The voltage-sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery

Although N- and P-type Ca2+ channels predominant in fast-secreting systems, Lc-type Ca2+ channels (C-class) can play a similar role in certain secreto ry cells and synapses. For example, in retinal bipolar cells, Ca2+ entry th rough the Lc channels triggers ultrafast exocytosis, and in pancreatic p-ce lls, evoked secretion is highly sensitive to Ca2+. These findings suggest t hat a rapidly release pool of vesicles colocalizes with the Ca2+ channels t o allow high Ca2+ concentration and a tight coupling of the Lc channels at the release site. In binding studies, we show that the Lc channel is physic ally associated with synaptotagmin (p65) and the soluble N-ethylmaleimide-s ensitive attachment proteins receptors: syntaxin and synaptosomal-associate d protein of 25 kDa. Soluble N-ethylmaleimide-sensitive attachent proteins receptors coexpressed in Xenopus oocytes along with the Lc channel modify t he kinetic properties of the channel. The modulatory action of syntaxin can be overcome by coexpressing p65, where at a certain ratio of p65/syntaxin, the channel regains its unaltered kinetic parameters. The cytosolic region of the channel, Lc(753-893), Separating repeats II-III of its alpha 1C sub unit, interacts with p65 and "pulls" down native p65 from rat brain membran es. LC753-893 injected into single insulin-secreting beta-cell, inhibits se cretion in response to channel opening, but not in response to photolysis o f caged Ca2+, nor does it affect Ca2+ current. These results suggest that L C753-893 competes with the endogenous channel for the synaptic proteins and disrupts the spatial coupling with the secretory apparatus. The molecular organization of the Lc channel and the secretory machinery into a multiprot ein complex (named excitosome) appears to be essential for an effective dep olarization evoked exocytosis.