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 C₂ 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, Ca²⁺ ions exhibit significant binding to polyE. We found that 40 μM of Ca²⁺ adequately competes with the polyE-MUN interaction to reduce polyE inhibition. This concentration is comparable to presynaptic local [Ca²⁺]_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 Ca²⁺ influx and/or posttranslational modifications in the MUN domain may underlie Munc13-1 function in neurotransmitter release and short-term plasticity.