Neurotransmitters acting via different G proteins inhibit N-type calcium current by an identical mechanism in rat sympathetic neurons.
ABSTRACT 1. We studied the mechanism of voltage-dependent inhibition of N-type calcium current by norepinephrine (NE) and vasoactive intestinal peptide (VIP) in adult rat superior cervical ganglion (SCG) neurons using the whole cell patch-clamp technique. 2. The voltage dependence of inhibition is manifest in the reversal of inhibition by strong depolarization. We tested the hypothesis that this voltage dependence results from disruption of G proteins binding to calcium channels. According to this hypothesis, the kinetics of calcium current reinhibition following a strong depolarization should become faster for higher concentrations of active G proteins. 3. Assuming that larger inhibitions result from higher concentrations of active G proteins, we used different concentrations of NE to alter the amplitude of inhibition and, thus, the active G protein concentration. We found that the kinetics of reinhibition at -80 mV following a depolarizing pulse to +80 mV were faster for larger inhibitions. 4. VIP induces voltage-dependent inhibition of N-current via a different G protein (Gs) than that of NE (Go). We found that the effect of VIP on reinhibition kinetics was identical to that produced by NE. 5. Combined application of NE and VIP did not greatly increase the amplitude of the inhibition but significantly increased the rate of reinhibition. Thus NE plus VIP appear to greatly increase the concentration of the molecule binding to the channel (G protein according to the hypothesis). 6. The kinetics of calcium current disinhibition during strong depolarization (step to +80 mV) did not change with the size of the inhibition induced by NE, VIP or application of NE and VIP together. 7. Both the concentration-dependent reinhibition kinetics and concentration-independent disinhibition kinetics are consistent with the hypothesis that active G proteins bind directly to N-type calcium channels to modulate their activity in rat sympathetic neurons.
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ABSTRACT: Whole-cell recordings have been used to extensively characterize the voltage-dependent inhibition of N-type calcium current induced by various neurotransmitters. Results from these studies have yielded several predictions on the effect of inhibition on N-channel gating, namely delayed channel opening and inhibition-induced reluctant openings. Previous single N-channel studies observed delayed channel opening but failed to find reluctant openings. However, strong depolarizations may be necessary to see reluctant openings, but this was not tested. We have examined N-channel gating at voltages depolarized to those used previously and found a neurotransmitter-induced open state that has properties predicted for the reluctant open state. The openings had lower open probability (P(o)) and brief open times compared to the dominant gating state observed in control (high P(o)). These reluctant events were reduced after strong depolarizing pulses used to reverse inhibition. The threshold voltage for activation of reluctant events was approximately 30 mV depolarized to that of the normal gating state (high P(o)). However, an action potential will provide sufficient depolarization to open reluctant N-channels.The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 06/2000; 20(9):3115-28. · 6.75 Impact Factor
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ABSTRACT: N-type voltage-gated calcium channel activity in rat superior cervical ganglion neurons is modulated by a variety of pathways. Activation of heterotrimeric G-proteins reduces whole-cell current amplitude, whereas phosphorylation by protein kinase C leads to an increase in current amplitude. It has been proposed that these two distinct pathways converge on the channel's pore-forming alpha(1B) subunit, such that the actions of one pathway can preclude those of the other. In this study, we have characterized further the actions of PKC on whole-cell barium currents in neonatal rat superior cervical ganglion neurons. We first examined whether the effects of G-protein-mediated inhibition and phosphorylation by PKC are mutually exclusive. G-proteins were activated by including 0.4 mM GTP or 0.1 mM GTP-gamma-S in the pipette, and PKC was activated by bath application of 500 nM phorbol 12-myristate 13-acetate (PMA). We found that activated PKC was unable to reverse GTP-gamma-S-induced inhibition unless prepulses were applied, indicating that reversal of inhibition by phosphorylation appears to occur only after dissociation of the G-protein from the channel. Once inhibition was relieved, activation of PKC was sufficient to prevent reinhibition of current by G-proteins, indicating that under phosphorylating conditions, channels are resistant to G-protein-mediated modulation. We then examined what effect, if any, phosphorylation by PKC has on N-type barium currents beyond antagonizing G-protein-mediated inhibition. We found that, although G-protein activation significantly affected peak current amplitude, fast inactivation, holding-potential-dependent inactivation, and voltage-dependent activation, when G-protein activation was minimized by dialysis of the cytoplasm with 0.1 mM GDP-beta-S, these parameters were not affected by bath application of PMA. These results indicate that, under our recording conditions, phosphorylation by PKC has no effect on whole-cell N-type currents, other than preventing inhibition by G-proteins.The Journal of General Physiology 04/2000; 115(3):277-86. DOI:10.1085/jgp.115.3.277 · 4.57 Impact Factor
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ABSTRACT: The membrane-delimited and voltage-dependent inhibition of N-type Ca2+ channels is mediated by Gbeta gamma subunits. Previously, exogenous excess GDP-bound GalphaoA has been shown to dramatically attenuate the norepinephrine (NE)-mediated Ca2+ current inhibition by sequestration of Gbeta gamma subunits in rat superior cervical ganglion (SCG) neurons. In the present study, we determined whether the attenuation of NE-mediated modulation is specific to GalphaoA or shared by a number of closely related (Galphatr, GalphaoB, Galphai1, Galphai2, Galphai3, Galphaz) or unrelated (Galphas, Galphaq, Galpha11, Galpha16, Galpha12, Galpha13) Galpha subunits. Individual Galpha subunits from different subfamilies were transiently overexpressed in SCG neurons by intranuclear injection of mammalian expression vectors encoding the desired protein. Strikingly, all Galpha subunits except Galphaz 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 Galpha subunits overexpressed in neurons. Galphas expression produced either enhancement or attenuation of the VIP-mediated modulation-an 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 Galpha subunits. Taken together, these data suggest that a wide variety of Galpha subunits are capable of forming heterotrimers with endogenous Gbeta gamma subunits mediating voltage-dependent Ca2+ channel inhibition. In conclusion, coupling specificity in signal transduction is unlikely to arise as a result of restricted Galpha/Gbeta gamma interaction.The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 07/1999; 19(12):4755-61. · 6.75 Impact Factor