Control of neurotransmitter release by two distinct membrane-binding faces of the Munc13-1 C(1)C(2)B region

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.