Control of neurotransmitter release by two distinct membrane-binding faces of the munc13-1 c₁c₂b region

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C₁ domain, a Ca²⁺/PIP₂-binding C₂B domain, a MUN domain and a C₂C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane through distinct interactions of the C₁C₂B region with the plasma membrane: i) one involving a polybasic face that is expected to yield a perpendicular orientation of Munc13-1 and hinder release; and ii) another involving the DAG-Ca²⁺-PIP₂-binding face that is predicted to result in a slanted orientation and facilitate release. Here we have tested this model and investigated the role of the C₁C₂B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair Ca²⁺-independent liposome bridging and fusion in in vitro reconstitution assays, and synaptic vesicle priming in primary murine hippocampal cultures. A K720E mutation in the polybasic face and a K706E mutation in the C₂B domain Ca²⁺-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca²⁺-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C₁-C₂B region of Munc13-1 plays a central role in vesicle priming and support the notion that two distinct faces of this region control neurotransmitter release and short-term presynaptic plasticity.