Abstract
Cognate soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins are now known to associate the secretory 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 β-cells, t-SNARE proteins, 25-kDa synaptosomal-associated protein (SNAP-25), and syntaxin 1A, independently inhibit the L-type Ca2+ channel (LCa). However, when both are present, they actually exhibit stimulatory actions on the LCa. This suggests that the positive regulation of the LCa 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 LCa. These SNARE proteins were recently shown to bind the Lc753–893 domain corresponding to the II and III intracellular loop of the α1C subunit of the LCa. In this study, using patch-clamp methods on primary pancreatic β-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 198–206) domains, on the LCa, particularly at the Lc753–893 domain. Intracellular application of SNAP-251–206 in primary β-cells decreased LCa currents by ∼15%. The reduction in LCa currents was counteracted by coapplication of Lc753–893. Overexpression or injection of wild-type SNAP-25 in HIT cells reduced LCa currents by ∼30%, and this inhibition was also blocked by the recombinant Lc753–893 peptide. Expression of BoNT/A surprisingly caused an even greater reduction of LCa 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-251–197 increased LCa currents (by 19% at 10 mV), and these effects were blocked by the Lc753–893 peptide. In contrast, injection of SNAP-25198–206 peptide into untransfected cells inhibited LCa currents (by 47%), and more remarkably, these inhibitory effects dominated over the stimulatory effects of SNAP-251–197 overexpression (by 34%). Therefore, the SNARE protein SNAP-25 possesses distinct inhibitory and stimulatory domains that act on the LCa. 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 LCa. 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 LCa 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 LC subtype Ca2+ channel activity in both primary β-cells and insulinoma HIT-T15 cells.
