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The Journal of Neuroscience, June 15, 1999, 19(12):4755-4761

Sequestration of G-Protein Subunits by Different G-Protein  Subunits Blocks Voltage-Dependent Modulation of Ca²⁺ Channels 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

The membrane-delimited and voltage-dependent inhibition of N-type Ca²⁺ channels is mediated by G subunits. Previously, exogenous excess GDP-bound G_oA has been shown to dramatically attenuate the norepinephrine (NE)-mediated Ca²⁺ current inhibition by sequestration of G subunits in rat superior cervical ganglion (SCG) neurons. In the present study, we determined whether the attenuation of NE-mediated modulation is specific to G_oA or shared by a number of closely related (G_tr, G_oB, G_i1, G_i2, G_i3, G_z) or unrelated (G_s, G_q, G₁₁, G₁₆, G₁₂, G₁₃) G subunits. Individual G subunits from different subfamilies were transiently overexpressed in SCG neurons by intranuclear injection of mammalian expression vectors encoding the desired protein. Strikingly, all G subunits except G_z nearly blocked basal facilitation and NE-mediated modulation. Likewise, VIP-mediated Ca²⁺ current inhibition, which is mediated by cholera toxin-sensitive G-protein, was also completely suppressed by a number of G subunits overexpressed in neurons. G_s expression produced either enhancement or attenuation of the VIP-mediated modulationan effect that seemed to depend on the expression level. The onset of the nonhydrolyzable GTP analog, guanylylimidodiphosphate-mediated facilitation was significantly delayed by overexpression of different GDP-bound G subunits. Taken together, these data suggest that a wide variety of G subunits are capable of forming heterotrimers with endogenous G subunits mediating voltage-dependent Ca²⁺ channel inhibition. In conclusion, coupling specificity in signal transduction is unlikely to arise as a result of restricted G/G interaction.

Key words: calcium channels; G subunit; G subunit; G-proteins; voltage-dependent inhibition; intranuclear injection; sympathetic neuron; coupling specificity

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Modulation of N-type Ca²⁺ channels by neurotransmitters occurs via multiple pathways. The most common modulatory pathway involves activation of pertussis toxin (PTX)-sensitive heterotrimeric G-proteins, resulting in a distinct form of membrane-delimited and voltage-dependent inhibition (Hille, 1994). Recently, G subunits have been shown to mediate the voltage-dependent inhibition of Ca²⁺ currents (Herlitze et al., 1996; Ikeda, 1996). The idea that G subunits directly interact with Ca²⁺ channels has been supported by the molecular identification of potential G binding motifs on the intracellular I-II loop (De Waard et al., 1997; Zamponi et al., 1997; Furukawa et al., 1998) and C terminus (Qin et al., 1997). In addition, a recent study suggests that the N terminus of the Ca²⁺ channel 1 subunit is also essential for the G-protein-mediated modulation (Page et al., 1998).

With regard to the interaction with Ca²⁺ channels, there seem to be few functional differences among different G combinations. For example, the Ca²⁺ currents were tonically inhibited by various G combinations, including G₁₂, G₁₃, G₁₇ (Ikeda, 1996), and G₂₃ (Herlitze et al., 1996) when overexpressed in sympathetic neurons [but see García et al. (1998)]. These observations are similar to the finding that G-protein-activated inwardly rectifying K⁺ (GIRK) channels were activated by different G combinations, with the exception of ₁₁ (Wickman et al., 1994). Furthermore, various G subunits are involved in coupling receptors to ion channels. Activation of different receptors activate GIRK channels via different G proteins (Lim et al., 1995; Ruiz-Velasco and Ikeda, 1998). Likewise, although G_o appears to be dominantly coupled to receptors mediating the voltage-dependent inhibition of Ca²⁺ channels in neuronal tissues, G_i (Ewald, 1989; Toselli et al., 1989) and G_s (Zhu and Ikeda, 1994) can also participate in this pathway. Overall, these observations argue against the idea that coupling specificity resides at the G/effector level and thus requires restricted G/ combinations.

To confirm the modulatory role of G in the neurotransmitter-mediated inhibition of Ca²⁺ channel currents, the stoichiometry between G and G subunits has been disrupted by overexpressing G_oA in sympathetic neurons (Ikeda, 1996). In this experiment, excess GDP-bound G_oA significantly blocked NE-mediated Ca²⁺ current inhibition by creating conditions favoring heterotrimer formation. In the present study, the same strategy was used to test whether the block of norepinephrine (NE)-mediated inhibition is specific to G_oA or shared by members in the same (G_i, i.e., G_oB, G_i1, G_i2, G_i3, G_tr, and G_z) or different G subfamilies (G_s, G_q/11, and G₁₂). In superior cervical ganglion (SCG) neurons, the parallel pathways using PTX-sensitive G_o/i and cholera toxin (CTX)-sensitive G_s converge to the voltage-dependent modulation of Ca²⁺ channels (Zhu and Ikeda, 1994). Thus, a complementary set of experiments was performed to test whether VIP-mediated inhibition is blocked by overexpression of the G subunits described above. Our data showed that a wide variety of G subunits can interact with the G subunits involved in NE-, VIP-, and guanylylimidodiphosphate [Gpp(NH)p]-mediated current inhibition. These results suggest that coupling specificity is unlikely to arise as a result of restricted G/ interactions.

Some preliminary data have been published previously in abstract form (Jeong and Ikeda, 1997).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Vectors and chemicals. The clones for G-protein G_oA, G_oB, G_q, G₁₁, G₁₆, G₁₂, and G₁₃ were generously provided by Dr. M. I. Simon (California Institute of Technology). The clones for G_s and G_i1-i3 were kind gifts from Dr. R. Reed (Johns Hopkins Medical School). The clone for the wild-type G_z was a generous gift from Dr. H. R. Bourne (University of California San Francisco). The pEGFP-N1 N-terminal fusion vector was purchased from Clontech Laboratories (Palo Alto, CA). The vectors were propagated in either XL-1 Blue or MC1061/p3 Escherichia coli, (Stratagene, Cambridge, UK) as appropriate, and purified using Qiagen (Chatsworth, CA) miniprep or maxiprep columns. Chemicals used in experiments were obtained as follows: Gpp(NH)p and NE from Sigma (St. Louis, MO); VIP from Bachem (Torrance, CA).

Dissociation of SCG neurons. SCG neurons were enzymatically dissociated as described previously (Ikeda, 1991; Zhu and Ikeda, 1993). 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 incubated with 1 mg/ml collagenase type D, 0.35 mg/ml trypsin (Boehringer Mannheim Biochemicals, Indianapolis, IN), and 0.1 mg/ml DNase type I (Sigma) in 10 ml of modified Earle's balance salt solution (EBSS, pH 7.4) in a 25 cm² tissue culture flask. The EBSS was modified by adding 3.6 gm/l glucose and 10 mM HEPES. The flask was then placed in a shaking water bath at 35°C for 1 hr. After incubation, neurons were dissociated by vigorous shaking of the flask. After centrifugation at 50 × g for 5 min, the dispersed neurons were resuspended in MEM containing 10% fetal calf serum, 1% glutamine, and 1% penicillin-streptomycin solution (all from Life Technologies, Grand Island, NY). Neurons were then plated onto polystyrene culture dishes (35 mm) coated with poly-D-lysine and maintained in the humidified atmosphere of a 95% air-5% CO₂ incubator at 37°C. All neurons were used within 24 hr after intranuclear injection of vectors.

Intranuclear injection of vectors. Vectors encoding particular proteins were directly injected into the nucleus of SCG neurons as described previously (Ikeda, 1996, 1997). Briefly, the cDNAs from stock solutions (~1.0 µg/µl) were diluted in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4) to a final concentration of 100 ng/µl (per subunit). A reporter vector (pEGFP-N1) encoding a double mutant (F64L:S65T) variant of the green fluorescent protein (GFP) was also included (5 ng/µl) to facilitate later identification of neurons receiving a successful nuclear injection. After centrifugation (16,000 × g for 20 min) to remove undissolved particles, the cDNA-containing solution was loaded into a fine borosilicate micropipette and injected into the nucleus of SCG neurons with an Eppendorf 5242 microinjector and 5171 micromanipulator system (Madison, WI) using injection pressure and duration of 120-200 hPa and 0.3 sec, respectively. Neurons successfully expressing particular proteins were easily identified 14-24 hr later by the observation of the fluorescence produced by the GFP under an inverted microscope (Diaphot, Nikon, Japan) equipped with an epifluorescence unit (B-2A filter cube, Nikon).

Electrophysiology. A culture dish containing dissociated SCG neurons was placed on an inverted phase-contrast microscope (Nikon) and superfused at a flow rate of 1-2 ml/min with an external solution as described below. Ionic currents were recorded using a whole-cell variant of the patch-clamp technique (Hamill et al., 1981) as described previously (Ikeda, 1991; Ikeda et al., 1995). Patch electrodes were fabricated from a borosilicate glass capillary (1.65 mm outer diameter, 1.2 mm inner diameter; Corning 7052, Garner Glass Co. Claremont, CA). The patch electrodes were coated with Sylgard 184 (Dow Corning, Midland, MI) and fire-polished on a microforge, and they had resistances of 1.5-2.5 M when filled with an internal solution described below. The bath was grounded by an Ag/AgCl pellet connected via a 0.15 M NaCl/agar bridge. The cell membrane capacitance and series resistance were always compensated (typically >80%) electronically using a patch-clamp amplifier (Axopatch-200; Axon Instruments, Foster City, CA). Voltage protocol generation and data acquisition were performed using custom data acquisition software on a Macintosh Quadra series computer equipped with a MacAdios II data acquisition board (G. W. Instruments, Somerville, MA). Current traces were generally low-pass-filtered at 5 KHz using the four-pole Bessel filter in the clamp amplifier, digitized at 2-5 kHz, and stored on the computer hard drive for later analysis. Ca²⁺ current traces were corrected for linear leakage current as determined from hyperpolarizing pulses. All experiments were performed at room temperature (21-24°C). Drugs were 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. Data were presented as means ± SEM. ANOVA followed by post hoc Dunnett's test, as appropriate, were performed to determine statistical significance. p < 0.05 was considered significant.

Solutions. For Ca²⁺ current recording, the external solution contained (in mM): 145 tetraethylammonium (TEA)-methanesulfonate (MS), 10 HEPES, 10 CaCl₂, 15 glucose, 0.0001 tetrodotoxin (TTX) (pH adjusted to 7.4 with TEA-OH, osmolality 318 mOsm/kg H₂O). Patch pipette contained (in mM) 120 N-methyl-D-glucamine (NMG)-MS, 20 TEA-MS, 20 HCl, 11 EGTA, 1 CaCl₂, 10 HEPES, 4 MgATP, 0.3 Na₂GTP, 14 creatine phosphate (pH adjusted to 7.2 with TEA-MS, osmolality 297 mOsm/kg H₂O). In experiments designed to activate the overexpressed G subunits, 0.5 mM Gpp(NH)p was included in the internal solution. For M-type K⁺ channel current (M-current) measurement, the external solution contained (in mM): 150 NaCl, 2.5 KCl, 10 HEPES, 1 MgCl₂, 2 CaCl₂, and 15 glucose (pH adjusted to 7.4 with NaOH, osmolality 320 mOsm/kg H₂O). The internal solution contained (in mM): 150 KCl, 0.1 K₄BAPTA, 10 HEPES, 4 MgATP, and 0.1 Na₂GTP (pH adjusted to 7.2 with KOH, osmolality 300 mOsm/kg H₂O).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Overexpression of different G subunits significantly blocks NE-mediated Ca²⁺ current inhibition

We first determined whether different G subunits are able to interact with the G subunits released on activation of ₂-adrenergic receptors (₂-ARs). To address this question, G subunits from different subfamilies were transiently overexpressed in SCG neurons by intranuclear injection of mammalian expression vectors encoding the desired proteins. Figure 1 illustrates typical whole-cell Ca²⁺ current records obtained in the absence or presence of 10 µM NE from SCG neurons previously injected with selected subunits from four G-subfamilies. The Ca²⁺ currents were evoked by 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). From the current traces, we measured facilitation of Ca²⁺ currents in the absence of agonist (basal facilitation) as well as percent inhibition of Ca²⁺ currents produced by the agonist. Facilitation was defined as the ratio of the postpulse to prepulse current amplitude measured isochronally at 10 msec after the start of the test pulse. In the control neuron, expressing only GFP, Ca²⁺ currents were tonically facilitated (~1.24) by the strong conditioning pulse in the absence of agonist (Fig. 1A). Previously, basal facilitation in SCG neurons has been shown to arise from a small degree of tonic G-protein activation (Ikeda, 1991). Application of 10 µM NE produced a typical voltage-dependent inhibition of Ca²⁺ currents characterized by kinetic slowing and an increased prepulse facilitation (from 1.24 to 2.84) (Elmslie et al., 1990). Expression of GFP had little effect on the magnitude of either basal facilitation or NE-mediated Ca²⁺ current inhibition (1.28 ± 0.02, n = 19 and 57 ± 2%, n = 15, respectively, for uninjected neurons; 1.32 ± 0.02, n = 39 and 61 ± 2%, n = 30, respectively, for injected neurons) (Fig. 2A,B). In contrast, basal facilitation was absent after overexpression of G subunits. In addition, a slight inactivation was evident after the large depolarizing conditioning pulse (Figs. 1B-F, 2A). These results are consistent with the sequestration of endogenous free G by overexpressed GDP-bound G subunits. As shown previously (Ikeda, 1996), the injection of the cDNA encoding for G_oA nearly abolished NE-mediated Ca²⁺ current inhibition (Fig. 1B). Likewise, another PTX-sensitive G, G_i1, was able to disrupt the NE-mediated modulation when overexpressed (Fig. 1C). Strikingly, overexpression of other G-subfamily members, including G_s, G_q, and G₁₂, produced effects similar to those by G_oA and G_i1 (Fig. 1D-F). Figure 2B summarizes the effects of overexpressing various G subunits on NE-mediated Ca²⁺ current inhibition. With the exception of G_z, all tested G subunits nearly abolished NE-mediated modulation (Fig. 2B). In neurons overexpressing G_z, the NE-mediated modulation was not significantly different from that of control neurons (p > 0.05, n = 8). Previously, we have shown that G_z substitutes for G_o/i by coupling to G_o/i-coupled receptors, including ₂-ARs (Jeong and Ikeda, 1998). Taken together, these data suggest that various GDP-bound G subunits sequester Gs released on receptor activation and prevent the NE-mediated Ca²⁺ current inhibition.

View larger version (21K): [in this window] [in a new window]   Figure 1.   Heterologous overexpression of different G subunits abolished NE-mediated Ca2+ currents in SCG neurons. Superimposed current traces were recorded in the absence () or presence () of 10 µM NE from neurons transiently expressing GFP alone (A), GoA (B), Gi1 (C), Gs (D), Gq (E), and G12 (F). The Ca2+ currents were evoked by a double-pulse protocol consisting of two identical test pulses to +10 mV from a holding potential of 80 mV separated by a large depolarizing conditioning pulse to +80 mV (inset in A). Note that the basal facilitation in the absence of NE was seen in control neurons (by the bottom dotted line) and abolished by the overexpression of G subunits.

View larger version (32K): [in this window] [in a new window]   Figure 2.   Summary of the effects of different G subunits on basal facilitation in the absence of agonist (A) and percentage inhibition in the presence of 10 µM NE (B) and VIP (C). Data are presented as mean ± SEM, and numbers in parentheses indicate the number of neurons tested.

Overexpression of different G subunits significantly blocks VIP-mediated Ca²⁺ current inhibition

VIP has been shown to produce membrane-delimited and voltage-dependent inhibition of Ca²⁺ currents virtually identical to that seen with ₂-AR stimulation in SCG neurons (Zhu and Ikeda, 1994; Ehrlich and Elmslie, 1995). However, the modulation pathway used by VIP apparently uses a cholera toxin-sensitive G-protein, G_s, instead of G_o/i. Thus, we determined the ability of individual G subunits to attenuate VIP-mediated Ca²⁺ current inhibition when overexpressed in SCG neurons. Figure 2C summarizes the effects of overexpression of various G subunits on VIP-mediated Ca²⁺ current inhibition. In uninjected (n = 8) and GFP-injected neurons (n = 15), the mean percent inhibition produced by 10 µM VIP was 47 ± 3% and 44 ± 2%, respectively. However, in neurons expressing members of subfamilies unrelated to G_s, i.e., G_i, G_q, and G₁₂, the VIP-mediated Ca²⁺ current inhibition was nearly abolished. G_z did not reconstitute the VIP response confirming its specificity for G_o/G_i-coupled receptors (Jeong and Ikeda, 1998). When G_s was overexpressed, the VIP responses were variable and categorized into two groups by correlating NE responses tested in the same neurons. In one group, in which the NE response was minimally affected by overexpression of G_s, the VIP response was significantly enhanced when compared with that in control neurons (44 ± 2%, n = 15 for GFP control vs 59 ± 2%, n = 8 for G_s, p < 0.05). In another group, in which the NE response was completely blocked, however, overexpression of G_s significantly attenuated the VIP-mediated Ca²⁺ current inhibition (44 ± 2%, n = 15 for GFP control vs 29 ± 4%, n = 4 for G_s). When two groups of data were pooled, VIP produced 49 ± 5% (n = 12) inhibition, similar to the control value in neurons expressing G_s (Fig. 2C).

Modulation of M-type K⁺ channel currents is not blocked by overexpression of G subunits

Because all G subunits that were tested produced positive effects, we did not confirm the expression of proteins using immunochemical techniques. Instead, we tested whether the heterologously expressed G subunits had nonspecific effects on Ca²⁺ current modulation. In sympathetic neurons, inhibition of M-current has been shown to be mediated by the  subunit rather than the subunits of the G_q heterotrimer (Haley et al., 1998; Kammermeier and Ikeda, 1999). Thus, as a negative control for the specific effects of G subunits in sequestering G subunits, the muscarinic modulation of M-current was assessed in SCG neurons expressing different G subunits, including G_S, G_oA G₁₁, and G₁₂. The deactivation of M-current was evoked by a test pulse to 60 mV for 0.5 sec from a holding potential of 30 mV (Fig. 3A). In a control neuron expressing GFP, 10 µM muscarine produced a significant inhibition of the M-current (Fig. 3A). Overexpression of G_S, G₁₁, and G₁₂ subunits did not affect the muscarinic inhibition of the M-currents (Fig. 3A). In contrast, M-current inhibition was partially but significantly attenuated in neurons expressing G_oA (p < 0.05). As summarized in Figure 3B, 10 µM muscarine inhibited the M-current by 83 ± 2% (n = 8) in control neurons, and by 80 ± 8% (n = 5), 52 ± 11% (n = 7), 88 ± 1% (n = 6), and 79 ± 10% (n = 5), respectively, in neurons expressing G_S, G_oA, G₁₁, and G₁₂ subunits. Conversely, overexpression of G_q(Q209L), a GTPase-deficient and constitutively active form of G_q subunit, virtually eliminated the M-current and thereby the muscarinic modulation (n = 4; data not shown), consistent with a previous finding (Haley et al., 1998). Taken together, these data suggest that the overexpressed  subunits interact with G subunits rather than nonspecific interaction with ₂-ARs, Ca²⁺ channels, or other signaling proteins to block the modulation of Ca²⁺ currents.

View larger version (29K): [in this window] [in a new window]   Figure 3.   Heterologous overexpression of different G subunits failed to abolish the modulation of muscarine-sensitive K+ current (M-current) in SCG neurons. A, Superimposed current traces in the absence () or presence () of 10 µM muscarine recorded from neurons transiently expressing GFP alone (Control), Gs, GoA, G11, and G12. The deactivation of M-current was evoked by a test pulse to 60 mV for 0.5 sec from a holding potential of 30 mV (inset). B, Summary of effects of different G subunits on M-current inhibition. Data are presented as mean ± SEM, and numbers in parentheses indicate the number of neurons tested. *p < 0.05 (by post hoc Dunnett's test)

Overexpression of G subunits significantly delayed Gpp(NH)p-induced Ca²⁺ current inhibition

Overexpression of the G_oA subunit has been shown to significantly delay Gpp(NH)p-mediated facilitation in SCG neurons (Ikeda, 1996). The delay is thought to arise from the ability of Gpp(NH)p, a nonhydrolyzable GTP analog, to irreversibly activate G subunits, thereby overcoming the sequestration of G produced by overexpression of G_oA. Thus, we tested whether different G subunits could delay the Gpp(NH)p-mediated facilitation when overexpressed in SCG neurons. When uninjected control neurons were dialyzed with 0.5 mM Gpp(NH)p, the mean facilitation was increased from 1.16 ± 0.03 to 2.51 ± 0.11 (n = 6) within 4 min (Fig. 4). However, the onset of Gpp(NH)p-mediated facilitation was significantly delayed by overexpression of G_oA (n = 6). Likewise, another PTX-sensitive member of the G_i subfamily, G_i2, exerted a similar effect on the Gpp(NH)p-mediated facilitation (n = 2) (Fig. 4B). When PTX-insensitive G subunits such as G_s(n = 5), G_z(n = 4), G_q (n = 5), and G₁₃ (n = 5) were overexpressed, the onset of facilitation by the dialysis of Gpp(NH)p was nearly blocked.

View larger version (21K): [in this window] [in a new window]   Figure 4.   Heterologous overexpression of different G subunits significantly delayed Gpp(NH)p-mediated facilitation in SCG neurons. A, Representative Ca2+ current traces showing time-dependent change of facilitation in control (left panel) and GoA-injected (right panel) neurons. In both cases, 0.5 mM Gpp(NH)p was present in patch pipettes. B, time course of Gpp(NH)p-mediated facilitation in neurons expressing Gs, GoA, Gi2, Gz, Gq, and G13. The dotted line in B indicates the absence of facilitation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

One experimental way to affirm G-mediated signaling is to increase the normal G/G ratio by overexpressing G subunits. Excess GDP-bound G, with a high affinity for G subunits, may create conditions favoring heterotrimer formation and thus produce a "G sink" (Slepak et al., 1995). Suppression of G-mediated signaling by exogenous GDP-bound G_o has been demonstrated for different effectors such as adenylyl cyclase (Federman et al., 1992), GIRK channels (Ito et al., 1992; Reuveny et al., 1994; Krapivinsky et al., 1995),), phospholipase C (Katz et al., 1992), and phosphoinositide 3 kinase (Stephens et al., 1994). Likewise, in SCG neurons, overexpression of GDP-bound G_oA has been shown to eliminate NE-mediated Ca²⁺ current inhibition (Ikeda, 1996). A biochemical study in which ADP-ribosylation by PTX and GTPase activity were measured has demonstrated that recombinant G subunits (G_s, G_i1, G_i2, and G_o) were able to form heterotrimers with different G subunits (Ueda et al., 1994). Recently, analysis of immunoprecipitation combined with silver stain and immunoblotting has also demonstrated the random association of G subunits with different G subunits from tissue and cell extracts (Ueda et al., 1998). In addition, another in vitro experiment has shown the ability of five G subunits (G_i1, G_i2, G_o, G_s, and G_q) to associate with G subunits (₁ and ₂) (Fletcher et al., 1998). On the basis of these in vitro experiments, the interaction between G and G subunits seems to display little specificity. However, an apparent exception to this pattern is the structurally divergent G₅ subunit that appears to demonstrate specificity for G_q (Fletcher et al., 1998). To date, it is unclear whether the nonspecific interaction between G and G subunits occurs in native systems.

In the present study, we showed that the suppression of the NE-mediated Ca²⁺ current inhibition occurred with nearly all G subunits, regardless of G subfamily (G_s, G_o/i, G_q, and G₁₂). Interestingly, overexpression of different G subunits also eliminated the current inhibition mediated by cholera toxin-sensitive G_s activated on VIP receptor activation (Zhu and Ikeda, 1994). In addition, overexpression of GDP-bound G subunits significantly delayed the Gpp(NH)p-mediated modulation. Thus, the exogenous GDP-bound G seems to interact with G subunits released from many different endogenous heterotrimers (e.g., G_s and G_o/i). This result might not be surprising given the molecular similarity (80-90% identical in sequence) of the known G subunits (except ₅) (Watson et al., 1994) and the results of in vitro experiments. It is likely that all effective G subunits suppress the current modulation by sequestration of G subunits because M-current modulation (selected as a negative control), which is mediated by G_q (Haley et al., 1998), was not abolished by overexpression of G subunits. This explanation is also supported by the fact that the NE-mediated Ca²⁺ current inhibition was intact when G_o was coexpressed with G subunits (Jeong et al., 1998). Unexpectedly, M-current modulation was partially attenuated when G_oA was overexpressed in SCG neurons. This phenomenon remains to be investigated. However, it is possible that an extremely high level of GDP-bound G_o absorbs the G subunits from G_q heterotrimers coupled to receptors or alters the signaling pathway for M-current modulation by unknown mechanisms.

For the modulation by either NE or VIP there was an exception; that is, NE- and VIP-mediated modulation was not eliminated by overexpression of G_z and G_s, respectively. Additional experiments showed that G_z was able to reconstitute the voltage-dependent inhibition of Ca²⁺ currents by different G_o/G_i-coupled receptors when overexpressed in SCG neurons (Jeong and Ikeda, 1998). G_z did not reconstitute the VIP-mediated current inhibition because of lack of coupling to VIP receptors but was able to interact with the G responsible for VIP-mediated inhibition. It is unclear how G_z replaces endogenous G_o/i subunits in neurons. However, the reconstitution of NE-mediated current inhibition by G_z can be blocked by the extremely high expression of G_z (data not shown).

When G_s was overexpressed, we acquired two different groups of results: (1) the enhancement or (2) the attenuation in VIP-mediated modulation in comparison to control. These dual effects can be explained as follows. When the expression level was high (as indirectly judged by block of NE response in the same tested neurons), excess GDP-bound G_s acted as G sinks to reduce VIP-mediated inhibition. Conversely, when the expression level was low (as indirectly judged by the unaltered NE response in the same tested neurons), G_s was capable of forming heterotrimer complexes (G_s) with endogenous free G subunits (decreasing basal facilitation), and subsequently coupled to receptors. This is consistent with findings that expression of exogenous G_s proteins at a low level results in the enhanced modulation by increasing the number of receptor-G_s complexes (Bertin et al., 1994; Lim et al., 1995; Krumins and Barber, 1997). Studies using non-neuronal heterologous expression systems have generated similar results, i.e., expression of G subunits decreased the basal facilitation and increased G-protein-mediated modulation (Bourinet et al., 1996; Roche and Treistman, 1998). Thus, whether a certain exogenous G subunit increases or blocks receptor-mediated current modulation seems to be determined by specificity to a certain receptor and/or expression level of the subunit in the tested cells. Consequently, the question arises as to whether exogenous expression of G_o might enhance the NE-mediated Ca²⁺ current inhibition in SCG neurons. Unlike the voltage-dependent modulation of Ca²⁺ channels by G_s-coupled receptors (e.g., VIP response), the NE-mediated response appears saturated at maximal concentrations of agonist. This notion is supported by experiments in which the voltage-dependent inhibition of Ca²⁺ currents was unchanged after application of both VIP and ₂-AR agonists together when compared with application of ₂-AR agonist alone (Zhu and Ikeda, 1994; Ehrlich and Elmslie, 1995). Thus, it seems unlikely that exogenous G_o expressed at low levels would enhance the NE response even after heterotrimer formation with endogenous G (Herlitz et al., 1996).

The ability of heterologously expressed G subunits to delay the onset of Ca²⁺ current modulation (Fig. 4) is consistent with the notion of G buffering. The delay can be rationalized by assuming that basal GDP-GTP exchange results in the binding of Gpp(NH)p to both endogenous and heterologously expressed G subunits. Because Gpp(NH)p cannot be hydrolyzed, the majority of G subunits eventually attain the Gpp(NH)p bound state and are thus incapable of binding G with high affinity. Consequently, the eventual loss of G-GDP results in "release" of free G and thus voltage-dependent modulation. What is not clear from these data is why G_s and G_z produce such a dramatic effect in this assay when compared with their weak ability to attenuate agonist-mediated modulation (Fig. 2). Although we have no definitive explanation for these results, it can be speculated that expression levels, intrinsic GDP-GTP (or Gpp(NH)p) exchange rates (Fields and Casey, 1997), or compartmentalization (Neubig, 1994) may underlie these findings.

When compared with "G sinks" derived from effector molecules, for example the C terminus of ARK (Koch et al., 1994) or the QEHA peptide (Chen et al., 1995), G subunits may prove advantageous in regard to probing signaling pathways. First, it is assumed that G-GDP has a greater affinity for G than effector molecules. Although we are unaware of studies that directly compare these properties, it stands to reason that this is the case because the termination of G actions is thought to require association with G-GDP. If the affinity of effector molecules for G exceeded that of G-GDP, termination of signaling could not occur via this mechanism. Recently, a biochemical study has shown that a peptide containing the G-binding motif QXXER failed to inhibit interactions between GDP-bound G and G subunits (Chen et al., 1995), thus supporting this idea. Second, effectors such as ARK have been shown to interact with specific G isoforms (Koch et al., 1994), whereas G/G interactions appear relatively nonspecific. Thus, one would predict that overexpression of G-GDP would affect a broader range of G-mediated responses. It should be noted, however, that information concerning heterotrimer formation for the various G-protein subunits is incomplete. Moreover, the ability of ARK isoforms to specifically bind different G combinations may prove useful for identification purposes once these interactions are better defined. Thus, the information gained by overexpressing G subunits or effector molecule binding sites may prove complementary in regard to G signaling.

In summary, the present study showed that a wide variety of GDP-bound G subunits were able to sequester the G subunits, resulting in block of NE-, VIP-, and Gpp(NH)p-mediated modulation of N-type Ca²⁺ channels in SCG neurons. These results suggest that coupling specificity in signal transduction is unlikely to arise as a result of restricted G/G interaction. Although the interaction between G and G subunits is relatively nonspecific, a specific heterotrimer combination may be required for selective coupling of certain receptors to Ca²⁺ channels (for review, see Kalkbrenner et al., 1996). Thus, it is likely that the identity of G subunits may be one of the major determinants for the selective recognition of a given heterotrimer by a receptor.

	FOOTNOTES

Received Dec. 14, 1998; revised March 29, 1999; accepted April 1, 1999.

This study was supported by National Institutes of Health Grant GM 56180. We thank M. King for technical assistance and Drs. V. Ruiz-Velasco and P. Kammermeier for critical reading of an earlier version of this manuscript. We are grateful to Drs. M. I. Simon, R. Reed, and H. R. Bourne for providing cDNA clones.

Correspondence should be addressed to Dr. Stephen R. Ikeda, Laboratory of Molecular Physiology, Guthrie Research Institute, One Guthrie Square, Sayre, PA 18840.

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