Control of neurotransmitter release by two distinct membrane-binding faces of the Munc13-1 C1C2B region
Marcial Camacho, Bradley Quade, Thorsten Trimbuch, Junjie Xu, Levent Sari, Josep Rizo, Christian Rosenmund
Abstract
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 through distinct interactions of the C 1 C 2 B 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-Ca 2+ -PIP 2 -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 C 1 C 2 B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair Ca 2+ -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 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 two distinct faces of this region control neurotransmitter release and short-term presynaptic plasticity.