bias, experimental and control measurements were alternated whenever possible, and concurrent controls were always performed. Where appropriate, data are expressed as mean ± SE.

Plasmids and materials. The DNAs encoding G₂, G₄, and G₅ were cloned in pcDNA I plasmid, G₁ was cloned in pCDM8, G₃ was cloned in pCIS, G₃ was cloned in pCI (all from M. Simon, Caltech, Pasadena, CA), and GFP was cloned in pEGFP-N1 (Clontech, Palo Alto, CA). Although not in identical vectors, all G-protein-containing plasmids were driven by the cytomegalovirus promoter. DNA encoding the L_I-II loop of rat brain N-type calcium channel (rbB-I) _1B was subcloned into the pAS2-1 vector (Clontech) as an EcoRI-BamHI fragment (nt 1069-1449; aa 357-483) to express a GAL4 DNA-binding domain (GBD) hybrid protein. The bovine G-protein ₁ subunit and ₂ subunit genes were subcloned into the pACT vector (Clontech) to express GAL4-activation domain (GAD) hybrid proteins. The plasmids pACT-G₃, pACT-G₄, and pACT-G₅ were described previously (Yan et al., 1996). Plasmids were purified using commercial kits (Qiagen, Valencia, CA). The yeast strain was PJ69-4A (MAT a trp1-901 leu2-3,112 ura 3-52 his3-200 gal4 gal 80 GAL2-ADE2 LYS2:: GAL-HIS3 met2:: GAL7-lacZ) (a gift of Dr. Philip James, University of Wisconsin) (James et al., 1996). This strain contains three reporters (ADE2, HIS3, and LacZ) that can be activated as a result of protein-protein interaction by reconstituted GAL4 transactivator function.

Tetracesium-BAPTA was obtained from Molecular Probes, and all other salts were obtained from Sigma (St. Louis, MO).

Yeast two-hybrid assay. Plasmids were made expressing hybrid proteins consisting either of a specific G subunit coupled to a GAD or an intracellular loop of the ₁ N-type calcium channel fused to a GBD. A plasmid expressing one of the GBD hybrids and the plasmid expressing the GAD hybrid were cotransformed into yeast strain PJ69-4A (usually without introducing any mammalian G subunit). Cells were first plated on a tryptophan-free leucine-free medium to select Trp+ Leu+ transformed cells containing the GBD hybrid- and GAD hybrid-expressing plasmids. To detect protein-protein interactions, these transformants were then transferred onto medium that also lacked adenine and histidine.

Immunoblotting. To check the expression of G-protein subunit hybrids in yeast, transformed yeast cells were grown to saturation phase. Cultures (1.5 ml) were harvested and resuspended in 150 µl of Z buffer (in mM: 30 Na₂HPO₄, 35 NaHPO₄, 10 KCl, and 0.4 MgSO₄, pH 7.0) with 5% glycerol, 0.5 mM dithiothreitol, 1 µg/ml aprotinin, and 2 µg/ml leupeptin, followed by addition of 0.1 gm of 425-600 µm glass beads (Sigma). The cells were vortexed for 4 min and centrifuged for 10 min at 4°C. The supernatants were saved, and 20 µl cell lysates were fractionated on a SDS gel. The separated proteins were electrophoretically transferred to a nitrocellulose membrane, and the blot was probed with anti-GAD antibody (0.2 µg/ml; Clontech), followed by horseradish peroxidase-conjugated anti-mouse IgG (1:2000; Amersham, Piscataway, NJ). The GAD hybrid proteins were detected by the enhanced chemiluminescence method (Amersham).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

We begin with the typical NE-induced modulation of N-type calcium currents. In control SCG neurons, a 20 msec depolarizing test pulse elicited a rapidly activating inward Ca²⁺ current (Fig. 1A, left), which was primarily carried in N-type channels because the medium contained 2 µM nifedipine to block L-type channels. Perfusion with 10 µM NE reduced the inward current amplitude by ~60% and slowed its rate of activation. To monitor current facilitation, as well as amplitude, we clamped the membrane potential using the voltage protocol in Figure 1A. Inward Ca²⁺ currents were evoked every 10 sec with a pair of 20 msec depolarizing test pulses to +10 mV from a holding potential of 80 mV, one before (test pulse 1) and the other after (test pulse 2) a 25 msec prepulse to +125 mV. The depolarizing prepulse transiently relieves much of the NE-induced inhibition, and the resulting facilitation can be seen by comparing currents in test pulse 2 with those in test pulse 1. The "facilitation ratio," defined as the current during test pulse 2 (at 5-6 msec) divided by the current during test pulse 1, is a convenient measure of voltage-dependent membrane-delimited inhibition by certain G-proteins. In standard Ringer's solution, this facilitation ratio is near 1.1, and after treatment with NE, it rises to 1.6. 

View larger version (23K): [in this window] [in a new window]   Figure 1.   Injecting G2 DNA increases calcium current facilitation. A-E, Superimposed calcium current (ICa) traces during a 20 msec depolarization (test pulse) to +10 mV from a holding potential of 80 mV, in the absence (bottom trace) or presence (top trace) of NE (10 µM), before (left) or after (right) a 25 msec +125 mV prepulse. Successive panels show uninjected cells (A) and cells injected with increasing concentrations of G2 DNA (B-E). G2 DNA was coinjected with 100 ng/µl G3 DNA in all cases. F, Summary of facilitation ratios for injections of several concentrations of G2 and G3 DNA.

Dose-dependent suppression of I_Ca by overexpression of G₂

Previously, we had found that injection of RNA for G₂ subunits, with and without RNA for G₃, into the cytoplasm of SCG neurons inhibited Ca²⁺ currents, increased the facilitation ratio, and partially occluded the actions of NE (Herlitze et al., 1996). Ikeda (1996) reported even stronger effects with intranuclear injection of DNA. To optimize conditions for intranuclear injection here, we first determined the dose-response relationship for the action of G₂ by varying the concentration of G₂ DNA injected, which was always coinjected with 100 ng/µl G₃ and 100 ng/µl GFP DNA. Injection of G₂ plasmid at 10, 20, and 100 ng/µl induced progressively larger facilitation and kinetic slowing of activation in standard Ringer's solution (Fig. 1B-D). Injection at 600 ng/µl (Fig. 1E) gave about the same facilitation as 100 ng/µl. Figure 1F shows the dose-response relationship for development of facilitation after these injections of DNA for G₂ compared with that after injection of G₃. Quite clearly, G₂ has a much greater effect than G₃. Injection with 100 or 500 ng/µl G₃ DNA solution did not induce facilitation (Fig. 1F); indeed, injection of G₃ slightly reduced facilitation below control levels (facilitation ratio, 1.03 ± 0.02; n = 10; p < 0.05).

The current traces after injection of G₂ show that the progressive increase of facilitation was accompanied by a decreasing effectiveness of NE and a decrease in basal calcium current density. The increasing amounts of expressed G₂ subunits presumably occlude the actions of NE and block N-type calcium channels. The facilitation ratio is consistently a more sensitive indicator of G₂ actions than the occlusion of NE action or the basal current density. From the dose-response curves for these three actions (Fig. 2), we elected to use 100 ng/µl G plasmids in the next experiments.

View larger version (15K): [in this window] [in a new window]   Figure 2.   Concentration-response relationships for facilitation ratio, inhibition by NE, and ICa density. G2 DNA concentrations ([2]) are plotted on a logarithmic scale. Data were fitted to a Hill function, y = [(A1-A2)/{1+([2]/[2]0)n}] + A2, with best fitting values of the midpoints, [2]0 = 20.0, 156, and 103 ng/µl (vertical solid line); Hill coefficients, n = 2.0, 1.2, and 1.51; low-concentration asymptotes, A1 = 0.94, 57, and 12.7; and high-concentration asymptotes, A2 = 2.4, 1.9, and 1.4 for A, B, and C, respectively. The horizontal dashed line in A indicates the facilitation ratio for control cells (1.1), and the vertical dashed line indicates a theoretical injected G2 DNA concentration (7.0 ng/µl) that would be equivalent to this basal facilitation ratio.

The dashed arrow in Figure 2A represents an extrapolated G₂ DNA concentration (7 ng/µl) that would be equivalent to the control basal facilitation levels, assuming that the facilitation ratio in the total absence of endogenous G would be the fitted value 0.94.

Identification of the G subtypes that modulate Ca²⁺ currents

The apparent inability of G₃ to produce facilitation prompted us to undertake a more systematic study of the effectiveness of the various G-protein  subunits in calcium channel inhibition. As before, we measured facilitation, tonic inhibition, and occlusion of further inhibition by NE. Again in control cells, application of 10 µM NE strongly reduced the Ca²⁺ current magnitude (Fig. 3A, left). Figure 3, B and D, illustrates Ca²⁺ currents recorded from neurons that had been injected with 100 ng/µl DNA for G₁, G₂, or G₅, with GFP DNA but without coinjection of a G DNA. In the absence of agonist, the Ca²⁺ currents in neurons injected with DNAs for G₁ or G₂ displayed kinetic slowing (Fig. 3, left traces of each pair) and facilitation (right traces) virtually identical to that produced by application of NE in control cells. Coinjection with G₃ DNA did not change the results for G₁ (data not shown) or G₂ (Fig. 1). Only a small effect was observed in cells injected with G₅ DNA, whether alone (Fig. 3D) or in combination with G₃ DNA (data not shown). Finally, in neurons injected with DNAs for G₃₃, G₄₃, or CB1 (a truncated, nonmembrane spanning form of the rat cannabinoid receptor in the pcDNA3 expression vector), the Ca²⁺ current activation was similar to that of uninjected neurons (data not shown).

View larger version (28K): [in this window] [in a new window]   Figure 3.   Identification of the G subtypes that cause voltage-dependent modulation of Ca currents. Cells were injected with DNA for G-protein  and  subunits, as indicated, and currents were recorded by the double-pulse protocol. A-D, Current records for uninjected control cell (A) and neurons injected with 100 ng/µl G1, G2, or G5 DNA (B-D). E, Summary data for facilitation ratios; numbers above individual columns represent the number of tested cells, and the same numbers apply to F and G. Data plotted as mean ± SEM. *p < 0.05; two-tailed t test versus control. F, Percent of ICa inhibited by NE (during test pulse 1). G, ICa density during test pulse 1 in the absence of NE.

The quantitative results of these DNA injections for the various G subunits are summarized in Figure 3, E-G. The G₁ or G₂ injections with and without G₃ give indistinguishable, strong, and highly significant effects when compared with cells injected with the control plasmid CB1 or to uninjected cells. In these G₁- or ₂-injected neurons, the facilitation ratio is raised from a mean of 1.1 to 2.3, the inhibition by NE is lowered from 60 to 22%, and the basal calcium current density is lowered from 22 to 9 pA/pF. These actions are significant at the p < 0.005 level. Interestingly, both the depression of current and the increase in facilitation ratio are greater than those which occur with 10 µM NE on control cells. By comparison, the effects of injecting DNA for G₃, G₄, or G₅ are weak or negligible, even in the presence of G₃. Of these, G₅ appears the most active. Our results suggest that the different G subtypes have different efficacies in voltage-dependent inhibition of Ca²⁺ channels, with a rank order of effectiveness: G₁ = G₂ > G₅  G₄ = G₃.

Characterization of the weakly active G subtypes

We wanted to explore more fully the reasons for the weak effects of G₃, ₄, and ₅ DNA injections. Did the plasmids fail to induce proper synthesis of the corresponding proteins? Were the proteins really inactive on Ca²⁺ channels? Because convenient antibodies for verifying expression of specific G subunits in single injected cells are lacking, we chose less direct approaches. We looked with increased sensitivity for electrophysiological effects on Ca²⁺ channels.

We made two changes in the electrophysiological assay. One was in the concentration of GFP DNA coinjected into the cells. In some systems, it has been found that the overexpression of GFP or other proteins from one plasmid leads to less expression from a second plasmid. Thus, the most brightly fluorescing cells express lower levels of protein from the second plasmid (N. Davidson, personal communication). Therefore, we lowered the concentration of GFP DNA in the injection solution from 100 to 10 ng/µl in the next experiments. A second factor that may have limited our sensitivity to detect occlusion of the NE response is the high concentration of NE we had used (10 µM) in Figures 1, 2, and 3. This concentration is sufficiently above that needed to have a maximal NE action that a small shift of the sensitivity to NE could have been obscured. Therefore, we reduced the NE concentration to 2 µM, which is submaximal.

Figure 4, A-D, illustrates Ca²⁺ currents recorded from an uninjected neuron and from neurons injected with G₃, G₄, and G₅ DNAs. In uninjected neurons, 2 µM NE inhibited the Ca²⁺ current by 41 ± 4% (SEM; n = 6), compared with the 55% inhibition obtained with saturating NE (10 µM) (Figs. 1-3). There were statistically significant changes in at least one of the experimental parameters for each injected neuron, suggesting expression of each G subunit. Facilitation was increased in the neurons injected with G₅; the inhibition by 2 µM NE was reduced by injection of G₄ or G₅, and the calcium current density was almost doubled by injection of G₃. Further evidence that injection of G₃ DNA affects cellular responses is seen in Figure 5, in which neurons were injected with G₁ alone, G₃ alone, or a mixture of G₁ and G₃ DNAs. As we have seen before, the facilitation ratio is strongly increased by G₁ and not increased by G₃; however, coinjection of G₃ with the G₁ gives significantly less facilitation than with G₁ alone, consistent, for example, with competition between the G subunits for formation of active G complexes. These results show that G₃ and G₄ DNA injections do have specific electrophysiological effects, even if they do not mimic actions of NE on Ca²⁺ currents. Therefore, these G subunits are expressed at functionally significant levels in our injected neurons.

View larger version (21K): [in this window] [in a new window]   Figure 4.   Three weakly active G subtypes. A-D, Current records for uninjected control cell (A) and neurons injected with 100 ng/µl G3, G4, or G5 DNA (B-D). To accentuate possible weak actions by G3, G4, and G5, the concentration of the reporter plasmid (pEGFP-N1) was decreased tenfold to 10 ng/µl, and NE was decreased to 2 µM. E-G, Summary data for facilitation ratio, inhibition by NE, and current density in these cells. Note that the axes have been expanded to reveal smaller differences.

View larger version (21K): [in this window] [in a new window]   Figure 5.   Coinjection of G1 and G3 DNAs. A-C, Current traces for cells were injected with 100 ng/µl G1 (A) or G3 (B) or with 100 ng/µl G1 plus 100 ng/µl G3 DNAs (C). Same experimental conditions as in Figure 3. D, Mean facilitation ratios for these cells. * indicates data significantly different from control. ** indicates data for G1 plus G3 significantly different from control, G1, or G3.

G₁ and G₂ interact strongly with the ₁ subunit of N-type calcium channels

It has been demonstrated that L_I-II of the ₁ subunits of classes A, B, and E (P/Q-, N-, and R-type) calcium channels interact directly with the G₁ subunit (De Waard et al., 1997; Zamponi et al., 1997) and that this loop may mediate the inhibitory modulation by the activated G-protein (Herlitze et al., 1997; Page et al., 1997). Our results presented above suggest that this interaction is not the same for all of the G subunits. We sought to investigate the mechanism underlying the differential effects of the five different G subunit subtypes by testing the possibility that they interact with the channel with different affinities. We examined the interaction of L_I-II of _1B with the G subunits using the two-hybrid assay, an assay that can detect protein-protein interactions in vivo in yeast (Fields and Song, 1989).

Plasmids encoding the GBD-L_I-II hybrid and the various GAD-G hybrids were introduced into a yeast reporter strain in which interactions between two hybrid proteins result in transcriptional activation of two reporter genes, HIS3 and ADE2. These two reporter gene products are involved in histidine and adenine synthesis, respectively, and their activation allows the yeast cells to grow on medium lacking histidine and adenine. As shown in Figure 6A, coexpression of L_I-II with G₁ or G₂ results in robust growth of the yeast cells on the selection medium, whereas with G₅, the cells grow at a much slower rate. In the case of G₃ and G₄, no transformed yeast cells were able to grow in the absence of histidine and adenine. The same results were obtained in five experiments. Because transcription signals resulting from the protein-protein interactions in the two-hybrid assay generally correlate with the binding affinity observed from in vitro experiments (Li and Fields, 1993; Yan et al., 1996), our data suggest that the order of affinity for L_I-II of _1B is G₁ = G₂ > G₅  G₃, G₄.

View larger version (35K): [in this window] [in a new window]   Figure 6.   G1 and G2 interact strongly with the 1 subunit of N-type calcium channels. A, Yeast reporter strain PJ69-4A was cotransformed with plasmids encoding GBD-LI-II and the indicated GAD-G subunits. Cells were first plated onto synthetic sucrose medium SS/Trp/Leu to select cotransformants. The cotransformants were then replica-plated onto medium SS/Trp/Leu/Ade/His to select for colonies having successful GBD-GAD interactions. B, Expression of GAD-G hybrid proteins in yeast. Yeast lysates were subjected to SDS-PAGE and immunoblotting. The blot was probed with anti-GAD monoclonal antibody and horseradish peroxidase-conjugated to anti-mouse IgG. The proteins were detected by ECL. Yeast lysate without GAD hybrid proteins was loaded in lane 6 as a negative control. Total protein amounts in the 20 µl lysates quantified by the Bradford assay (Bio-Rad, Hercules, CA) were 1.94, 1.30, 0.72, 0.80, and 1.70 µg in lanes 1-5, respectively. Intensity of individual GAD-G bands on the immunoblot was determined by densitometry (Molecular Dynamics, Sunnyvale, CA), normalized to the protein amount in their respective lysates, and plotted as a percent of GAD-G3 to show the relative levels of these hybrid proteins.

To exclude the possibility that the tighter binding between G₁, G₂, and L_I-II is attributable to their more efficient recruitment of endogenous yeast G subunits, we also performed the assay in the presence of overexpressed rat G₃ subunit. The results obtained were the same (data not shown), suggesting that G subunits are not a determinant of the interaction between G and L_I-II in our assay. Hence, activation of the HIS3 and ADE2 genes in the assay might be the result of direct interactions between G₁, G₂, and G₅ with the calcium channel L_I-II loop region.

Because an apparent differential affinity of G subtypes for L_I-II of _1B observed in the two-hybrid experiments could also be attributable to differences of their protein level in the yeast cell, we examined their expression by immunoblotting. This method measures the level of expressed protein but does not test correct folding or association with G subunits. Immunoblots of the yeast cell lysates with anti-GAD antibody showed these GAD-hybrid proteins are expressed at similar levels (Fig. 6B, lanes 1-5). Densitometric scanning and normalization to the amount of total cellular protein in each extract showed that expression of G₃, G₄, and G₅ was higher than expression of G₁ and G₂. A second experiment gave the same result. These results support our conclusion that, in the yeast two-hybrid assay, G₁ and G₂ are more effective because they bind with higher affinity to L_I-II.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Our electrophysiological and protein-protein interaction experiments give concordant results for the  subunits of G-proteins. All G subunits are not equal. Mimicry and occlusion of membrane-delimited voltage-dependent inhibition of N-type Ca²⁺ currents and interaction with the L_I-II loop of _lB (N-type) calcium channels follow the sequence G₁ = G₂ > G₅  G₃, G₄. The agreement of the two approaches adds weight to the proposal that the functional target of voltage-dependent G interaction includes the L_I-II loop (De Waard et al., 1997; Herlitze et al., 1997; Page et al., 1997; Zamponi et al., 1997). The activity of overexpressed G₁ and G₂ has been documented before in SCG cells (Herlitze et al., 1996; Ikeda, 1996; Delmas et al., 1998). Unlike our results (Fig. 1), however, Ikeda (1996) and Delmas et al. (1998), who studied only G₁, found that a G subunit had to be coexpressed with G₁ to have an effect on calcium channels. We do not know any reason for this clear difference from our work. They tested only G₂, and we tested only G₃. If G dimers are needed for mimicking action of NE, the G subunits must have combined with endogenous G subunits when we did not inject G DNA. The endogenous complement of G subunits is not known for SCG neurons.

Although the vectors for G₃ and G₄ did not mimic voltage-dependent Ca²⁺ current inhibition by NE, they did alter electrophysiological properties of the cells. Thus, they were expressed. Unexpectedly, expression of G₃ led to a near doubling of the Ca²⁺ current density (Fig. 4). Because the percent of inhibition by NE was unchanged in these cells, the increased current density was primarily caused by an increase in N-type Ca²⁺ current. On the other hand, expression of G₄ led to a reduction in modulation by NE, without an increase in facilitation or a decrease in current density. These effects of G₃ and G₄ might have been caused by interactions with signaling systems in the cell that alter gene expression or act in other ways on the channel. They could also represent direct interactions of G₃ and G₄ with parts of the channel that do not have the binding specificity we found for the L_I-II loop. It is not likely that G₃ or G₄ failed to pair with coinjected G₃ subunit, because it has been found previously that this subunit pairs efficiently with the G₃ and G₄ subunits (Yan et al., 1996). Indeed, in comparison with G₂₃, the reporter activity in a yeast-two-hybrid assay was 55% for G₃₃, 72% for G₄₃, and 83% for G₅₃ (Yan et al., 1996).

The literature does not contain much information on specificity of the actions of G subunits. From our results, the strong membrane-delimited voltage-dependent modulation of N-type calcium channels seen in many neurons is almost certainly mediated by G₁ and/or G₂ subunits. A similar conclusion may be drawn for the activation of G-protein-coupled inward rectifier K⁺ (GIRK) channels. A two-hybrid screen with residues 1-83 of the GIRK1 channel showed interaction with G₁ and G₂ but not with G₃, G₄, or G₅ (Yan and Gautam, 1996), suggesting that G₁ and G₂ may be generally involved in membrane-delimited modulation of ion channels. Functional tests of G₃, G₄, or G₅ on GIRK channels have not been published.

Specificity has been investigated in a few other systems. Kleuss et al. (1991, 1992, 1993) used injection of antisense oligonucleotides directed against specific members of the G-protein heterotrimer to study agonist-induced inhibition of L-type calcium channels in GH₃ cells. They established that G_o1/₁/₄ mediates inhibition acting via m₄ muscarinic receptors, whereas G_o2/₃/₃ mediates inhibition by somatostatin receptors. The molecular mechanisms of these signaling pathways have not been worked out, and it is possible that the very high specificity lies at the level of the receptors rather than the (unknown) effector(s). This contrasts with work on adenylyl cyclase II in the two-hybrid system in which all  subunits interact to some degree, and the rank order of interaction was ₁ > ₂ > ₃ = ₅ > ₄ (Yan and Gautam, 1996). Interestingly, ₁ stimulates, whereas ₅ inhibits adenylyl cyclase II (Bayewitch, 1998). Both interactions were strong in the two-hybrid system.

Other systems in which G-protein subtype specificity has been examined include inhibition of adenylyl cyclase I (no difference between ₁ and ₅; Bayewitch, 1998), activation of MAP kinase (₁ more efficient than ₅; Zhang et al., 1996b), activation of phospholipase ₂ (no difference between ₁ and ₅; Zhang et al., 1996b), and binding to G-protein receptor kinases (GRKs) (₁ and ₂, but not ₃, bind to GRK2, and ₁, ₂, and ₃ all bind to GRK3; Daaka, 1997). Thus, in the case of classical G-protein effectors (adenylyl cyclase, phospholipase, and ion conducting channels) specificity of interactions with G-protein  subunit types has previously been noted only in ₅ compared with ₁. However, this is not entirely surprising considering that ₅ is a unique  subunit type that is only 53% identical to the rest and is primarily expressed in brain (Watson et al., 1994). The differential activity of ₁ and ₂ subunits compared with the ₃ and ₄ subunits seen here on Ca²⁺ channels is of interest, because these  subunit types are very similar. In fact, ₁ and ₂ are more similar to ₄ (~90% identity) than ₃ to ₄ (~80% identity) (Yan et al., 1996). The functional differences must, however, be caused by structural difference(s) between the two pairs of proteins. A scan of the sequences to identify residues common to G₁ and ₂, but different in ₃ and ₄, yields seven residues: R19, S31, N35, P39, A193, R197, and A305, using G₁ numbering. Most of these residues are near regions thought to interact with the G subunit. The molecular basis for the functional difference between ₁/₂ versus ₃/₄ may not be restricted to these residues, because ₃ and ₄ diverge individually with respect to the each other, as well as ₁ and ₂ subtypes at many positions in their amino acid sequences. This divergence is also consistent with the differential effects of ₃ compared with ₄ on Ca²⁺ channel properties (Fig. 4). A recent major study by Ford et al. (1998) has looked for residues in G₁ that affect interaction with five different effectors, including N-type calcium channels containing _1B subunits. They mutated 15 residues within the interaction domain for G subunits with G subunits and found eight that affected the calcium channel facilitation. However, because all of the 15 residues tested are identical among G₁, G₂, G₃, and G₄, these results do not help to explain why two of the subunits are so efficacious and two of them are ineffective on N-type calcium channels.

As mentioned before, the G₅ subunit stands out among G subunits. Unlike the others, it is reported to couple almost exclusively to the G_q subunit in a manner that can prevent the others from binding (Fletcher et al., 1998). In our experiments, injection of the vector encoding G₅ produced a small but reliable voltage-dependent inhibition of the Ca²⁺ current and partial occlusion of NE-induced inhibition (Fig. 4). Using a G₅ antibody (provided by M. Simon, Caltech) (Watson et al., 1994), we found that SCG neurons express G₅ (data not shown), as anticipated from its enrichment in brain, and therefore stimulation of G_q via m₁ muscarinic or angiotensin II receptors in SCG cells should release G₅ subunits. Together, these three results suggest that the small but fast component of calcium channel inhibition seen with receptors linked to G_q in SCG neurons (such as m₁ muscarinic receptors) (Zhou et al., 1997) could be attributable to G₅.

The putative sites of interaction of the G-protein  subunit with the calcium channel have been controversial (e.g., Dolphin, 1998). Typically, experiments examining this interaction have relied on overexpression of appropriately engineered calcium channels in cell lines or Xenopus oocytes or on the demonstration of protein-protein interactions with recombinant proteins in vitro. We have taken a complementary approach with the two-hybrid system, and our results strongly support the hypothesis that the interaction of G subunits with the linker between homologous domains I and II of the ₁ subunit determines the subunit specificity for voltage-dependent inhibition of N-type calcium channels by G-protein subunits. Although it is likely that G-protein  subunits interact with other regions of the calcium channel, further experimentation will be necessary to determine whether these additional interactions contribute to the voltage-dependent inhibition.

	FOOTNOTES

Received July 1, 1998; revised Aug. 26, 1998; accepted Aug. 27, 1998.

This work was supported by the W. M. Keck Foundation, an Alexander von Humboldt Stiftung Fellowship, DGAPA Universidad Nacional Autonoma de Mexico and Consejo Nacional de Ciencia y Tecnologia, and National Institutes of Health Grants NS01588, DA08934, DA00286, NS08174, NS22625, and GM46963. N.G. is an Established Investigator of the American Heart Association. We thank D. Anderson, S. Brown, and L. Miller for technical help.

Correspondence should be addressed to Dr. Bertil Hille, Department of Physiology and Biophysics, Box 357290, University of Washington, Seattle, WA 98195-7290.

Drs. Li, García-Ferreiro, and Hernández-Ochoa contributed equally to this work.

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