Modulation of N-Type Calcium Channel Activity by G-Proteins and Protein Kinase C

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 α1B 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-γ-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-γ-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-β-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.