A specific negatively charged sequence confers intramolecular regulation on Munc13-1 function in synaptic exocytosis

Munc13 family proteins are crucial for the secretion of neurotransmitters and hormones necessary for cell communication. They share a conserved C-terminal region that includes C 2 and the MUN domains, which facilitate membrane interactions and the assembly of soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complexes. Neuronal isoforms of Munc13 possess a variable N-terminal region that is essential for neurotransmitter release and short-term plasticity, although the precise functions of this region remain not fully understood. Here, we identified a negatively charged sequence within the N terminus of Munc13-1, termed polyE, which is specific to Munc13-1 among all Munc13 isoforms and potentially derived from a common ancestor of homeotherms. We found that polyE binds significantly to the MUN domain through charge–charge interactions, inhibiting MUN activity in promoting SNARE complex assembly. Disrupting the polyE–MUN interaction by introducing pseudophosphorylated mutations in the MUN domain alleviates this inhibition, thereby enhancing neurotransmitter release. Strikingly, Ca 2+ ions exhibit significant binding to polyE. We found that 40 μM of Ca 2+ adequately competes with the polyE–MUN interaction to reduce polyE inhibition. This concentration is comparable to presynaptic local [Ca 2+ ] i triggered by a single action potential. Taken together, these results indicate an autoinhibition conformation of Munc13-1 mediated by the polyE–MUN interaction. In addition, the relief of this autoinhibition conformation of Munc13-1 by presynaptic Ca 2+ influx and/or posttranslational modifications in the MUN domain may underlie Munc13-1 function in neurotransmitter release and short-term plasticity.