Modulation of L-type Ca2+ channels by distinct domains within SNAP-25

Cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are now known to associate the secretor vesicle with both the target plasma membrane and Ca2+ channels in order to mediate the sequence of events leading to exocytosis in neurons and neuroendocrine cells. Neuroendocrine cells, particularly insulin-secreting islet beta-cells, t-SNARE proteins, 25-kDa synaptosomal-associated protein (SNAP-25), and syntaxin 1A, independently inhibit the L-type Ca2+ channel (L-Ca). However, when both are present, they actually exhibit stimulatory actions on the L-Ca. This suggests that the positive regulation of the L-Ca is conferred by a multi-SNARE protein complex. We hypothesized an alternate explanation, which is that each of these SNARE proteins possess distinct inhibitory and stimulatory domains that act on the L-Ca. These SNARE proteins were recently shown to bind the Lc(753-893) domain corresponding to the II and III intracellular loop of the alpha1C subunit of the L-Ca. In this study, using patch-clamp methods on primary pancreatic beta-cells and insulinoma HIT-T15 cells, we examined the functional interactions of the botulinum neurotoxin A (BoNT/A) cleavage products of SNAP-25, including NH2-terminal (1-197 amino acids) and COOH-terminal (amino acid 198206) domains, on the L-Ca, particularly at the Lc(753-893) domain. Intracellular application of SNAP-25(1-206) in primary beta-cells decreased L-Ca currents by similar to15%. The reduction in L-Ca currents was counteracted by coapplication of Lc(753-893). Overexpression or injection of wildtype SNAP-25 in HIT cells reduced L-Ca currents by similar to30%, and this inhibition was also blocked by the recombinant Lc(753-893) peptide. Expression of BoNT/A surprisingly caused an even greater reduction of L-Ca currents (by 41%), suggesting that the BoNT/A cleavage products of SNAP-25 might possess distinct inhibitory and positive regulatory domains. Indeed, expression of SNAP-25(1-197) increased L-Ca currents (by 19% at 10 mV), and these effects were blocked by the Lc(753-893) peptide. In contrast, injection of SNAP-25(198-206) peptide into untransfected cells inhibited L-Ca currents (by 47%), and more remarkably, these inhibitory effects dominated over the stimulatory effects of SNAP-25(1-197) overexpression (by 34%). Therefore, the SNARE protein SNAP-25 possesses distinct inhibitory and stimulatory domains that act on the L-Ca. The COOH-terminal 197-206 domain of SNAP-25, whose inhibitory actions dominate over the opposing stimulatory NH2-terminal domain, likely confers the inhibitory actions of SNAP-25 on the L-Ca. We postulate that the eventual accelerated proteolysis of SNAP-25 brought about by BoNT/A cleavage allows the relatively intact NH2-terminal SNAP-25 domain to assert its stimulatory action on the L-Ca to increase Ca2+ influx, and this could in part explain the observed weak or inconsistent inhibitory effects of BoNT/A on insulin secretion. The present study suggests that distinct domains within SNAP-25 modulate L-C subtype Ca2+ channel activity in both primary beta-cells and insulinoma HIT-T15 cells.