Control of neurotransmitter release and presynaptic plasticity by re-orientation of membrane-bound Munc13-1

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C ₁ domain, a Ca ²⁺ /PIP ₂ -binding C ₂ B domain, a MUN domain and a C ₂ C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane in two different orientations mediated by distinct interactions of the C ₁ C ₂ B region with the plasma membrane: i) one involving a polybasic face that yields a perpendicular orientation of Munc13-1 and hinders release; and ii) another involving the DAG-Ca ²⁺ -PIP ₂ -binding face that induces a slanted orientation and facilitates release. Here we have tested this model and investigated the role of the C ₁ C ₂ B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair synaptic vesicle priming in primary murine hippocampal cultures, and Ca ²⁺ -independent liposome bridging and fusion in in vitro reconstitution assays. A K720E mutation in the polybasic face and a K706E mutation in the C ₂ B domain Ca ²⁺ -binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca ²⁺ -evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C ₁ -C ₂ B region of Munc13-1 plays a central role in vesicle priming and support the notion that re-orientation of Munc13-1 controls neurotransmitter release and short-term presynaptic plasticity.