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The Journal of Neuroscience, July 1, 1998, 18(13):4883-4890

Ca²⁺ Channel ₃ Subunit Enhances Voltage-Dependent Relief of G-Protein Inhibition Induced by Muscarinic Receptor Activation and G

John P. Roche and Steven N. Treistman

Department of Pharmacology and Molecular Toxicology and Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts 01655

	ABSTRACT

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The Ca²⁺ channel  subunit has been shown to reduce the magnitude of G-protein inhibition of Ca²⁺ channels. However, neither the specificity of this action to different forms of G-protein inhibition nor the mechanism underlying this reduction in response is known. We have reported previously that coexpression of the Ca²⁺ channel ₃ subunit causes M₂ muscarinic receptor-mediated inhibition of _1B Ca²⁺ currents to become more voltage-dependent. We report here that the ₃ subunit increases the rate of relief of inhibition produced by a depolarizing prepulse and also shifts the voltage dependency of this relief to more hyperpolarized voltages; these effects are likely to be responsible for the reduction of inhibitory response of _1B channels to G-protein-mediated inhibition seen after coexpression of the Ca²⁺ channel ₃ subunit. Additionally, the ₃ subunit alters the rate and voltage dependency of relief of the inhibition produced by coexpressed G₁₁, in a manner similar to the changes it produces in relief of M₂ receptor-induced inhibition. We conclude that the Ca²⁺ channel ₃ subunit reduces the magnitude of G-protein inhibition of _1B Ca²⁺ channels by enhancing the rate of dissociation of the G-protein subunit from the Ca²⁺ channel _1B subunit.

Key words: Ca²⁺ channels; G-proteins; _1A; _1B; Ca²⁺ channel  subunit; G-protein  subunit; G-protein subunit; voltage-dependent inhibition; Xenopus oocyte; muscarinic M₂ receptor; NEM

	INTRODUCTION

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G-protein-mediated inhibition of voltage-gated Ca²⁺ channels provides an important mechanism for regulating synaptic strength (Holz et al., 1986; Wheeler et al., 1994; Dittman and Regehr, 1996; Takahashi et al., 1996). Although many types of Ca²⁺ channels can undergo this class of inhibition, N-type Ca²⁺ current is the most frequently studied target of this modulation (Schultz et al., 1990; Anwyl, 1991; Dolphin, 1991; Hille, 1994). Various members of the seven membrane-spanning family of receptors, after binding neurotransmitter, transduce their signal via activation of a variety of heterotrimeric G-proteins. The activated G-proteins then either directly interact with the channel to cause inhibition, in a process known as membrane-delimited inhibition (Bean, 1989; Brown and Birnbaumer, 1990), or subsequently activate a second messenger cascade that ultimately acts on the channel to cause inhibition. N-type Ca²⁺ channels are inhibited via both a membrane-delimited pathway (Schultz et al., 1990; Anwyl, 1991; Dolphin, 1991; Hille, 1994) and a pathway requiring diffusible intracellular second messengers (Beech et al., 1992; Shapiro et al., 1994a).

Membrane-delimited G-protein inhibition encompasses both voltage-dependent and voltage-independent inhibition. Voltage-dependent inhibition exhibits two main characteristics in voltage-clamp studies: (1) slowed activation kinetics and (2) diminished inhibition at more depolarized voltages (Marchetti et al., 1986; Wanke et al., 1987; Bean, 1989; Kasai and Aosaki, 1989). The diminished inhibition at more depolarized voltages gives rise to a third characteristic of voltage-dependent inhibition, prepulse current facilitation (Elmslie et al., 1990; Ikeda, 1991; Lopez and Brown, 1991). Strongly depolarizing voltages are thought to cause a temporary dissociation of the G-protein from the Ca²⁺ channel (Bean, 1989; Lopez and Brown, 1991; Golard and Siegelbaum, 1993); thus, a current elicited during this period of G-protein dissociation will be facilitated compared with current elicited by the same test voltage step without a depolarizing prepulse.

Voltage-independent inhibition is characterized by equivalent current inhibition at all voltages, with no change in current kinetics during the inhibition. Frequently, voltage-independent G-protein inhibition requires intracellular signaling cascades and thus is not membrane-delimited (Beech et al., 1991, 1992; Bernheim et al., 1991; Shapiro et al., 1994a). However, there are instances of membrane-delimited voltage-independent inhibition (Shapiro and Hille, 1993; Diverse-Pierluissi et al., 1995; Wollmuth et al., 1995).

Voltage-dependent inhibition of N-type Ca²⁺ currents in rat superior cervical ganglion (SCG) sympathetic neurons (Herlitze et al., 1996; Ikeda, 1996), as well as _1A Ca²⁺ channel currents expressed in tsA-201 cells (Herlitze et al., 1996), is mediated by the G-protein subunit. G, however, seems not to be responsible for voltage-dependent inhibition of N-type currents in embryonic chick dorsal root sympathetic ganglion neurons (Diverse-Pierluissi et al., 1995). The G-protein subunit is capable of binding to at least two regions of the intracellular loop between transmembrane regions I and II of _1A and _1B Ca²⁺ channels (De Waard et al., 1997; Zamponi et al., 1997); the same intracellular loop contains the binding region for the Ca²⁺ channel  subunit (Pragnell et al., 1994). Mutations that reduce in vitro G-protein subunit binding to this region of the Ca²⁺ channel also block some characteristics of voltage-dependent G-protein inhibition of _1A current (De Waard et al., 1997), although similar mutations do not affect somatostatin-induced inhibition of _1B currents (Zhang et al., 1996). The critical amino acids within _1A responsible for G binding are not the same as those critical for Ca²⁺ channel  subunit binding (De Waard et al., 1997), suggesting that direct competition for a binding site on the ₁ subunit is unlikely.

The Ca²⁺ channel  subunit reduces the magnitude of G-protein inhibition of both _1A and _1B Ca²⁺ channels expressed in Xenopus oocytes (Roche et al., 1995), as well as Ca²⁺ currents in rat dorsal root ganglion neurons (Campbell et al., 1995). Speculation on the mechanism underlying this reduction in sensitivity to G-protein inhibition includes: (1) direct competition between the Ca²⁺ channel ₃ subunit and the G-protein for the same site on the Ca²⁺ channel ₁ subunit (McAllister-Williams and Kelly, 1995; Roche et al., 1995; Bourinet et al., 1996; Clapham, 1996), (2) steric blockade of the G-protein binding site (Roche et al., 1995; Bourinet et al., 1996), and (3) a Ca²⁺ channel  subunit-induced increase in the GTPase activity of the G-protein (Campbell et al., 1995). Examination of M₂ muscarinic receptor-induced inhibition of _1B currents in Xenopus oocytes revealed that not only is the magnitude of the G-protein inhibition reduced after coexpression of the Ca²⁺ channel ₃ subunit but the portion of inhibited current that is voltage-dependent is increased as well (Roche and Treistman, 1998). Here, we examine possible mechanisms that underlie the increase in voltage-dependence and discuss whether this mechanism can explain the reduction in the magnitude of M₂ receptor-induced inhibition of _1B currents after coexpression of the Ca²⁺ channel ₃ subunit. We address these questions using _1B Ca²⁺ channels coexpressed with muscarinic M₂ receptors in Xenopus oocytes. The coexpressed M₂ receptor couples to the endogenous pertussis toxin-sensitive G-proteins of the Xenopus oocyte (Lechleiter et al., 1991). We also coexpress G-protein  and subunits individually to determine the G-protein subunit mediating inhibition of both _1B and _1B3 Ca²⁺ channel currents and to assess the influence of the Ca²⁺ channel ₃ subunit on the direct actions of these G-protein subunits on _1B Ca²⁺ channels.

	MATERIALS AND METHODS

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Expression plasmids and oocyte preparation. Capped RNA transcripts encoding full-length _1A (XbaI-linearized/SP6 RNA polymerase; gift of Dr. Y. Mori, University of Cincinnati Medical Center), _1B (SalI/SP6; gift of Dr. Y. Fujita, Kyoto University), and ₃ (NotI/T7; gift of Dr. Edward Perez-Reyes, Loyola University Medical Center) calcium channel subunits as well as the muscarinic M₂ receptor (EcoRI/BglII; gift of Dr. Wolfgang Sadee, University of California San Francisco) and G-protein i₂ (gift of Dr. Randall Reed, HHMI, Baltimore, MD) and ₁₁ (gift of Drs. Melvin Simon and Anna Aragay, California Institute of Technology, Pasadena, CA) subunits were synthesized using the mMESSAGE mMACHINE in vitro transcription kit (Ambion, Austin, Texas). Xenopus laevis stage V-VI oocytes were removed and treated with collagenase (Sigma type IV; Sigma, St. Louis, MO) to remove the follicular layer. The oocytes were then injected with cRNA encoding _1B in combination with M₂ in a ratio of 2:1 or in combination with both M₂ and ₃ (2:2:1). The concentration of all individual RNAs before injection was 0.1 µg/µl, with the exception of the G-protein  and subunit RNA that was 0.5 µg/µl, and 20-60 nl of RNA mixed at the above ratios was injected. The oocytes were maintained in culture at 18°C for at least 2 d in ND-96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, and 5 mM HEPES, pH 7.5) supplemented with 2.5 mM sodium pyruvate and 2 mg/ml gentamycin.

Electrophysiological recording and experimental treatments. Two-electrode voltage-clamp currents were recorded using a Dagan CA-1 amplifier. Oocytes were clamped at a holding potential of 80 mV, and various electrophysiological protocols were used, as noted. Currents were filtered at 1 or 10 kHz, and a p/4 leak subtraction technique was used. Inhibition of current amplitude was determined by measurements of the peak current attained at any point during the 250 msec test pulse. Analysis was done off-line, using pClamp software version 6.0.2 (Axon Instruments, Foster City, CA). Electrodes contained 3 M KCl and had resistances of 0.5-2 M. Oocytes were placed in a 1 ml chamber and perfused at a rate of 0.5 ml/min. All recordings were made at room temperature using bath solutions containing (in mM): Ba(OH)₂, 10; NaOH, 50; CsOH, 2; TEA-OH, 20; N-methyl-D-glucamine, 20; and HEPES, 5, titrated to pH 7.5 with methanesulfonic acid. In all experiments, 20 nl of a 100 mM stock solution of K₃-1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) (Sigma) was injected at least 2 hr before the experiment. The final concentration of BAPTA inside the oocyte was estimated to be between 2 and 5 mM, assuming an oocyte volume of 1 µl. For experiments using N-ethylmaleimide (NEM) (Aldrich, Milwaukee, WI), the NEM was dissolved in the external solution to a final concentration of 200 µM and was applied to the oocyte for 2 min. Acetylcholine (ACh) (Sigma) was stored as a 10 mM stock solution in water and dissolved in the recording medium to a final concentration of 50 µM.

	RESULTS

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Ca²⁺ channel ₃ subunit modulates voltage dependence of M₂-mediated inhibition

A protocol designed to remove tonic G-protein inhibition of _1B Ca²⁺ channels allowed study of the isolated muscarinic M₂ receptor-induced G-protein inhibition of these channels. Briefly, we exposed the oocyte to 50 µM ACh. Immediately after removal of the ACh, there is a large rebound of current amplitude, resulting from temporary loss of tonic G-protein inhibition (Roche and Treistman, 1998). During the period in which tonic inhibition is abolished, ascertained by the loss of prepulse facilitation, the current remains sensitive to muscarinic receptor-induced inhibition. Loss of tonic inhibition occurred, in most cases, after a single 1 min application of ACh; on occasion, multiple ACh applications were required to remove tonic inhibition completely. Using this protocol, we have demonstrated that expression of the Ca²⁺ channel ₃ subunit reduced the magnitude of muscarinic M₂ receptor-induced inhibition (Fig. 1A,B). However, the reduction in magnitude of inhibition was voltage-dependent, with substantial reductions of G-protein inhibition at voltages more positive than 0 mV, and no effect on calcium current inhibition during voltage steps to 10 or 0 mV (Fig. 1C). In addition to the reduced inhibition, a depolarizing prepulse during muscarinic inhibition elicits greater relief of G-protein inhibition after coexpression of the Ca²⁺ channel ₃ subunit (Fig. 1D).

View larger version (33K): [in this window] [in a new window]   Figure 1.   The Ca2+ channel 3 subunit modifies the voltage dependence of muscarinic-induced G-protein inhibition. A, B, Representative records of 1B and 1B3 Ca2+ currents for the control situation (Con), as well as after the application of 50 µM ACh and before (+ACh,PP) and after (+ACh,+PP) a depolarizing prepulse to +100 mV for 75 msec. Oocytes were held at 80 mV and stepped to a test potential of +10 mV for 250 msec. The M2 receptor is coupling to G-proteins that are endogenous to the oocyte. C, Inhibition of current amplitude at various test potentials for both the 1B (open) and 1B3 (filled) Ca2+ currents. D, Relief of M2 receptor-induced inhibition by a depolarizing prepulse to +100 mV for 75 msec. The prepulse was given 20 msec before the test pulse. Facilitation was measured as the percentage of inhibited current that was reversed by the prepulse voltage protocol.

The Ca²⁺ channel ₃ subunit increases the rate of voltage-dependent relief of G-protein inhibition of _1B currents

Voltage-dependent relief of G-protein inhibition of N-type currents is thought to result from temporary dissociation of the G-protein from the Ca²⁺ channel (Lopez and Brown, 1991; Golard and Siegelbaum, 1993). Thus, the heightened relief of the inhibited current by depolarizing voltages after coexpression of the Ca²⁺ channel ₃ subunit suggests that the rate of G-protein dissociation has changed. An increase in the G-protein dissociation rate could explain the reduced inhibition of current by M₂ receptor activation when the Ca²⁺ channel ₃ subunit is coexpressed, because the inhibition would be more easily reversed by moderate voltages, such as those in the range normally used to activate the Ca²⁺ channel.

This model was tested by increasing the duration or voltage of the prepulse incrementally and determining the rate of current facilitation of _1B Ca²⁺ currents both with and without Ca²⁺ channel ₃ subunit associated with the _1B channel. The Ca²⁺ channel ₃ subunit dramatically decreased the duration of the prepulse necessary for maximal facilitation from ~160 msec to <20 msec; a single exponential fit to the data revealed a decrease in the time constant of relief by a voltage step to +100 mV from 67 msec in the absence of ₃ auxiliary subunit to 6.9 msec after coexpression of the Ca²⁺ channel ₃ subunit (Fig. 2A,B).

View larger version (36K): [in this window] [in a new window]   Figure 2.   Modulation of time and voltage dependency of prepulse facilitation by the Ca2+ channel 3 subunit. A, B, Facilitation of current amplitude after G-protein inhibition induced by application of acetylcholine. The prepulse was to +75 mV for varying periods of time, as indicated. The test potential was +10 mV. The data were fit with a single exponential, revealing time constants of 67 msec for the 1B currents and 6.9 msec for the 1B3 currents. C, D, Facilitation of current amplitude with a 75 msec prepulse of varying voltage, as indicated. The test potential was +10 mV. These data were fit with a Boltzmann curve, revealing V1/2 values of 52 mV for the 1B currents and 39 mV for the 1B3 currents.

We also tested for changes in the voltage dependence of prepulse facilitation. This protocol was similar to the duration protocol used previously except, in this case, the voltage of the prepulse step was increased incrementally, while maintaining a fixed prepulse duration. The data were fitted with Boltzmann curves, revealing a V_1/2 for current facilitation of 52 mV for the _1B current and 39 mV after coexpression of the Ca²⁺ channel ₃ subunit (Fig. 2C,D). Thus, the rate of reversal of G-protein inhibition, as well as the voltage that is necessary to reverse the G-protein inhibition of the Ca²⁺ channel, has decreased after coexpression of the Ca²⁺ channel ₃ subunit.

G-protein subunit mediates inhibition of _1B currents

There is evidence that the voltage-dependent form of G-protein inhibition is mediated by the G-protein subunit for N-type currents in rat SCG neurons (Herlitze et al., 1996; Ikeda, 1996). However, it is also clear that this is not the case in chick dorsal root ganglion neurons, in which the subunit mediates a voltage-independent form of inhibition (Diverse-Pierluissi et al., 1995). We coexpressed subunits of a heterotrimeric G-protein with _1B and _1B3 Ca²⁺ channels to determine the G-protein subunit mediating voltage-dependent inhibition in our system, first examining the tonic inhibition of Ca²⁺ currents produced by exogenously expressed G-proteins. Although _1B currents display a large degree of tonic G-protein-mediated inhibition from G-proteins endogenous to the oocyte, activation of a coexpressed M₂ receptor results in both a further decrease in current amplitude and a slowing of activation kinetics (Roche and Treistman, 1998), suggesting that we should be able to detect any further G-protein inhibition induced by coexpression of a G-protein subunit. We first examine the results of G coexpression and then the influence of the G subunit.

Coexpression of the G-protein subunit slowed current activation kinetics in comparison with current in oocytes in which no exogenous subunits were expressed (Fig. 3A), similar to the slowing of activation kinetics seen after muscarinic receptor-induced inhibition. Coexpression of the G-protein subunit complex significantly increased the time necessary to reach peak current levels from 31.5 ± 2.2 to 70.0 ± 24.0 msec (p  0.05, Student's t test) (Fig. 3A,E). Facilitation of current amplitude by depolarizing prepulses dramatically increased after coexpression of the G-protein subunit. Figure 3B shows the currents elicited both before (PP) and after (+PP) a depolarizing prepulse for oocytes expressing the G-protein subunit (+G). The mean facilitation of current amplitude was significantly increased from 108 ± 11 to 183 ± 10% after coexpression of the G-protein subunit (p  0.05, Student's t test) (Fig. 3B,C). Slowed activation kinetics and increased prepulse facilitation are both consistent with increased voltage-dependent G-protein inhibition.

View larger version (37K): [in this window] [in a new window]   Figure 3.   Effects of coexpressed G-protein  and subunits on 1B Ca2+ currents. A, Representative currents elicited by a voltage step from a holding potential of 80 mV to a test voltage of +10 mV. Control currents are labeled Con, whereas the currents elicited after application of 50 µM ACh are labeled +ACh. B, Representative currents elicited using the voltage protocol illustrated in oocytes coexpressing the G-protein (G) or  (Gi2) subunits. Currents elicited without the prepulse are labeled PP, whereas the currents elicited after a voltage step to +100 mV are labeled +PP. C, Summary of mean voltage-dependent facilitation before and after G-protein subunit coexpression. D, Summary of the inhibition of peak current amplitude by the initial application of ACh (Initial) and during the rebound phase induced after previous ACh application (Reb), as well as after coexpression of the G-protein (+G) and  (+Gi2) subunits. E, Elapsed time from the beginning of the voltage step to the peak amplitude of the elicited current before (black) and after (gray) application of 50 µM acetylcholine. This was done for 1B alone (1B), as well as after the coexpression of G-protein (+G) or  (+Gi2). Sample size indicated above bars.

We next examined the effect of overexpression of the subunit on M₂-mediated inhibition. The magnitude of inhibition of current amplitude after activation of the M₂ receptor was reduced after coexpression of the G-protein subunit, from a value of 51 ± 3% inhibition for oocytes that were tonically inhibited but expressed no exogenous G-protein subunits (data not shown) to a value of 37 ± 8% inhibition after coexpression of the G-protein subunit (Fig. 3D). This partial occlusion of the M₂-mediated inhibition is consistent with a common pathway for M₂- and exogenous subunit-mediated inhibition. Further support for this conclusion is provided by examination of another measure of G-protein inhibition, the slowing of I_Ba activation kinetics measured as the time-to-peak current. The effects of M₂ activation and exogenous G were nonadditive, with similar values for maximal slowing obtained by M₂ receptor activation in the absence and presence of coexpressed G (Fig. 3E). These data suggest a common pathway, consistent with voltage-dependent G-protein inhibition of _1B Ca²⁺ currents mediated by the G-protein subunit.

G-protein  subunit blocks tonic G-protein inhibition

If G mediates the voltage-dependent inhibition of _1B currents, we might expect that coexpression of the G-protein  subunit would block G-protein inhibition by acting as a "sink" for free subunit. Such an effect of exogenous G-protein  subunit on G-protein signaling has been suggested previously (Ikeda, 1996). Coexpression of G resulted in a significant decrease in the amount of tonic inhibition. We have shown previously that application of the alkylating agent NEM causes a potentiation of current amplitude (Roche et al., 1995), resulting from uncoupling of the basally active G-protein population. Coexpression of the G-protein  subunit should also eliminate potentiation of current amplitude by NEM, if the exogenous G-protein  subunit has blocked the tonic G-protein pathway. This is, indeed, the case. Potentiation of current amplitude by application of NEM to oocytes expressing _1B currents and no exogenous G-protein subunits was 225 ± 25%, whereas the potentiation was reduced to 29 ± 9% after coexpression of the G-protein  subunit (data not shown). These data are consistent with the assumption that the G-protein  subunit acts as a sink for the tonically active subunit, thus blocking the inhibition mediated by the G-protein subunit. The G-protein  subunit did not, however, buffer M₂ receptor-induced inhibition (79 ± 1.8% inhibition for control vs 86 ± 2.4% inhibition after coexpression of Gi₂) (Fig. 3A,D).

Figure 3B shows representative currents elicited before and after a depolarizing prepulse to +100 mV, demonstrating the loss of prepulse facilitation after coexpression of G. Facilitation of current amplitude was reduced from 108 ± 11% facilitation for oocytes that expressed no exogenous G-protein subunits to 23 ± 10% facilitation after coexpression of exogenous G-protein subunit (Fig. 3C). This loss of prepulse current facilitation is another indicator of the loss of voltage-dependent G-protein inhibition, supporting the conclusion that G mediates voltage-dependent inhibition of _1B Ca²⁺ current.

Influence of Ca²⁺ channel ₃ subunit on G-protein subunit-mediated inhibition

The Ca²⁺ channel  subunit has been shown to significantly modify G-protein modulation of Ca²⁺ channels, and we examined its influence on G-induced inhibition. The expression of exogenous G-protein subunit was also effective in mediating voltage-dependent inhibition of _1B3 Ca²⁺ currents. Similar to our results for the _1B currents, the activation kinetics of the _1B3 currents was significantly slowed by coexpression of the G-protein subunit. Figure 4A shows representative currents in the presence of exogenous G-protein subunits, demonstrating the slowed activation kinetics of the _1B3 currents after coexpression of the G-protein subunit. In addition, the G-protein subunit also occludes the M₂ receptor-mediated inhibition (Fig. 4A,C), again suggesting that -induced inhibition is acting via the same mechanism as M₂-induced inhibition.

View larger version (31K): [in this window] [in a new window]   Figure 4.   Effects of G-protein  and subunit coexpression on 1B3 Ca2+ currents. A, Currents elicited by a voltage step from a holding potential of 80 mV to a test voltage of +10 mV. Control currents are labeled Con, whereas the currents elicited after application of 50 µM ACh are labeled +ACh. B, Representative currents elicited both before (PP) and after (+PP) a depolarizing prepulse to +100 mV with and without coexpression of G-protein (+G) and  (+Gi2) subunits. C, Summary of the inhibition of peak current amplitude by the initial application of ACh for 1B3 alone and after coexpression of the G-protein  (+Gi2) and (+G) subunits. D, Summary of prepulse facilitation of current amplitude before and after coexpression of G-protein subunits. Sample size indicated above bars.

There was very little facilitation of _1B3 current amplitude by depolarizing prepulses in the absence of exogenous G-protein subunits (Fig. 4D). However, after we coexpressed the G-protein subunit, the facilitation of current amplitude by depolarizing prepulses was significantly increased. Figure 4B shows representative _1B3 currents, elicited before and after a depolarizing prepulse to +100 mV, in the absence and presence of coexpressed G. Prepulse facilitation using this protocol increased from 4 ± 4% when no exogenous G-protein subunits were expressed to 57 ± 8% after coexpression of the G-protein subunit (Fig. 4D), indicative of a substantial increase in the amount of voltage-dependent inhibition.

Influence of Ca²⁺ channel ₃ subunit on the ability of G-protein  subunit to block tonic G-protein inhibition

As with the _1B current, the G-protein  subunit caused a small but significant increase in the magnitude of M₂-mediated inhibition of _1B3 current (Fig. 4C), from 55 ± 2.3% inhibition in oocytes that expressed no exogenous G-proteins to 68 ± 2% inhibition in oocytes that expressed exogenous G subunit. Although the G-protein  subunit did not reduce the magnitude of M₂-induced inhibition, the G-protein  subunit did block a small tonic inhibition, evidenced by a decrease in the small amount of facilitation that was seen in the control (Fig. 4D).

The Ca²⁺ channel ₃ subunit modulates the voltage sensitivity of G-protein subunit-induced inhibition

A model for membrane-delimited voltage-dependent inhibition in which the G-protein subunit binds directly to the _1B Ca²⁺ channel has recently received experimental support (De Waard et al., 1997; Zamponi et al., 1997). Modulation of the inhibition mediated by exogenous G by coexpression of the Ca²⁺ channel ₃ subunit, therefore, would likely result from changes in the effectiveness of the interaction of G with the Ca²⁺ channel. We examined the influence of the Ca²⁺ channel ₃ subunit on the rate of voltage-dependent relief of G-protein subunit-mediated inhibition. Figure 5 demonstrates that the Ca²⁺ channel ₃ subunit also dramatically increases the rate of relief of the inhibition produced by the coexpressed G subunit. A single exponential fit to the data revealed a shift in the rate at which the G-protein -induced inhibition is reversed by depolarizing prepulses, from a time constant of 58 msec for _1B alone to 6 msec after coexpression of the Ca²⁺ channel ₃ subunit (Fig. 5A,B). This was similar to the increase in the rate of current facilitation produced by the Ca²⁺ channel ₃ subunit for M₂ receptor-induced inhibition of _1B and _1B3 currents (67 and 7 msec, respectively).

View larger version (35K): [in this window] [in a new window]   Figure 5.   Effects of G-protein subunit coexpression on rate and voltage dependence of prepulse facilitation. A, B, Exponential fit of prepulse facilitation by 100 mV prepulse of varying duration in the presence of the G-protein subunit coexpressed with 1B or 1B3. C, D, Boltzmann fit of facilitation of 1B current amplitude by a 75 msec prepulse to varying voltages in the presence of the G-protein subunit coexpressed with 1B or 1B3.

Figure 5, C and D, shows also shows the voltage dependence of the relief of G-protein subunit-induced current inhibition. A Boltzmann fit of the data revealed an ~10 mV leftward shift in voltage sensitivity, similar to the shift in voltage-dependent relief of M₂ receptor-induced inhibition. Thus, the Ca²⁺ channel  subunit increases the rate and decreases the voltage necessary for facilitation of G-inhibited Ca²⁺ currents.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Our data demonstrate that the rate of reversal of M₂-mediated inhibition by depolarizing prepulses dramatically increases and the voltage necessary for reversal decreases after coexpression of the Ca²⁺ channel ₃ subunit. We have also confirmed that the G-protein subunit mediates the inhibition of N-type currents and have extended this observation to include both _1B and _1B3 Ca²⁺ currents. In addition, we have demonstrated that the Ca²⁺ channel ₃ subunit increases the rate and decreases the voltage necessary for voltage-dependent reversal of G-induced inhibition. This results in voltage-dependent relief of inhibition at the moderate voltages used to activate the channel during voltage-clamp experiments and likely explains the reduction in the magnitude of G-protein inhibition of _1B current after coexpression of the Ca²⁺ channel ₃ subunit. Although the leftward shift in the voltage dependence of facilitation is most likely the result of more rapid unbinding of the G-protein in the presence of the Ca²⁺ channel ₃ subunit, caution should be used when interpreting this shift, because the Ca²⁺ channel ₃ subunit also causes a 10 mV leftward shift of peak current in the I-V relation (Roche and Treistman, 1998). Because reversal of G-protein inhibition is thought to result from a conformational change in the channel, produced by voltage, the apparent steeper voltage dependence of activation produced by the Ca²⁺ channel ₃ subunit may contribute to the leftward shift in voltage dependence of facilitation.

Recent findings suggest that some characteristics of voltage-dependent inhibition are a result of the G-protein subunit binding to its consensus site next to the Ca²⁺ channel  subunit binding site (De Waard et al., 1997). The critical amino acids responsible for binding of the Ca²⁺ channel  subunit are not critical for G-protein subunit binding and vice versa. The close proximity of the two sites, however, make modification of the G-protein binding site by the bound Ca²⁺ channel ₃ subunit a likely possibility. However, it should be noted that some groups have reported that the G consensus binding sequence on the I-II loop of the Ca²⁺ channel is not responsible for mediating the effects of G-proteins (Zhang et al., 1996; Qin et al., 1997). Adjacent proximity of the G and calcium channel ₃ binding sites within the protein is not a requirement for the model we are proposing. The most reasonable interpretation, combining the information from mutagenesis studies and the results presented here, is that the bound ₃ subunit enhances the G dissociation rate and thus reduces the magnitude of G-protein inhibition of _1B Ca²⁺ channels.

It is interesting that coexpression of the G-protein  subunit eliminates tonic G-protein inhibition but not M₂-mediated inhibition of _1B Ca²⁺ channel current. The differential effect of G might be explained by a variety of mechanisms. One possibility is that the endogenous free subunit, a portion of which is responsible for tonic inhibition, exists at levels that saturate the exogenous free  subunit, so that the expressed G subunit cannot buffer the additional subunit liberated by activation of the muscarinic receptor. In support of this mechanism, we find that M₂-mediated inhibition is only partially blocked by NEM, an agent that uncouples the G-protein  subunit from receptor activation (Jakobs et al., 1982; Nakajima et al., 1990), when no exogenous G-protein subunits are present. However, after coexpression of the NEM-sensitive Gi (Shapiro et al., 1994b), the M₂-mediated inhibition is almost entirely blocked by NEM (data not shown). This result is predicted by a model in which exogenous G subunits form inactive heterotrimers with the tonically active endogenous G subunits. These newly formed heterotrimers are then activated after binding of an agonist to the M₂ receptor, liberating the G subunit, and overwhelming the buffering capacity of the coexpressed G subunits.

Regulation of responsiveness to G-proteins at the level of the ultimate target may be a widely used mechanism, enabling a channel or other protein to regulate its sensitivity to modulation while maintaining its basal properties. This mechanism may be necessary in situations in which a modulatory signal is greatly amplified or when the signal has a large number of ultimate targets. In such situations, downregulation of the receptor itself may have unwanted consequences or may be ineffective because of amplification of the signal.

Functional Ca²⁺ channels may exist in the absence of a component auxiliary  subunit (De Waard and Campbell, 1995). Additionally, a recent report (Qin et al., 1997) suggests that a second calcium channel  subunit binding site is located on the C terminal of _1A, _1B, and _1E calcium channels and that this site is responsible for the antagonism of G-protein inhibition of these channels by the calcium channel  subunit. This site is distinct from that believed to be responsible for high expression and insertion of channels. Thus, it is possible that differential occupancy of this second site by the channel  subunit could serve a regulatory function, consistent with our observations with cloned channels. The increased voltage sensitivity of the inhibition observed after coexpression of the Ca²⁺ channel ₃ subunit may play an important role in the regulation of transmitter release in response to high-frequency or long-duration action potentials (Brody et al., 1997), in which depolarization of the presynaptic terminal would reach levels sufficient to relieve G-protein inhibition of Ca²⁺ channels controlling release.

	FOOTNOTES

Received Nov. 18, 1997; revised April 15, 1998; accepted April 16, 1998.

This work was supported by the National Institutes of Health Grants AA05542 and AA08003 to S.N.T. and a National Institutes of Health predoctoral fellowship to J.P.R. We thank Dr. Ann Rittenhouse for careful reading of this manuscript and Andy Wilson and Lynda Zorn for expert technical assistance.

Correspondence should be addressed to Dr. Steven N. Treistman, Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical Center, Worcester, MA 01655.

Dr. Roche's present address: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195. 

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