Neurotransmitters acting via different G proteins inhibit N-type calcium current by an identical mechanism in rat sympathetic neurons

Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112-2699, USA.
Journal of Neurophysiology (Impact Factor: 2.89). 01/1996; 74(6):2251-7.
Source: PubMed


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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    • "Superior cervical ganglion (SCG) neurons were acutely isolated from adult Sprague-Dawley rats (150 –350 g) as described previously (Ehrlich and Elmslie 1995). Briefly, rats were anesthetized with ether Y. S. Goo and W. Lim contributed equally to this work. "
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    ABSTRACT: Ca2+ -dependent inactivation (CDI) has recently been shown in heterologously expressed N-type calcium channels (CaV2.2), but CDI has been inconsistently observed in native N-current. We examined the effect of Ca2+ on N-channel inactivation in rat sympathetic neurons to determine the role of CDI on mammalian N-channels. N-current inactivated with fast (tau approximately 150 ms) and slow (tau approximately 3 s) components in Ba2+. Ca2+ differentially affected these components by accelerating the slow component (slow inactivation) and enhancing the amplitude of the fast component (fast inactivation). Lowering intracellular BAPTA concentration from 20 to 0.1 mM accelerated slow inactivation, but only in Ca2+ as expected from CDI. However, low BAPTA accelerated fast inactivation in either Ca2+ or Ba2+, which was unexpected. Fast inactivation was abolished with monovalent cations as the charge carrier, but slow inactivation was similar to that in Ba2+. Increased Ca2+, but not Ba2+, concentration (5-30 mM) enhanced the amplitude of fast inactivation and accelerated slow inactivation. However, the enhancement of fast inactivation was independent of Ca2+ influx, which indicates the relevant site is exposed to the extracellular solution and is inconsistent with CDI. Fast inactivation showed U-shaped voltage dependence in both Ba2+ and Ca2+, which appears to result from preferential inactivation from intermediate closed states (U-type inactivation). Taken together, the data support a role for extracellular divalent cations in modulating U-type inactivation. CDI appears to play a role in N-channel inactivation, but on a slower (sec) time scale.
    Journal of Neurophysiology 10/2006; 96(3):1075-83. DOI:10.1152/jn.01294.2005 · 2.89 Impact Factor
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    • "The intracellular pathway mediating voltage-dependent inhibition appears to involve direct interaction between G␤␥ and the N-channel (Herlitze et al., 1996; Ikeda, 1996; De Waard et al., 1997; Zamponi et al., 1997; Delmas et al., 1998; Stephens et al., 1998) (but see, Diversé-Pierluissi et al., 1995, 1997). The voltage dependence appears to result from the transient disruption of G␤␥-N-channel coupling, because the kinetics of reinhibition after strong depolarization become faster with higher concentrations of active G-proteins (Golard and Siegelbaum, 1993; Elmslie and Jones, 1994; Ehrlich and Elmslie, 1995; Zamponi and Snutch, 1998). Boland and Bean (1993) interpreted the willing–reluctant model in terms of G-protein binding and unbinding, with reluctant gating corresponding to the G-protein-bound channel and G-protein unbinding leading to the willing state. "
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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. · 6.34 Impact Factor
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    • "The rate of reinhibition of currents facilitated by prepulse application is related to the concentration of activated G-proteins. This was demonstrated with increasing concentrations of GTP-␥ -S (Lopez and Brown, 1991), neurotransmitter (Erlich and Elmslie, 1995), or free intracellular G ␤␥ (Zamponi and Snutch, 1998). Each of these studies showed that higher levels of G-protein activity resulted in faster rates of reinhibition following facilitation, thereby suggesting that prepulses lead to dissociation of the G-protein from the channel. "
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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 · 4.79 Impact Factor
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