Vesicle priming and recruitment by ubMunc13-2 are differentially regulated by calcium and calmodulin.

Ca2+ regulates multiple processes in nerve terminals, including synaptic vesicle recruitment, priming, and fusion. Munc13s, the mammalian homologs of Caenorhabditis elegans Unc13, are essential vesicle-priming proteins and contain multiple regulatory domains that bind second messengers such as diacylglycerol and Ca2+/calmodulin (Ca2+/CaM). Binding of Ca2+/CaM is necessary for the regulatory effect that allows Munc13-1 and ubMunc13-2 to promote short-term synaptic plasticity. However, the relative contributions of Ca2+ and Ca2+/CaM to vesicle priming and recruitment by Munc13 are not known. Here, we investigated the effect of Ca2+/CaM binding on ubMunc13-2 activity in chromaffin cells via membrane-capacitance measurements and a detailed simulation of the exocytotic machinery. Stimulating secretion under various basal Ca2+ concentrations from cells overexpressing either ubMunc13-2 or a ubMunc13-2 mutant deficient in CaM binding enabled a distinction between the effects of Ca2+ and Ca2+/CaM. We show that vesicle priming by ubMunc13-2 is Ca2+ dependent but independent of CaM binding to ubMunc13-2. However, Ca2+/CaM binding to ubMunc13-2 specifically promotes vesicle recruitment during ongoing stimulation. Based on the experimental data and our simulation, we propose that ubMunc13-2 is activated by two Ca2+-dependent processes: a slow activation mode operating at low Ca2+ concentrations, in which ubMunc13-2 acts as a priming switch, and a fast mode at high Ca2+ concentrations, in which ubMunc13-2 is activated in a Ca2+/CaM-dependent manner and accelerates vesicle recruitment and maturation during stimulation. These different Ca2+ activation steps determine the kinetic properties of exocytosis and vesicle recruitment and can thus alter plasticity and efficacy of transmitter release.

Tomosyn inhibits priming of large dense-core vesicles in a calcium-dependent manner.

Neurotransmitter release is a multistep process that is coordinated by a large number of synaptic proteins and depends on proper protein-protein interactions. Using morphological, capacitance, and amperometric measurements, we investigated the effect of tomosyn, a Syntaxin-binding protein, on the different kinetic components of exocytosis in adrenal chromaffin cells. Overexpression of tomosyn decreased the release probability and led to a 50% reduction in the number of fusion-competent vesicles. The number of docked vesicles and the fusion kinetics of single vesicles were not altered suggesting that tomosyn inhibits the priming step. Interestingly, this inhibition is partially relieved at elevated calcium concentration. Calcium ramp experiments supported the latter finding and indicated that the reduction in secretion is caused by a shift in the calcium-dependence of release. These results indicate that secretion is not entirely blocked but occurs at higher calcium concentrations. We suggest that tomosyn inhibits the priming step and impairs the efficiency of vesicle pool refilling in a calcium-dependent manner.