Complexin Clamps Asynchronous Release by Blocking a Secondary Ca2+ Sensor via Its Accessory α Helix

Complexin activates and clamps neurotransmitter release; impairing complexin function decreases synchronous, but increases spontaneous and asynchronous synaptic vesicle exocytosis. Here, we show that complexin-different from the Ca2+ sensor synaptotagmin-1-activates synchronous exocytosis by promoting synaptic vesicle priming, but clamps spontaneous and asynchronous exocytosis similar to synaptotagmin-1-by blocking a secondary Ca2+ sensor. Activation and clamping functions of complexin depend on distinct, autonomously acting sequences, namely its N-terminal region and accessory alpha helix, respectively. Mutations designed to test whether the accessory alpha helix of connplexin clamps exocytosis by inserting into SNARE-complexes support this hypothesis, suggesting that the accessory alpha helix blocks completion of trans-SNARE-complex assembly until Ca2+ binding to synaptotagmin relieves this block. Moreover, a juxtamembranous mutation in the SNARE-protein synaptobrevin-2, which presumably impairs force transfer from nascent trans-SNARE complexes onto fusing membranes, also unclamps spontaneous fusion by disinhibiting a secondary Ca2+ sensor. Thus, complexin performs mechanistically distinct activation and clamping functions that operate in conjunction with synaptotagmin-1 by controlling trans-SNARE-complex assembly.