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 Ca ²⁺/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.