Effect of G protein heterotrimer composition on coupling of neurotransmitter receptors to N-type Ca²⁺ channel modulation in sympathetic neurons

Abstract

Voltage-dependent (VD) inhibition of N-type Ca²⁺ channels is mediated primarily by neurotransmitter receptors that couple to pertussis toxin (PTX)-sensitive G proteins (such as G_o and G_i). To date, however, the composition of heterotrimeric complexes, i.e., specific Gαβγ combinations, capable of coupling receptors to N-type Ca²⁺ channels has not been defined. We addressed this question by heterologously expressing identified Gαβγ combinations in PTX-treated rat sympathetic neurons and testing for reconstitution of agonist-mediated VD inhibition. The heterologously expressed Gα subunits were rendered PTX-insensitive by mutating the codon specifying the ADP ribosylation site. The following results were obtained from this approach. (i) Expression of Gα_oA, Gα_oB, and Gα_i2 (along with Gβ₁γ₂) reconstituted VD inhibition mediated by α₂-adrenergic, adenosine, somatostatin, and prostaglandin E₂ receptors. Conversely, expression of Gα_i1 and Gα_i3 was ineffective at restoring coupling. (ii) Coupling efficiency, as determined from the magnitude of reconstituted Ca²⁺ current inhibition, depended on both the receptor and Gα subtype. The following rank order of coupling efficiency was observed: Gα_oA = Gα_oB > Gα_i2 for α₂-adrenergic receptor; Gα_i2 > Gα_oA = Gα_oB for adenosine and prostaglandin E₂ receptors; and Gα_oB = Gα_i2 > Gα_oA for the somatostatin receptor. (iii) In general, varying the Gβγ composition of Gα_oA-containing heterotrimers had little effect on the coupling of α₂-adrenergic receptors to the VD pathway. Taken together, these results suggest that multiple, diverse Gαβγ combinations are capable of coupling neurotransmitter receptors to VD inhibition of N-type Ca²⁺ channels. Thus, if exquisite Gαβγ-coupling specificity exists in situ, it cannot arise solely from the inherent inability of other Gαβγ combinations to form functional signaling complexes.

The minimal G protein-signaling pathway is composed of a seven-transmembrane spanning receptor, a heterotrimeric G protein complex (i.e., Gαβγ), and an effector. A specific form of neurotransmitter-mediated N-type Ca²⁺ channel modulation, termed voltage-dependent (VD) inhibition, appears to require only these elements. The accumulated evidence suggests that VD inhibition is membrane-delimited, does not require an enzymatic component (e.g., kinase or phosphatase) (1), and is mediated by interaction of “free” Gβγ with the α₁-subunit (α_1B) of the N-type Ca²⁺ channel (2–5). Moreover, VD inhibition is the most common form of N-type Ca²⁺ channel modulation, is easily identifiable from distinct biophysical characteristics (e.g., “kinetic slowing” and prepulse facilitation), and most likely plays a crucial role in regulating neurotransmitter release from presynaptic nerve terminals (1, 5). Although general aspects of this signaling pathway have been well studied, the impact of heterotrimeric G protein composition, i.e., the specific Gα, Gβ, and Gγ isoforms, on coupling receptors to VD N-type Ca²⁺ channel modulation has been explored little.

The question of how G protein heterotrimer composition influences signaling pathways arises because there are a large number of potential discrete Gαβγ combinations. At present, about 20 Gαs, 5 Gβs, and 11 Gγs have been identified in mammals. Consequently, around 1,100 potential G protein heterotrimer combinations are possible. However, several factors serve to limit the number of combinations relevant to a given signaling pathway. First, several of the Gα and Gγ subunits have a very restricted tissue distribution that confines their potential contribution mainly to sensory transduction systems (6). Second, some combinations of Gβ and Gγ do not appear to form functional heterodimers, at least when produced in vitro (7–10). Third, it has been reported that Gβ₅-containing heterodimers couple exclusively to Gα_q/11 family subunits (11). Finally, agents such as pertussis toxin (PTX) modify a distinct subset of Gα subunits (12). Thus, if the PTX-sensitivity of a signaling pathway is known, the number of possible combinations is reduced further. Unfortunately, even after improbable heterotrimer combinations are eliminated, there often can remain >100 candidate heterotrimer compositions.

For the simplest systems, i.e., those composed of receptor, G protein, and effector, coupling specificity can be examined at three different junctions. First, the ability of a defined G protein heterotrimer composition to interact with a receptor can be examined. This usually is investigated in heterologous systems in which the ability of defined G protein heterotrimers to affect ligand affinity is measured (13, 14). Successful coupling of the G protein to receptor is indicated by an increase in ligand affinity. Second, the ability of activated G protein components, i.e., GTP-bound Gα or Gβγ subunits, to interact with an effector can be examined. For GIRK-type K⁺ channels and N-type Ca²⁺ channels, “free” Gβγ appears to be the component responsible for activation and VD inhibition, respectively (5, 15). G protein/effector interaction thus can be determined by applying in vitro manufactured Gβγ (16) or heterologously overexpressing Gβγ (17) and determining whether tonic channel modulation occurs. Third, one can attempt to express a defined Gαβγ combination and determine whether receptor activation leads to effector modulation. In the following experiments, native G protein coupling was disrupted by treating the sympathetic neurons with PTX. Reconstitution then was attempted by heterologously expressing a PTX-insensitive mutant of Gα (18–21) along with Gβ and Gγ. Successful reconstitution was determined by receptor-mediated VD inhibition of N-type Ca²⁺ channels. The most parsimonious view of receptor–effector coupling predicts that the intersection of data sets describing receptor/G protein and G protein/effector coupling will dictate the outcome of receptor/G protein/effector reconstitution experiments. Accordingly, because only limited specificity has been observed between receptor/G protein and G protein/effector (e.g., Gβγ and N-type Ca²⁺ channels), successful reconstitution of coupling by numerous combinations of Gαβγ might be expected. However, antisense knockout of individual G protein subunits in GH3 cells suggests that exquisitely specific Gαβγ combinations mediate native coupling of receptors to L-type Ca²⁺ channels (22–24). Thus, reconstitution experiments complement the knockout experiments by examining which Gαβγ combinations are capable of supporting coupling rather than which Gαβγ combinations mediate native coupling. Taken together, these data should lend insight into factors regulating receptor–effector-coupling specificity.

Materials and Methods

Dissociation of Sympathetic Neurons.

Superior cervical ganglion (SCG) neurons were enzymatically dissociated from adult male Wistar rats as described previously (25, 26). After washing twice, the dissociated neurons were resuspended in minimum essential medium (MEM) containing 10% FCS, 1% glutamine, and 1% penicillin-streptomycin solution (all from GIBCO). Neurons then were plated onto polystyrene culture dishes (35 mm), coated with poly-l-lysine, and maintained in a humidified atmosphere of 95% air/5% CO₂ at 37°C. All neurons were used within 24 hr after intranuclear injection of vectors. PTX-pretreated neurons were incubated overnight (16–20 hr) with 500 ng/ml PTX (List Biological Laboratories, Campbell, CA).

Construction and Expression of PTX-Insensitive Mutants of Gα Subunits.

Vectors encoding wild types of mouse Gα_oA,B, bovine Gβ₁, human Gβ_2,3, mouse Gβ_4,5, and bovine Gγ_2,3 were from M. I. Simon (California Institute of Technology, Pasadena, CA); rat Gα_i1–3 were from R. R. Reed (Johns Hopkins Medical School, Baltimore, MD); and mouse Gγ₄ was from N. Gautam (Washington University School of Medicine, St. Louis). The coding region for each subunit was subcloned into the mammalian expression vector, pCI (Promega). To generate PTX-insensitive mutants of the Gα_o/i subunits, the codon specifying the fourth amino acid, cysteine (C), from the carboxyl terminus was mutated to code for a glycine (G). The coding region of each Gα subunit was amplified by PCR with Pfu polymerase. Site-directed mutagenesis was performed by incorporating the altered base into the reverse primer. For Gα_oA, cysteine-to-serine (S) or -isoleucine (I) mutations also were generated. The resultant PCR products were subcloned into the mammalian expression vector, pCI. The sequence of each construct was verified with an automated DNA sequencer (ABI 310; PE Applied Biosystems). The PTX-insensitive Gα mutants were coexpressed with different Gβ and Gγ subunits by using intranuclear injection methods as described previously (2, 25). The cDNAs encoding Gα, Gβ, and Gγ subunits (10 mM Tris/1 mM EDTA, pH 7.4) were injected at a ratio (weight) of ≈0.3–0.5:1:1.

Electrophysiology and Data Analysis.

Ca²⁺ channel currents were recorded by using the whole-cell variant of the patch–clamp technique (27) as described previously (26, 28, 29). To isolate Ca²⁺ currents, patch electrodes were filled with a solution containing 120 mM N-methyl-d-glucamine (NMG) methanesulfonate (MS), 20 mM TEA-MS, 20 mM HCl, 11 mM EGTA, 1 mM CaCl₂, 10 mM Hepes, 4 mM MgATP, 0.3 mM Na₂GTP, and 14 mM creatine phosphate (pH 7.2, 297 mOsM/kg H₂O). External recording solution contained 145 mM tetraethylammonium (TEA)-MS, 10 mM Hepes, 10 mM CaCl₂, 15 mM glucose, and 0.0003 mM tetrodotoxin (TTX) (pH 7.4, 318 mOsM/kg H₂O). All experiments were performed at room temperature (21–24°C). Data were presented as means ± SEM where appropriate. Student's t test (unpaired) or ANOVA followed by a posthoc test, as appropriate, was applied to the data to determine statistical significance. P < 0.05 was considered significant.

Results

Reconstitution of α₂-Adrenergic Receptor Coupling by PTX-Insensitive Mutants in SCG Neurons.

Norepinephrine (NE) produces VD inhibition of N-type Ca²⁺ currents via α₂-adrenergic receptors (α₂-ARs) in SCG neurons (30). The signaling pathway underlying the modulation is PTX-sensitive (31) and likely involves Gα_o (32). We thus examined the ability of PTX-insensitive Gα_o mutants to couple α₂-ARs to N-type Ca²⁺ channels. The PTX-insensitive mutant (C351G) codes for glycine instead of cysteine at position 351, the site for PTX-catalyzed ADP ribosylation in the Gα_o (33). Ca²⁺ currents were evoked from a holding potential of −80 mV with a double-pulse protocol consisting of two identical test pulses to +10 mV separated by a large, depolarizing conditioning pulse to +80 mV (see Fig. 1A). In control neurons expressing only GFP, 10 μM NE inhibited Ca²⁺ currents by 62 ± 2% (n = 26) (Fig. 1 A and E). NE-induced Ca²⁺ current inhibition displayed the hallmarks of VD inhibition (see ref. 5), i.e., slowed activation kinetics in the prepulse and an enhanced postpulse amplitude (Fig. 1 A and D). Facilitation, defined as the ratio of the postpulse to prepulse current amplitude, increased from 1.17 ± 0.01 to 2.36 ± 0.07 (n = 26) after NE application. Overnight treatment of the neurons with PTX (500 ng/ml) greatly attenuated NE-induced Ca²⁺ current inhibition (Fig. 1 B and E), implicating the involvement of a PTX-sensitive G protein(s) in the signaling pathway. Coexpression of Gα_oA(C351G) with Gβ₁ and Gγ₂ subunits (denoted Gβ₁γ₂ hereafter) reconstituted the NE-induced Ca²⁺ current inhibition in PTX-treated neurons (Fig. 1C). The success of the reconstitution experiments was critically dependent on establishing a functional stoichiometric match between the Gα and Gβγ subunits expressed in SCG neurons. For example, if expression of Gα greatly exceeded that of Gβγ, basal facilitation and NE-induced Ca²⁺ current inhibition were ablated. This result presumably reflects sequestration of free Gβγ subunits (both endogenous and exogenous) by the GDP-bound Gα (2, 34). In contrast, if expression of Gβγ exceeded that of Gα, a significant tonic inhibition (as indicated by increased basal facilitation) was observed. This result presumably reflects interaction of “free” or excess Gβγ with N-type Ca²⁺ channels (2, 3, 17). To obtain criteria for judging the “balanced condition,” we examined the relationship between Ca²⁺ current inhibition (%) and basal facilitation ratio (BFR) for 49 cells expressing G_oA(C351G)β₁γ₂ (data not shown). When the BFR was <1, reconstitution did not occur. In cells showing successful reconstitution, the BFR was always >1 (ranging from 1.02 to 1.46). Although reconstitution was apparent in cells with a BFR >1.5 (1.66–2.59), we did not include these neurons in the analysis. Thus, we defined a basal facilitation range of 1.02–1.5 as indicating adequate “balance” between Gα and Gβγ subunits. In neurons expressing Gα_oA(C351G)β₁γ₂, only about 20% of the neurons (9 of 49) fulfilled this criteria. For these neurons, NE application inhibited Ca²⁺ currents by 52 ± 3% and increased prepulse facilitation from 1.15 ± 0.05 to 1.90 ± 0.09 (n = 9) after PTX treatment (Fig. 1 D and E).

Figure 1.

Reconstitution of α₂-AR coupling to N-type Ca²⁺ channels by expression of PTX-insensitive Gα_oA mutants. Time course of current inhibition and superimposed current traces in the absence (1 and 3) or presence (2 and 4) of 10 μM NE recorded from control neurons (no PTX) (A), control (500 ng/ml PTX, overnight) (B), PTX-pretreated neurons coexpressing Gα_oA(C351G) and Gβ₁γ₂ (C). The cDNAs encoding PTX-insensitive Gα mutants and Gβ₁γ₂ were injected directly into nuclei of SCG neurons. The Ca²⁺ current was evoked every 10 s by a double-pulse voltage protocol (see Inset in A) consisting of two identical test pulses (+10 mV from a holding potential of −80 mV) separated by a large, depolarizing conditioning pulse to +80 mV. The amplitudes of currents generated by pre- (●) and post- (○) pulses were plotted. (D) Summary of prepulse facilitation in the absence or presence of NE. Prepulse facilitation was calculated by the ratio of the postpulse to prepulse current amplitude measured isochronally at 10 ms after the start of the test pulses. (E) Summary of NE-induced Ca²⁺ current inhibition. The C-terminal cysteine residue of Gα_oA also was mutated to serine or isoleucine. Inhibition (%) was calculated by using the amplitudes of currents determined isochronally 10 ms after the start of the prepulse. In both D and E, data are presented as mean ± SEM and numbers in parentheses indicate the number of neurons tested.

The nature of amino acid residue at the site of PTX-catalyzed ADP ribosylation has been shown to influence the maximal stimulation of Gα_i1 by α₂-ARs (35, 36). In addition, Gα_z, a naturally occurring PTX-insensitive Gα (which contains an isoleucine at the equivalent site), has been shown to efficiently couple α₂-ARs to N-type Ca²⁺ channels in SCG neurons (26). Accordingly, we tested whether reconstitution of NE-induced Ca²⁺ current inhibition was altered by the expression of Gα_oA(C351I) or Gα_oA(C351S) when compared with Gα_oA(C351G). Coexpression of Gα_oA(C351I) or Gα_oA(C351S) with Gβ₁γ₂ resulted in a 31 ± 4% (n = 9) and 24 ± 7% (n = 5) inhibition of Ca²⁺ current, respectively, after NE application (Fig. 1E). These data indicated that the C→G mutation, under the experimental conditions utilized in these studies, produced the greatest current inhibition and, thus, presumably, coupling efficiency. Therefore, the C→G mutation was used to render all other Gαs used in the study as PTX-insensitive. When compared with native coupling, NE-induced Ca²⁺ current inhibition via Gα_oA(C351G) occurred with about 2-fold less potency [EC₅₀ of 2.35 ± 0.51 (n = 3) vs. 1.25 ± 0.38 μM (n = 6), data not shown] but similar maximal inhibition [50 ± 9% (n = 3) vs. 59 ± 4% (n = 6), data not shown].

Effect of Gβγ Composition on Coupling Between α₂-ARs and Ca²⁺ Channels.

In GH₃ cells, somatostatin and muscarinic receptors couple to L-type Ca²⁺ channel modulation via very specific Gαβγ compositions as indicated by antisense experiments (22–24). Likewise, α₂-ARs have been shown to discriminate among Gα_i heterotrimers containing different Gβγ combinations (14). Thus, we tested whether various Gβγ combinations influenced the ability to reconstitute coupling between α₂-ARs and N-type Ca²⁺ channels in PTX-treated neurons. For these experiments, five different Gβγ combinations (Gβ₁γ₂, Gβ₁γ₃, Gβ₂γ₂, Gβ₃γ₄, and Gβ₄γ₄) were selected based on previous studies done by Schultz and colleagues (37). When these Gβγ combinations were coexpressed with Gα_oA(C351G) in PTX-treated SCG neurons, the VD inhibition of Ca²⁺ currents by NE was robust (Fig. 2A). On average, G_oA(C351G) heterotrimers containing Gβ₁γ₂, Gβ₁γ₃, Gβ₂γ₂, Gβ₃γ₄, or Gβ₄γ₄ produced 52 ± 3% (n = 9), 49 ± 9% (n = 5), 43 ± 4% (n = 10), 45 ± 4% (n = 5), and 45 ± 9% (n = 6) inhibition of Ca²⁺ currents, respectively (Fig. 2B). The NE-induced Ca²⁺ current inhibition was not significantly different among the tested Gβγ combinations (P > 0.05). These results suggest that a specific Gβγ combination is not required for coupling between α₂-ARs and N-type Ca²⁺ channels. As a negative control, we tested Gβ₅γ₂, a Gβγ combination thought to selectively interact with Gα_q subunits (11). Expression of β₅γ₂ alone produced a modest basal facilitation, supporting the functional expression of this Gβγ (V. Ruiz-Velasco and S.R.I., unpublished data). Coexpression of Gα_oA(C351G) with β₅γ₂ produced a tonic inhibition (as judged by kinetic slowing and basal facilitation) similar to that produced by the expression of β₅γ₂ alone and failed to reconstitute NE-induced Ca²⁺ current inhibition (n = 6, Fig. 2). These data suggest that, under the conditions used here, heterologously expressed Gα_oA(C351G) does not, to a significant extent, form functional heterotrimers with natively expressed Gβγ.

Figure 2.

Specific Gβγ combinations are not required for coupling between α₂-ARs and N-type Ca²⁺ channels. (A) Superimposed current traces in the absence or presence of 10 μM NE recorded from neurons expressing Gα_oA(C351G) and different Gβγ combinations (β₁γ₂, β₁γ₃, β₂γ₂, β₃γ₄, β₄γ₄, β₅γ₂). (B) Summary of VD Ca²⁺ current inhibition by NE under conditions described above. Note that tonic inhibition is apparent in neurons expressing Gα_oA(C351G) β₅γ₂. Data are presented as mean ± SEM, and numbers in parentheses indicate the number of neurons tested.

Specificity of Interaction Between Gα Subunits and α₂-ARs for Ca²⁺ Channel Modulation.

Additional members of the PTX-sensitive Gα_i/o family were tested to determine their ability to reconstitute VD inhibition of N-type Ca²⁺ channels. PTX-insensitive mutants of Gα_oB, Gα_i1, Gα_i2, and Gα_i3, generated as for Gα_oA, were coexpressed with Gβ₁γ₂ subunits in PTX-treated SCG neurons. Fig. 3 illustrates representative traces and summary data acquired from neurons expressing different PTX-insensitive heterotrimer compositions. NE application produced VD inhibition of Ca²⁺ currents in neurons expressing Gα_oB(C351G) and Gα_i2(C352G) (Fig. 3). In contrast, NE-induced Ca²⁺ current inhibition was negligible in neurons expressing Gα_i1(C351G) or Gα_i3(C351G) (Fig. 3). The functional expression of Gα_i1(C351G) and Gα_i3(C351G) was indirectly confirmed by observing a lack of basal facilitation when these constructs were expressed without Gβγ subunits (data not shown, n = 3 for each mutant type of Gα_i). Furthermore, the Gα_i1(C351G) and Gα_i3(C351G) constructs used in this study are capable of coupling m₂-muscarinic receptors to GIRK channel activation in Xenopus oocytes (C. Doupnik, personal communication). On average, NE produced 48 ± 6% (n = 5) and 30 ± 3% (n = 7) inhibition of Ca²⁺ currents in neurons expressing Gα_oB(C351G) and Gα_i2(C352G), respectively (Fig. 3B). Taken together, these results suggest that Gα_oA/B and Gα_i2, when coexpressed with Gβ₁γ₂, are capable of coupling α₂-ARs to VD N-type Ca²⁺ channel inhibition. Under the present experimental conditions, expression of Gα_o isoforms resulted in larger NE-induced inhibitions when compared with neurons expressing Gα_i.

Figure 3.

Contribution of both Gα_o and Gα_i to coupling between α₂-ARs and N-type Ca²⁺ channels with some degree of specificity. (A) Superimposed current traces in the absence or presence of 10 μM NE recorded from neurons coexpressing PTX-insensitive mutants (C351G) of different Gα subunits (Gα_oB and Gα_i1-i3) and Gβ₁γ₂. (B) Summary of VD Ca²⁺ current inhibition by NE. Data are presented as mean ± SEM, and numbers in parentheses indicate the number of neurons tested.

Differential Coupling Between Gα Subunits and Other Receptors.

In SCG neurons, it is well established that several different neurotransmitters produce VD inhibition of N-type Ca²⁺ channels via a pathway utilizing PTX-sensitive G proteins (1). To determine the specificity of coupling between other neurotransmitter receptors and Gα_o/i-containing heterotrimers, we carried out experiments analogous to those described above. We selected adenosine (38), prostaglandin E₂ (PGE₂) (39), and somatostatin (40) because of their large and robust effects on N-type Ca²⁺ channels. Fig. 4 summarizes the effects of adenosine (10 μM), PGE₂ (1 μM), and somatostatin (300 nM) on Ca²⁺ currents in neurons expressing Gα_oA(C351G)β₁γ₂, Gα_oB(C351G)β₁γ₂, and Gα_i2(C352G)β₁γ₂. Inhibition of Ca²⁺ currents was 35 ± 2% (n = 19) after adenosine administration and was prevented by PTX pretreatment, as reported previously (38). In PTX-treated neurons, expression of Gα_i2(C352G) partially reconstituted the VD inhibition of Ca²⁺ currents (25 ± 5%, n = 8). Expression of Gα_oA(C351G) and Gα_oB(C351G) had smaller effects than Gα_i2(C352G), reconstituting inhibition to 14 ± 3% (n = 7) and 14 ± 4% (n = 5), respectively. PGE₂-induced Ca²⁺ current inhibition was 37 ± 3% (n = 12) and was prevented mostly by the PTX pretreatment. When Gα_oA(C351G), Gα_oB(C351G) and Gα_i2(C352G) were expressed in PTX-pretreated neurons, PGE₂ inhibited the Ca²⁺ current by 35 ± 6% (n = 3), 30 ± 3% (n = 5), and 50 ± 5% (n = 7), respectively. It should be noted that the PGE₂-induced Ca²⁺ current inhibition in Gα_i2(C352G)-expressing cells (PTX-treated) exceeded the inhibition occurring through native G proteins. Activation of somatostatin receptor produced 53 ± 3% and 3 ± 1% inhibition (n = 26) of Ca²⁺ currents in uninjected control and PTX-treated neurons, respectively. Expression of Gα_oB(C351G) and Gα_i2(C352G) reconstituted the Ca²⁺ current inhibition to 24 ± 5% (n = 5) and 28 ± 5% (n = 7), respectively. However, the somatostatin-induced Ca²⁺ current inhibition was only marginally restored (10 ± 2%, n = 8) by expression of Gα_oA(C351G). Expression of either Gα_i1(C352G)β₁γ₂ or Gα_i3(C352G)β₁γ₂ failed to restore coupling of all tested receptors to N-type Ca²⁺ channels (data not shown).

Figure 4.

Summary of differential couplings between Gα subunits and other receptors. The coupling specificity was assessed in PTX-pretreated neurons coexpressing Gα(C351G) and Gβ₁γ₂. The VD inhibition of N-type Ca²⁺ channels was induced by application of adenosine (10 μM), PGE₂ (1 μM), and somatostatin (0.3 μM) by using the voltage protocol described in Fig. 1. Data are presented as mean ± SEM, and numbers in parentheses indicate the number of neurons tested.

Taken together, these data suggest that neurotransmitter receptors natively expressed in SCG neurons are capable of coupling to N-type Ca²⁺ channels via heterotrimers containing Gα_oA, Gα_oB, and Gα_i2, but not Gα_i1 or Gα_i3.

Discussion

In these experiments, we investigated the contribution of G protein heterotrimer composition to functional coupling between neurotransmitter receptors and N-type Ca²⁺ channels. The impetus for the experiments arises from two seemingly paradoxical observations. On one hand, the combinatorial potential for different G protein heterotrimer compositions is quite large. On the other hand, receptor-mediated Ca²⁺ channel modulation has been proposed to occur with exquisite coupling selectivity. For example, Kleuss et al. (22–24), using antisense knockout techniques, have provided evidence that inhibition of L-type Ca²⁺ channels in GH₃ cells occurs via coupling to Gα_oAβ₃γ₄ and Gα_oBβ₁γ₃ for M₄-muscarinic and somatostatin receptors, respectively. In regard to these studies, it should be noted that the modulatory pathway underlying inhibition of L-type Ca²⁺ channels in GH₃ cells has not been defined and may not be analogous to the pathway mediating VD modulation of N-type Ca²⁺ channels. The strategy utilized in our experiments relied on uncoupling natively expressed G proteins with PTX followed by heterologous expression of a defined G protein heterotrimer composition. The approach was adopted from earlier studies in which PTX-insensitive Gα mutants were stably transfected into clonal cell lines (18–21). In stably transfected cells, there apparently is a reestablishment of Gα:Gβγ stoichiometry in which the heterologously expressed PTX-insensitive Gα pairs with native and, thus, undefined Gβγ. Hence, these studies evaluated only the contribution of Gα to receptor/effector coupling. In contrast, we have shown previously that transient (ca. 24 hr) expression of either Gα or Gβγ in SCG neurons results in functional alterations consistent with a stoichiometric mismatch between Gα and Gβγ (2, 34). Apparently, the compensatory changes that occur in clonal cells stably transfected with Gα subunits do not occur in neurons, at least during the time scale of our experiments (see ref. 41), therefore allowing us to examine the contribution of the G protein heterotrimer to coupling.

There were several advantages to the strategy we employed. First, coupling was investigated against a neuronal background and, thus, only G protein subunits required heterologous expression. Receptors and channels were natively expressed and presumably existed in a physiologically relevant environment. The level of receptor expression, in particular, has been shown to influence G protein-coupling specificity (42). Second, measurement of N-type Ca²⁺ channel current facilitation provided a functional indicator of Gα:Gβγ stoichiometry. This ability was crucial for the successful reconstitution of responses. In addition, the ability to express either Gα or Gβγ alone and to determine functional consequences (decreased or increased basal facilitation, respectively) provided a positive control for subunit expression. Third, SCG neurons are a well characterized and standard model system for studying the VD form of N-type Ca²⁺ channel inhibition (1). Numerous receptor systems have been shown to produce robust and reproducible Ca²⁺ channel inhibition via a PTX-sensitive pathway. Thus, we were able to compare G protein coupling with several different receptors under identical experimental conditions.

As with all experimental methods, there are potential limitations that need to be addressed as well. First, we had no way of determining the absolute level of G protein subunit expression. As mentioned above, basal Ca²⁺ current facilitation can be used as a functional measure of relative Gα:Gβγ expression. We attempted to equalize expression of the G protein subunits by subcloning only the ORF of Gα and Gβ subunits into the expression vector, thereby avoiding the potential influence of untranslated regions on gene expression (e.g., ref. 43). Additionally, the start codon was placed in a Kozak consensus sequence (44) when possible. Nonetheless, differences in translational efficiency (e.g., codon bias) or protein half-life would result in different expression levels. Second, in regard to effects on coupling specificity, the consequences of mutating the site of ADP ribosylation are not known. Clearly, the carboxyl terminus of Gα is a major determinant of receptor/G protein-coupling specificity. For example, altering the last 3 aa residues of Gα has dramatic effects on Gα coupling (45). Milligan and colleagues (46) have shown that C→G mutants of Gα_i couple to α_2A-ARs with specificity similar to wild-type Gα_i. However, agonist potency and efficacy were decreased in the Gα_i mutant-expressing cells. Thus, some caution is required when interpreting data derived from cells expressing PTX-insensitive Gα subunits. It seems likely that no single experimental approach to coupling specificity will provide definitive answers—hence, data from multiple diverse approaches will be required to reach a consensus view of the process.

Given these caveats, what have these experiments revealed? First, expression of Gα_oA(C351G) along with Gβ₁ and Gγ₂ successfully reconstituted NE-induced Ca²⁺ current inhibition after PTX pretreatment. The Gα_oA isoform was chosen for the initial trials because previous studies have demonstrated that Gα_o is abundant in SCG neurons and involved with NE-induced Ca²⁺ channel modulation (32). A second finding was that Gα influenced coupling to a greater extent than Gβγ. Although only a limited number of different Gβγ combinations were coexpressed with Gα_oA(C351G), all but one, Gβ₅γ₂, resulted in robust reconstitution of NE-induced Ca²⁺ current inhibition. The inability of Gβ₅γ₂ to reconstitute coupling is consistent with the finding that Gβ₅ containing Gβγ subunits forms heterotrimers exclusively with the Gα_q/11 family (11) and provides evidence that the expressed Gα was not significantly recombining with native Gβγ subunits. In addition, the degree of basal facilitation observed in these experiments (Fig. 2A Lower Right) likely reflects the modest ability of “free” Gβ₅-containing Gβγ subunits to inhibit the N-type Ca²⁺ channel (17). Conversely, the findings that Gβ₃- and Gβ₄-containing heterotrimers reconstituted coupling are at odds with the results of Garcia et al. (17), who reported that overexpression of Gβ₃ or Gβ₄ (either alone or with Gγ) produced no significant tonic inhibition of N-type Ca²⁺ channels. Results from our laboratory, however, indicate that both Gβ₃- and Gβ₄-containing Gβγ subunits produce robust Ca²⁺ channel modulation when overexpressed in SCG neurons (V. Ruiz-Velasco and S.R.I., unpublished data). It is possible that different levels of expression underlie these discrepant findings. In contrast to the minimal effects of altering the Gβγ composition, changing the Gα subunit resulted in large effects on NE-mediated coupling efficiency. Expression of either isoform of Gα_o resulted in Ca²⁺ channel inhibition similar to that observed under native conditions. Conversely, expression of Gα_i2 produced significant but less inhibition (when compared with Gα_o) whereas expression of Gα_i1 or Gα_i3 failed to reconstitute coupling. In general, studies of α_2A-ARs in clonal cells lines have shown that the receptor couples to all three Gα_i subtypes, Gα_oA, and even Gα_s/Gα_q (42, 46, 47). Thus, the apparent Gα specificity demonstrated in sympathetic neurons is of interest. There are two possible junctures at which specificity could occur. First, heterotrimers containing Gα_i1/3 may not couple to α₂-AR in SCG neurons, suggesting that additional factors present in the neuronal expression system restrict receptor/G protein interactions. Although it is possible that Gα_i1/3 requires a Gβγ combination other than Gβ₁γ₂ to effectively couple to α₂-ARs, this seems unlikely because Gα_i1β₁γ₂ effectively couples to α_2A-AR in in vitro systems (14). Second, heterotrimers containing Gα_i1/3 may couple to receptors but be incapable of producing VD modulation of N-type Ca²⁺ channels after receptor activation. In support of this idea, Delmas et al. (41) have shown that expression of the PTX-insensitive Gα_i mutant, Gα_i1(C351I), in SCG neurons failed to reconstitute VD N-type Ca²⁺ channel modulation by NE. Interestingly, the expression of Gα_i1(C351I) coupled adrenergic receptors to a voltage-independent pathway, which probably would not be observed under our experimental conditions (whole-cell vs. perforated patch recordings). Because Gα_i1/3 containing heterotrimers did not successfully reconstitute Ca²⁺ channel inhibition by any receptor tested in our experiments, we were unable to distinguish between these two possibilities.

Finally, we have shown that different, natively expressed receptors couple with varying degrees of efficiency to heterotrimers containing Gβ₁γ₂ and different PTX-insensitive Gα subunits. Here, the term “efficiency” is used in a descriptive sense and without mechanistic implications—i.e., the observed differences in coupling efficiency might have arisen from alterations in agonist efficacy, potency, or both. Our results demonstrate that the apparent coupling preferences displayed by these receptors could not have arisen solely from differences in the magnitude of G protein expression. Because all of these experiments were performed with Gβ₁γ₂, it is not clear whether altering Gβγ subunits would convey another level of specificity. However, it is clear from these data that the identity of Gα subunit influences coupling between receptors and N-type Ca²⁺ channels. Taken together, these data present a picture of G protein-coupling specificity that falls somewhere between the promiscuous coupling observed in biochemical studies and the exquisitely selective coupling reported from antisense knockout studies. It is easy to envision how modest differences in the expression levels of G protein subunits could convey functional selectivity greater than that observed for our heterologous studies. However, if receptor coupling to N-type Ca²⁺ channels in situ occurs via a specific heterotrimer composition (as has been proposed for L-type channels), then additional elements must exist to convey such specificity.

Abbreviations

α₂-AR

α₂-adrenergic receptor

NE

norepinephrine

PGE₂

prostaglandin E₂

PTX

pertussis toxin

SCG

superior cervical ganglion

VD

voltage-dependent