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The Journal of Neuroscience, June 15, 2000, 20(12):4489-4496

Endogenous Regulator of G-Protein Signaling Proteins Modify N-Type Calcium Channel Modulation in Rat Sympathetic Neurons

Seong-Woo Jeong and Stephen R. Ikeda

Laboratory of Molecular Physiology, Guthrie Research Institute, Sayre, Pennsylvania 18840

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Experiments using heterologous overexpression indicate that regulator of G-protein signaling (RGS) proteins play important roles in G-mediated ion channel modulation. However, the roles subserved by endogenous RGS proteins have not been extensively examined because tools for functionally inhibiting natively expressed RGS proteins are lacking. To address this void, we used a strategy in which G_oA was rendered insensitive to pertussis toxin (PTX) and RGS proteins by site-directed mutagenesis. Either PTX-insensitive (PTX-i) or both PTX- and RGS-insensitive (PTX/RGS-i) mutants of G_oA were expressed along with G₁ and G₂ subunits in rat sympathetic neurons. After overnight treatment with PTX to suppress natively expressed G subunits, voltage-dependent Ca²⁺ current inhibition by norepinephrine (NE) (10 µM) was reconstituted in neurons expressing either PTX-i or PTX/RGS-i G_oA. When compared with neurons expressing PTX-i G_oA, the steady-state concentration-response relationships for NE-induced Ca²⁺ current inhibition were shifted to lower concentrations in neurons expressing PTX/RGS-i G_oA. In addition to an increase in agonist potency, the expression of PTX/RGS-i G_oA dramatically retarded the current recovery after agonist removal. Interestingly, the alteration in current recovery was accompanied by a slowing in the onset of current inhibition. Together, our data suggest that endogenous RGS proteins contribute to membrane-delimited Ca²⁺ channel modulation by regulating agonist potency and kinetics of G-protein-mediated signaling in neuronal cells.

Key words: calcium channel; G-protein; G; G; intranuclear injection; RGS protein; sympathetic neuron; voltage-dependent inhibition

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Biochemical studies indicate that members of the regulator of G-protein signaling (RGS) family of proteins serve as GTPase-activating proteins (GAPs) for G_i and G_q but not for G_s (for review, see Dohlman and Thorner, 1997; Koelle, 1997; Berman and Gilman, 1998). Recent evidence implicates RGS proteins as key components in G-mediated modulation (Wickman and Clapham, 1995; Ikeda and Dunlap, 1999) of inwardly rectifying K⁺ (GIRK) and N-type Ca²⁺ channels. The potential roles of RGS proteins in G-mediated ion channel modulation were deduced from the discrepancy between the fast kinetics of modulation in vivo and the relatively slow intrinsic GTPase activities of G_o/i in vitro. For example, after agonist removal, muscarinic (M₂) receptor-activated GIRK currents in atrial myocytes deactivate ~40-fold faster than the GTP hydrolysis rate of pertussis toxin (PTX)-sensitive G subunits in vitro (Breitwieser and Szabo, 1988; Kurachi, 1995; Shui et al., 1995). Logically, these discrepancies might be reconciled by invoking the ability of natively expressed RGS proteins to stimulate the intrinsic GTPase of G subunits thereby accelerating termination of G-mediated signaling by favoring reformation of the heterotrimeric state. In support of this notion, heterologous expression of RGS proteins has been shown to accelerate the activation and deactivation kinetics of GIRK currents in Xenopus oocytes (Doupnik et al., 1997; Saitoh et al., 1997, 1999; Kovoor et al., 2000). In addition, heterologous expression of RGS proteins have been shown to alter the magnitude and kinetics of N-type Ca²⁺ channel inhibition (Jeong and Ikeda, 1998; Melliti et al., 1999). To date, however, the ability of natively expressed RGS proteins to influence G-mediated ion channel modulation in neurons has not been established primarily because few tools are available for inhibiting endogenous RGS proteins.

In the present study, we addressed this void in our knowledge by developing a strategy based on a combination of previously identified point mutations in G subunits. First, G_oA was rendered insensitive to the actions of PTX by mutating the C-terminal cysteine in which PTX-mediated ADP ribosylation occurs (Milligan, 1988). In this way, natively expressed G subunits could be uncoupled from receptors by PTX treatment, thereby isolating the action of the mutated G subunit (Taussig et al., 1992; Hunt et al., 1994; Senogles 1994; Kozasa et al., 1996; Jeong and Ikeda, 2000). Second, point mutations were introduced into the switch I and II regions of G_oA that have been identified previously to prevent RGS binding and GAP activity without altering GDP release and basal GTP hydrolysis in other G subunits (Lan et al., 1998; Natochin and Artemyev, 1998a) (but see DiBello et al., 1998). Using this strategy, we examined alterations in the magnitude and kinetics of voltage-dependent (VD) N-type Ca²⁺ current inhibition reconstituted in PTX-treated rat sympathetic neurons expressing the G_oA mutants. Our results indicate that endogenous RGS proteins in sympathetic neurons play an integral role in controlling the magnitude and speed of neurotransmitter-induced N-type Ca²⁺ channel modulation.

Parts of this paper have been published previously in abstract form (Jeong and Ikeda, 1999a).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Preparation of sympathetic neurons. Superior cervical ganglion (SCG) neurons were enzymatically dissociated as described previously (Ikeda, 1997). Briefly, adult (200-350 gm) male Wistar rats were decapitated using a laboratory guillotine. The SCG were dissected free of the carotid bifurcation and placed in cold (4°C) HBSS. The ganglia were desheathed, cut into small pieces, and transferred to the oxygenated Earle's balance salt solution (EBSS), pH 7.4, containing 0.6 mg/ml collagenase type D (Boehringer Mannheim, Indianapolis, IN), 0.4 mg/ml trypsin (TRL type; Worthington, Lakewood, NJ), and 0.1 mg/ml DNase Type I (Sigma, St. Louis, MO) in a 25 cm² tissue culture flask. The EBSS was modified by adding 3.6 gm/l glucose and 10 mM HEPES. After incubation for 60 min in a water bath shaker at 35°C, neurons were dispersed by vigorous shaking of the flask. After centrifugation twice for 6 min at 50 × g, the neurons were resuspended in Minimum Essential Medium (Mediatech, Inc., Herndon, VA) containing 10% fetal calf serum (Atlanta Biologicals, Atlanta, GA) and 1% glutamine/penicillin-streptomycin solution (Life Technologies, Grand Island, NY). Neurons were then 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. As appropriate, neurons were incubated overnight (16-20 hr) with 500 ng/ml PTX (List Biologic, Campbell, CA).

Construction and expression of RGS-insensitive mutants of G subunits. Previously, we have generated a PTX-insensitive G_oA (PTX-i G_oA) by introducing a cysteine (C) to glycine (G) mutation in the residue 4 from the C terminus (C351G) (Jeong and Ikeda, 2000). Using PTX-i G_oA as a template, additional point mutations (DiBello et al., 1998; Lan et al., 1998; Natochin and Artemyev, 1998a) were introduced to render G_oA both PTX- and RGS-insensitive (PTX/RGS-i G_oA). The following mutations were introduced into G_oA(C351G) using the GeneEditor site-directed mutagenesis kit (Promega, Madison, WI) per instructions of the manufacturer: G184S, S207D, and G184S/S207D. The following primers were used in constructing the mutations: G184S, 5'-GTCAAAACAACTTCCATCGTAGAAACCCAC-3'; and S207D, 5'-TCGGGGGCCAGCGAGATGAACGCAAGAAGTG-3'. The sequence of each construct was verified with an automated DNA sequencer (ABI 310; Applied Biosystems, Foster City, CA). Either PTX-i G_oA or PTX/RGS-i G_oA subunits were coexpressed with G₁ and G₂ subunits (denoted G₁₂, hereafter) by intranuclear microinjection as described previously (Ikeda, 1996, 1997). All G-protein subunits were expressed from the cytomegalovirus promoter-driven vector, pCI (Promega). Neurons were used within 24 hr after intranuclear injection of vectors. Injected neurons were identified by fluorescence from coexpressed jellyfish green fluorescent protein (EGFP; Clontech, Palo Alto, CA).

Electrophysiology. Ca²⁺ channel currents were recorded using the whole-cell variant of the patch-clamp technique (Hamill et al., 1981) as described previously (Ikeda, 1991; Ikeda et al., 1995). To isolate Ca²⁺ currents, patch electrodes were filled with a solution containing (in mM); 120 N-methyl-D-glucamine methanesulfonate (MS), 20 tetraethylammonium (TEA)-MS, 20 HCl, 11 EGTA, 1 CaCl₂, 10 HEPES, 4 MgATP, 0.3 Na₂GTP, and 14 creatine phosphate, pH 7.2 (297 mOsm/kg H₂O). The external recording solution contained (in mM): 145 TEA-MS, 10 HEPES, 10 CaCl₂, 15 glucose, and 0.0003 tetrodotoxin, pH 7.4 (318 mOsm/kg H₂O). All experiments were performed at room temperature (21-24°C). Norepinephrine (NE) was applied to single neurons via a gravity-fed fused silica capillary tube connected to an array of seven polyethylene tubes. The outlet of the perfusion system was located within 100 µm of the cell. Drug application was started by switching the control external solution to a drug solution. Complete solution exchange occurred within <1 sec.

Data analysis. The concentration-response curves were plotted as values normalized to the inhibition produced at the maximal [NE] (30 µM). The concentration-response curves were fit to the following Hill equation: I = 1/[1 + (k/[NE])ⁿ], where I, k, [NE], and n are normalized inhibition, a constant, NE concentration, and Hill factor, respectively. The EC₅₀ values were calculated using the following relationship: EC₅₀ = kⁿ. The "on-off" times of the NE-induced Ca²⁺ current inhibition were approximated by fitting a polynomial function to the time courses and interpolating the appropriate values (i.e., t_0.5 and t_rise). All curve fitting was performed with the IGOR PRO data analysis package (WaveMetrics, Lake Oswego, OR). Data are presented as means ± SEM. Student's t test (unpaired) or ANOVA followed by a post hoc Dunnett's test, as appropriate, were applied to the data to determine statistical significance. p < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reconstitution of NE-induced Ca²⁺ current inhibition after expression of PTX- and RGS-insensitive G_oA subunits

As a first step toward elucidating the potential roles of endogenous RGS proteins in Ca²⁺ channel modulation, we tested whether heterologous expression of PTX/RGS-i G_oA subunits reconstituted NE-induced VD Ca²⁺ current inhibition in SCG neurons after uncoupling of endogenous G_o/i-proteins by PTX treatment. G_oA was selected to be rendered RGS-insensitive because G_o has been implicated as the primary signaling element coupling ₂-adrenergic receptors (₂-ARs) to N-type Ca²⁺ channels (Caulfield et al., 1994) in sympathetic neurons.

Figure 1 illustrates the effects of PTX/RGS-i G_oA subunits on NE-induced Ca²⁺ current inhibition when expressed in SCG neurons. 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 (Fig. 1A). In uninjected control neurons, NE (10 µM) inhibited Ca²⁺ currents by 63 ± 2% (n = 30) (Figs. 1A, 2). NE-induced Ca²⁺ current inhibition displayed the hallmarks of VD inhibition (Ikeda and Dunlap, 1999), i.e., slowed activation kinetics in the prepulse and an enhanced postpulse amplitude (Figs. 1A, 2). Prepulse facilitation ratio (PFR), defined as the ratio of the postpulse to prepulse current amplitude, increased from 1.20 ± 0.02 to 2.40 ± 0.10 (n = 30) after NE application. This form of N-type Ca²⁺ channel modulation has been shown to be mediated by the G subunit (Herlitze et al., 1996; Ikeda, 1996).

View larger version (39K): [in this window] [in a new window]   Figure 1.   Reconstitution of 2-AR coupling to N-type Ca2+ channels by expression of PTX/RGS-i GoA mutants. Time courses of current inhibition and superimposed current traces in the absence (a, c) or presence (b, d) of 10 µM NE recorded from uninjected control neurons (no PTX) (A), uninjected PTX-treated control neurons (500 ng/ml PTX, overnight) (B), PTX-treated neurons expressing a PTX-i GoA mutant, GoA(C351G) (C), and PTX-treated neurons expressing PTX/RGS-i GoA mutants, GoA(G184S: C351G) (D), GoA(S207D: C351G) (E), or GoA(G184S: S207D: C351G) (F). The cDNAs encoding G mutants and G12 were directly injected into nuclei of SCG neurons. The Ca2+ current was evoked every 10 sec 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 prepulses (filled circles) and postpulses (open circles) were plotted. Note that the expression of RGS-insensitive G mutants retarded onset of the steady-state current inhibition and the current recovery after agonist withdrawal.

View larger version (35K): [in this window] [in a new window]   Figure 2.   Summary of prepulse facilitation and Ca2+ current inhibition in the presence of NE. Prepulse facilitation was calculated by the ratio of the postpulse to prepulse current amplitude measured isochronally at 10 msec after the start of the test pulses. Inhibition (percentage) was calculated using the amplitudes of currents determined isochronally 10 msec after the start of the prepulse. Data are presented as mean ± SEM, and numbers in parentheses indicate the number of neurons tested. ANOVA (p < 0.0001) followed by Dunnett's t test; uninjected control versus PTX control, CG, GS:CG, SD:CG, and GS:SD:CG. *p < 0.05; **p < 0.01.

Overnight treatment of the neurons with PTX (500 ng/ml) greatly attenuated NE-induced Ca²⁺ current inhibition (8 ± 1%, n = 25) (Figs. 1B, 2), confirming the involvement of a PTX-sensitive G-protein(s) in the signaling pathway (Hille, 1994). To reconstitute NE-induced Ca²⁺ current inhibition in PTX-treated neurons, either PTX-i G_oA or PTX/RGS-i G_oA subunits were coexpressed with G₁₂ in SCG neurons using an intranuclear microinjection technique. As shown previously (Jeong and Ikeda, 2000), "balanced" expression of G_oA mutants and G₁₂ was critical for successful reconstitution on the time scale used in these experiments (<24 hr after injection of cDNA). Briefly, if expression of G mutants greatly exceeded that of G, basal facilitation and NE-induced Ca²⁺ current inhibition were ablated. This effect likely reflects sequestration of free G subunits (both endogenous and exogenous) by the GDP-bound G (Ikeda, 1996; Jeong and Ikeda 1999b). In contrast, if expression of G exceeded that of G, a significant tonic inhibition (as indicated by increased basal facilitation) was observed. This result presumably reflects interaction of "free" or excess G with N-type Ca²⁺ channels (Herlitze et al., 1996; Ikeda, 1996; García et al., 1998; Ruiz-Velasco and Ikeda, 2000). Thus, we have defined a basal PFR range of 1.02-1.50 as indicating adequate balance between PTX-i G_oA and G subunits (Jeong and Ikeda, 2000). In general, we applied the same criteria for PTX/RGS-i G_oA mutants. In a few neurons expressing G_oA(G184S:C351G), however, reconstitution of Ca²⁺ current inhibition occurred with basal PFR of <1. NE (10 µM) inhibited Ca²⁺ currents by 52 ± 3% (n = 9) when G_oA(C351G) was coexpressed with G₁₂ in PTX-treated neurons. The characteristics of the Ca²⁺ current inhibition reconstituted by the PTX-i G_oA were basically similar to those of uninjected controls neurons in terms of voltage dependence of the inhibition (PFR increased from 1.15 ± 0.05 to 1.90 ± 0.09 after NE application) and relatively rapid on-off kinetics of NE action. These results are consistent with previous findings using the G_oA(C351G) mutant (Jeong and Ikeda, 2000). Similarly, coexpression of the PTX/RGS-i G_oA subunits G_oA(G184S:C351G) or G_oA(S207D:C351G), along with G₁₂, resulted in successful reconstitution of NE-induced Ca²⁺ current inhibition (Fig. 1). These results demonstrate that introduction of the additional point mutations did not affect the ability of G_oA(C351G) to form heterotrimers or couple receptors to Ca²⁺ channels. On average, NE (10 µM) inhibited Ca²⁺ currents by 69 ± 1 (n = 6) and 70 ± 2% (n = 8) in neurons expressing G_oA(G184S:C351G) and G_oA(S207D:C351G), respectively (Fig. 2). It should be noted that these magnitudes of inhibition were significantly larger than those of uninjected or PTX-i G_oA-expressing neurons (p < 0.05). The voltage dependence of the reconstituted Ca²⁺ current inhibition remained intact as evidenced by the kinetic slowing of current activation and increase in PFR after NE application [1.01 ± 0.02 to 2.23 ± 0.06 for G_oA(G184S:C351G); 1.19 ± 0.05 to 2.26 ± 0.07 for G_oA(S207D:C351G)] (Fig. 2). However, the most striking feature produced by expression of the PTX/RGS-i G_oA subunits was a sluggish recovery from inhibition after agonist removal (Fig. 1D,E). Interestingly, the onset of agonist action also appeared slower when compared with control neurons (see Fig. 4). Coexpression of RGS8 (1 ng/µl DNA) with G_oA(G184S:C351G) in a limited number of neurons (n = 2) resulted in qualitatively similar results to those obtained in neurons expressing only G_oA(S207D:C351G) (data not shown). In particular, the recovery from inhibition after agonist removal remained very slow, indicating an inability of RGS8 to reverse this effect. In addition to G_oA(G184S:C351G) and G_oA(S207D:C351G), we generated a construct that contained both the G184S and S207D mutations. When expressed along with G₁₂, G_oA(G184S:S207D:C351G) reconstituted VD Ca²⁺ current inhibition (62 ± 2%; PFR, 2.37 ± 0.07, n = 7) (Figs. 1F, 2). The time course of current recovery after agonist removal was dramatically retarded in neurons expressing this mutant. It is not clear whether this effect reflects an intrinsically greater "resistance" to the effects of RGS proteins or a secondary effect of the combined mutations. Together, these data demonstrate that expression of the PTX/RGS-i G_oA subunits (together with G₁₂) reconstituted functional coupling of ₂-ARs to N-type Ca²⁺ channels in PTX-treated neurons. In addition, the data are consistent with biochemical data showing that the G184S and S207D mutations confer resistance to the GAP effects of exogenous RGS proteins. Thus, additional studies were undertaken to quantify the alterations in Ca²⁺ channel inhibition and time course.

Expression of PTX/RGS-i G_oA subunits produces a leftward shift in concentration-response curves

As noted above, Ca²⁺ current inhibitions produced by a single concentration of NE (10 µM) were modestly enhanced in neurons expressing PTX/RGS-i G_oA subunits when compared with neurons expressing PTX-i G_oA. These results are consistent with previous studies demonstrating that increasing levels of RGS proteins attenuate agonist-induced Ca²⁺ current inhibition in SCG neurons (Jeong and Ikeda, 1998) and HEK293 cells (Melliti et al., 1999). In the latter study, rightward shifts in steady-state concentration-response curves were observed after heterologous expression of RGS proteins. Therefore, it was predicted that expression of PTX/RGS-i G_oA subunits would shift concentration-response curves in the opposite direction, i.e., leftward, if the G subunit mutations interfered with RGS protein interactions. To test this possibility, we measured Ca²⁺ current inhibition over a wide range of NE concentrations (0.1-30 µM). Figure 3A illustrates representative time courses of the current inhibition in neurons expressing G_oA(C351G) and G_oA(G184S:C351G) after sequential application of increasing concentrations of NE. Figure 3B summarized the steady-state concentration-response curves for neurons expressing PTX-i G_oA (control) or PTX/RGS-i G_oA. When compared with uninjected neurons (non-PTX-treated), NE-induced Ca²⁺ current inhibition in neurons expressing PTX-i G_oA, i.e., G_oA(C351G), was approximately twofold less potent but equally efficacious (data not shown). In neurons expressing PTX/RGS-i G_oA subunits, the steady-state concentration-response curves were shifted leftward with a rank order of potency (EC₅₀) as follows: G_oA(S207D:C351G) (0.28 µM, n = 5) > G_oA(G184S:C351G) (0.41 µM, n = 6) > G_oA(G184S:S207D:C351G) (0.50 µM, n = 4) > G_oA(C351G) (2.35 µM, n = 4). The maximal inhibitions produced by NE (30 µM) in neurons expressing G_oA(C351G), G_oA(G184S:C351G), G_oA(S207D:C351G), and G_oA(G184S:S207D:C351G) were 52 ± 6, 67 ± 1, 63 ± 3, and 55 ± 2%, respectively. In addition, the Hill factor for the concentration-response curves ranged between 1.5 and 1.8. Together, these data indicate that endogenous RGS proteins influence the agonist potency, and to a minor extent, efficacy, in sympathetic neurons.

View larger version (25K): [in this window] [in a new window]   Figure 3.   Expression of PTX/RGS-i GoA mutants shifts concentration-response relationships of NE-induced Ca2+ current inhibition to lower concentrations. A, Representative time courses of Ca2+ current inhibitions acquired from sequential applications of NE over a wide range of concentrations (0.1-30 µM) in neurons expressing either GoA(C351G) or GoA(G184S: C351G). Currents were evoked by single 50 msec test pulses to +10 mV from a holding potential of 80 mV. B, Average concentration-response curves in neurons expressing either PTX-i GoA or PTX/RGS-i GoA mutants. Inhibition (percentage) was normalized to that at the maximal NE concentration (30 µM). Data are presented as mean ± SEM, and numbers in parentheses indicate the number of neurons tested. The concentration-response curves were fit to the Hill equation: I = 1/[1 + (k/[NE])n], where I, k, [NE], and n are normalized inhibition, a constant, NE concentration, and Hill factor, respectively. The n values for GoA(C351G), GoA(G184S: C351G), GoA(S207D: C351G), and GoA(G184S: S207D: C351G) were 1.5, 1.8, 1.6, and 1.7, respectively.

Expression of PTX/RGS-i G_oA subunits alters the kinetics of NE-induced Ca²⁺ current inhibition

It has been well established that RGS proteins, acting as GAPs, accelerate G-catalyzed GTP hydrolysis in vitro (for review, see Dohlman and Thorner, 1997; Koelle, 1997; Berman and Gilman, 1998). These biochemical results implicate RGS proteins in controlling the kinetics of G-protein signaling. In recent electrophysiological studies, heterologously expressed RGS proteins have been shown to accelerate recovery of Ca²⁺ channels from the inhibition mediated by G_z (Jeong and Ikeda, 1998) and G_o/i (Melliti et al., 1999), and deactivation of GIRK channels (Doupnik et al., 1997; Saitoh et al., 1997; Kovoor et al. 2000). As described in Figure 1, the recovery of Ca²⁺ channels from inhibition was slowed, consistent with a disruption of endogenous RGS actions on expressed PTX/RGS-i G_oA subunits. Therefore, additional experiments were undertaken to quantify these effects. Figure 4A illustrates time courses of NE-induced Ca²⁺ current inhibition onset and recovery in neurons expressing PTX-i G_oA (control) or PTX/RGS-i G_oA subunits. For these experiments, the Ca²⁺ currents were evoked every 2 or 5 sec by single 20 msec test pulses to +10 mV from a holding potential 80 mV. In neurons expressing PTX-i G_oA, NE (10 µM) produced a rapid onset of inhibition and current recovery after agonist removal similar to that observed in uninjected neurons (Fig. 4C) (Zhou et al., 1997). Conversely, the current recovery was dramatically retarded in neurons expressing PTX/RGS-i G_oA (Figs. 4A,B). To compare the time course of current recovery, the half recovery time (t_0.5) was determined for the relaxation phase after agonist removal (Fig. 4B). A time constant was not calculated because the recovery time course under these circumstances was clearly nonexponential. The mean t_0.5 for G_oA(C351G) (control), G_oA(G184S:C351G), G_oA(S207D:C351G), and G_oA (G184S:S207D:C351G) -expressing neurons was 9 ± 1 (n = 19), 59 ± 5 (n = 16), 47 ± 5 (n = 14), and 130 ± 13 sec (n = 10), respectively (Fig. 4C, top). Interestingly, decreases in the off rate seemed to be accompanied by decreases in the on rate. Indeed, the onset of the steady-state current inhibition was significantly retarded in neurons expressing PTX/RGS-i G_oA when compared with neurons expressing PTX-i G_oA (Fig. 4A). Because the rate of current inhibition onset was relatively rapid in relationship to the frequency of test pulses (therefore limiting temporal resolution), the onset of the NE-induced Ca²⁺ current inhibition was estimated as a 10-90% rise time (t_rise) instead of t_0.5. As summarized in Figure 4C, mean t_rise for G_oA(C351G), G_oA(G184S:C351G), G_oA(S207D:C351G), and G_oA(G184S:S207D:C351G) was 3.4 ± 0.1, 15.0 ± 3.0, 8.0 ± 1.7, and 11.8 ± 1.3 sec, respectively. It should be noted that the slowing of current inhibition onset was not proportional to the slowing of current recovery. Together, these data suggest that endogenous RGS proteins play an important role in controlling the lifetime of Ca²⁺ current inhibition after receptor activation in neurons.

View larger version (37K): [in this window] [in a new window]   Figure 4.   Effects of expression of PTX/RGS-i GoA mutants on kinetics of NE-mediated current inhibition. A, Time courses of NE-induced Ca2+ current inhibitions and the current recovery after agonist removal when neurons heterologously expressed GoA(C351G) (top), GoA(G184S: C351G) (middle), or GoA(G184S: S207D: C351G) (bottom), along with G12. The currents were evoked every 2 sec by single 20 msec test pulses to +10 mV from a holding potential of 80 mV. The superimposed current traces in the insets represent those before (a) and during (b) agonist applications. B, Comparison of the current recovery rates after removal of agonist among GoA(C351G), GoA(G184S:C351G), or GoA(G184S:S207D:C351G). The current amplitudes were normalized, and the dashed line indicates complete recovery to the current amplitude before agonist application. C, Summary of half recovery time (t0.5) and onset time (trise) for the steady-state current inhibition. The trise was estimated as a 10-90% rise time. Both parameters were approximated by fitting a polynomial function to the time courses and interpolating the appropriate values (i.e., t0.5 and trise). Curve fitting were performed with the IGOR data analysis package (WaveMetrics). Data are presented as mean ± SEM, and numbers in parentheses indicate the number of neurons tested. ANOVA (p < 0.0001) followed by Dunnett's t test; uninjected control versus CG, GSCG, SDCG, and GSSDCG. *p < 0.05; **p < 0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Increasing evidence suggests that RGS proteins play an integral role in G-protein signaling. To date, however, information regarding the functional roles of RGS proteins is limited primarily to in vitro biochemical assays and heterologous overexpression experiments. Thus, the role played by natively expressed RGS proteins in G-protein signaling remains unclear. A reason for this void in our knowledge is the lack of experimental tools for uncoupling endogenous RGS proteins from G-protein signaling pathways. Thus, the development of the better tools represents a major challenge for understanding the physiological roles subserved by RGS proteins. In this regard, several potential strategies are apparent. First, genetically ablating specific RGS proteins, either acutely (e.g., antisense techniques) or stably (e.g., knock-out mice), might lend insight into endogenous RGS protein functions. However, >20 mammalian RGS proteins have been identified so far, and it is likely that even single cells contain multiple different RGS protein subtypes (Gold et al., 1997; Kardestuncer et al., 1998). Moreover, of the RGS proteins studied to date, only limited G specificity (i.e., G_i/o and G_q families) was observed in regard to GAP activity (for review, see Dohlman and Thorner, 1997; Koelle, 1997; Berman and Gilman, 1998). Thus, it seems likely that redundancy of RGS protein actions might confound studies in which single RGS proteins are eliminated. In one study, however, dialysis of neurons with an antibody directed at the RGS4 C terminus appeared to produce a specific result (Diverse-Pierlussi et al., 1999), indicating that this approach may be a fruitful one. A second strategy is to produce a dominant negative mutant RGS protein. Unfortunately, the means of creating such a mutant are not apparent, because mutations that alter GAP activity also disrupt G binding (Srinivasa et al., 1998). Finally, it might be possible to construct a mutant G subunit that sequesters RGS proteins. It has been shown that RGS4 interacts with a GTPase-deficient mutant of G_i1, G_i1(R178C) (Berman et al., 1996b). In preliminary experiments, however, expression of G_oA(R178C) resulted in apparent sequestration of G (Ikeda, 1996; Jeong and Ikeda, 1999b), presumably arising from a population of GDP-bound G_oA(R178C) (S.-W. Jeong and S. R. Ikeda, unpublished data).

The strategy used in this study originated from the seminal findings of several groups. First, a founding member of the RGS family, SST2, has been shown to desensitize G-mediated pheromone signaling via direct interaction with GPA1, a G subunit in Saccharomyces cerevisiae (Dohlman et al., 1996). Biochemical and functional observations demonstrated that a GPA1 mutant (gpa1^sst) phenotypically mimicked the loss of SST2, suggesting that the mutant was insensitive to RGS GAP activity (DiBello et al., 1998). Subsequent sequencing of the G mutant revealed that Gly302, a conserved amino acid in the G family, was mutated to Ser. Homologous mutations in mammalian G subunits (G_o/i and G_q) resulted in an equivalent phenotype, thus demonstrating the generality of the substitution (DiBello et al., 1998; Lan et al., 1998). Second, mutation of Ser202 to Asp in transducin was shown to abolish interaction with a human retinal RGS isoform, hRGSr, in vitro (Natochin and Artemyev, 1998a,b; Posner et al., 1999). Mutation of Ser202 was arrived at by comparing the sequences of RGS-sensitive (i.e., G_i/o and G_q/11) and -insensitive G subunits (i.e., G_s and G₁₂) (Berman et al., 1996a; Huang et al., 1997). A problem with using RGS-insensitive G mutants for investigating the functional roles of endogenous RGS proteins in neurons is suppressing the activity of endogenous G subunits. To this end, we adopted the method used by several laboratories (Taussig et al., 1992; Hunt et al., 1994; Senogles 1994; Kozasa et al., 1996) and subsequently studied in detail by Milligan and colleagues (Wise et al., 1997; Bahia et al., 1998) in which G subunits are rendered PTX-insensitive by mutating a Cys residue in the C terminus. We have shown previously that heterologously expressed G-protein heterotrimers (Gs) containing PTX-i G subunits functionally replace (as determined from VD Ca²⁺ channel inhibition) natively expressed G-proteins (G_o/i) after PTX treatment of SCG neurons (Jeong and Ikeda, 2000). Thus, the strategy we used combined these two sets of mutations.

Success in using this method relied on reconstituting VD Ca²⁺ channel inhibition in PTX-treated neurons expressing PTX/RGS-i G_oA subunits. Because the crystal structure of G_i1-GDP-AlF₄ indicates that the mutated Gly and Ser residues are located near G contact sites within the switch I and II regions, respectively (Lambright et al., 1996), there was concern that heterotrimer formation might be disrupted by the mutations. Preliminary experiments, however, indicated that overexpression of PTX/RGS-i G_oA (without coexpression of G) attenuated NE-induced Ca²⁺ current inhibition in a manner consistent with the sequestration of G subunits (data not shown) (Ikeda, 1996; Jeong and Ikeda, 1999b), thus providing evidence for in situ heterotrimer formation. Consistent with this observation, successful reconstitution of VD Ca²⁺ channel inhibition was observed for all PTX/RGS-i G_oA subunits (Fig. 1) whenever a functional stoichiometric match (Jeong and Ikeda, 2000) between PTX/RGS-i G_oA and G₁₂ subunits was achieved.

Major features of the NE-induced Ca²⁺ current inhibition reconstituted in neurons expressing PTX/RGS-i G_oA subunits were (1) a leftward shift in the NE concentration-response relationships, (2) a dramatically retarded recovery time course after agonist removal, and (3) an increase in the time to reach steady-state current inhibition after agonist application. Some of these results can be rationalized by the failure of endogenous RGS proteins to interact with PTX/RGS-i G_oA subunits (in the transition conformation) and, consequently, the resulting loss of RGS-mediated GTPase acceleration. In this situation, G-protein heterotrimer reformation is limited by the relatively slow intrinsic GTPase activities of G_oA subunits (Higashijima et al., 1987; Lan et al., 1998). Consequently, the concentration of free Gs around the Ca²⁺ channel would remain relatively high for a prolonged time after agonist removal, thus retarding recovery of channels from inhibition. Moreover, because steady-state concentrations of G-GTP (and hence free G) should be determined by the relative rates of G-GTP formation and destruction, the increase in agonist potency and modest increase in efficacy can be accommodated by this interpretation as well. These results are consistent with several previous studies in which RGS actions were increased by heterologously overexpressing RGS proteins. For example, expression of RGS4 in COS-7 cells produced a rightward shift in the concentration-response relationship for activation of mitogen-activated protein kinase (Yan et al., 1997). In HEK293 cells, Melliti et al. (1999) showed that expression of RGS3T, RGS3, and RGS8 altered the magnitude and kinetics of N-type Ca²⁺ channel inhibition, as well as produced a rightward shift of the steady-state concentration-response curve. In SCG neurons, Jeong and Ikeda (1998) have shown that overexpression of RGS4 and RGS10 accelerate the recovery of NE-induced inhibition of Ca²⁺ channels reconstituted with G_z. In that study, overexpression RGS10 also attenuated NE-induced Ca²⁺ current inhibition in control SCG neurons, suggesting a rightward shift in the concentration-response curve. Conversely, RGS proteins appear to accelerate deactivation of GIRK currents without an apparent shift in the concentration-response relationship (Doupnik et al., 1997; Saitoh et al., 1997). Although the reason for this difference remains unclear, it possibly suggests that, in addition to GAP functions, RGS proteins subserve other roles, such as increasing G availability (Bünemann and Hosey, 1998) or effecting receptors/G-proteins coupling (Zeng et al., 1998; Xu et al., 1999).

The slowing of the on rate of steady-state Ca²⁺ current inhibition seen in this study is more difficult to explain within in the framework of established RGS functions. Multiple steps, such as agonist binding to receptor, receptor/G-protein interaction, GDP/GTP exchange rate, and G interaction with the channel, could contribute to this rate. A possibility that should be considered is that the G mutations used in our studies have additional actions, such as altering the receptor-mediated GDP/GTP exchange rate. However, in vitro experiments indicate that basal GDP/GTP exchange rates are not significantly altered by these G mutations (Lan et al., 1998). Moreover, the current results are consistent with previous studies in which overexpression of RGS proteins exerted opposite effects, i.e., acceleration of the onset of steady-state Ca²⁺ current inhibition (Jeong and Ikeda, 1998; Melliti et al. 1999) and GIRK activation (Doupnik et al., 1997; Saitoh et al., 1997). Two possibilities might explain this phenomenon. First, RGS proteins may directly or indirectly stimulate GDP/GTP exchange via unknown mechanisms, although at present there is little evidence to support this view (Dohlman and Thorner, 1997; Koelle, 1997; Berman and Gilman, 1998). Second, RGS proteins may affect the receptor/G-protein coupling independently of the GAP activity associated with the RGS core domain (Zeng et al., 1998; Xu et al., 1999), thereby altering the rate of Ca²⁺ channel inhibition. In this regard, it should be mentioned that the term RGS "insensitivity," in the current context, refers solely to the GAP activity of RGS proteins and does not preclude effects that may be conferred by domains lying outside of the RGS core domain. In fact, preliminary data indicate that the kinetics of GIRK channel activation/deactivation after expression of PTX/RGS-i G_oA subunits are altered by noncore RGS domains (Jeong and Ikeda, unpublished data).

Previously, Zhou et al. (1997) have shown that N-type Ca²⁺ channels in SCG neurons recovered from NE-induced inhibition after agonist removal with a rate constant (between 0.09 and 0.18 sec¹) much higher than that reported for the intrinsic GTP hydrolysis rate (~0.03 sec¹) of G_o determined in vitro (Higashijima et al., 1987). Our results indicate that the actions of endogenous RGS proteins might account for this discrepancy. Thus, in addition to receptors, G-proteins and N-type Ca²⁺ channels, RGS proteins must be added to the membrane-delimited pathway describing VD channel modulation. How RGS proteins contribute to the more integrative functions of N-type Ca²⁺ channels, such as presynaptic inhibition resulting from neurotransmitter release, remains to be determined. It is possible that the strategy used here, i.e., the combined use of PTX- and RGS-insensitive G subunits, will be useful in defining the functional roles of the endogenous RGS proteins in different systems.

	FOOTNOTES

Received Dec. 30, 1999; revised March 14, 2000; accepted April 7, 2000.

This study was supported by National Institutes of Health Grant GM 56180 (S.R.I.). We thank M. King for excellent technical assistance and Dr. M. I. Simon (California Institute of Technology, Pasadena, CA) for G-protein plasmids.

Correspondence should be addressed to Dr. Stephen R. Ikeda, Laboratory of Molecular Physiology, Guthrie Research Institute, One Guthrie Square, Sayre, PA 18840. E-mail: sikeda{at}inet.guthrie.org.

Dr. Jeong's present address: Department of Physiology, Yonsei University, Wonju College of Medicine, Wonju, Korea.

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				Q. Wang, M. Liu, B. Mullah, D. P. Siderovski, and R. R. Neubig Receptor-selective Effects of Endogenous RGS3 and RGS5 to Regulate Mitogen-activated Protein Kinase Activation in Rat Vascular Smooth Muscle Cells J. Biol. Chem., July 5, 2002; 277(28): 24949 - 24958. [Abstract] [Full Text] [PDF]

				J. Perroy, G. J. Gutierrez, V. Coulon, J. Bockaert, J.-P. Pin, and L. Fagni The C Terminus of the Metabotropic Glutamate Receptor Subtypes 2 and 7 Specifies the Receptor Signaling Pathways J. Biol. Chem., November 30, 2001; 276(49): 45800 - 45805. [Abstract] [Full Text] [PDF]

				S.-W. Jeong and S. R Ikeda Differential regulation of G protein-gated inwardly rectifying K+ channel kinetics by distinct domains of RGS8 J. Physiol., September 1, 2001; 535(2): 335 - 347. [Abstract] [Full Text] [PDF]

				H. Zhong and R. R. Neubig Regulator of G Protein Signaling Proteins: Novel Multifunctional Drug Targets J. Pharmacol. Exp. Ther., June 1, 2001; 297(3): 837 - 845. [Abstract] [Full Text]

				K. Melliti, U. Meza, and B. A Adams RGS2 blocks slow muscarinic inhibition of N-type Ca2+ channels reconstituted in a human cell line J. Physiol., April 15, 2001; 532(2): 337 - 347. [Abstract] [Full Text] [PDF]

				C. B. Cooper, M. I. Arnot, Z.-P. Feng, S. E. Jarvis, J. Hamid, and G. W. Zamponi Cross-talk between G-protein and Protein Kinase C Modulation of N-type Calcium Channels Is Dependent on the G-protein beta Subunit Isoform J. Biol. Chem., December 22, 2000; 275(52): 40777 - 40781. [Abstract] [Full Text] [PDF]

				K.-L. Lan, H. Zhong, M. Nanamori, and R. R. Neubig Rapid Kinetics of Regulator of G-protein Signaling (RGS)-mediated Galpha i and Galpha o Deactivation. Galpha SPECIFICITY OF RGS4 AND RGS7 J. Biol. Chem., October 20, 2000; 275(43): 33497 - 33503. [Abstract] [Full Text] [PDF]

				H. Chen and N. A. Lambert Endogenous regulators of G protein signaling proteins regulate presynaptic inhibition at rat hippocampal synapses PNAS, November 7, 2000; 97(23): 12810 - 12815. [Abstract] [Full Text] [PDF]

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