Abstract
Complexins are small soluble proteins that bind to assembling SNARE complexes during synaptic vesicle exocytosis, which in turn mediates neurotransmitter release. Complexins are required for clamping of spontaneous "mini " release and for the priming and synaptotagmin-dependent Ca(2+) triggering of evoked release. Mammalian genomes encode four complexins that are composed of an N-terminal unstructured sequence that activates synaptic exocytosis, an accessory α-helix that clamps exocytosis, an essential central α-helix that binds to assembling SNARE complexes and is required for all of its functions, and a long, apparently unstructured C-terminal sequence whose function remains unclear. Here, we used cultured mouse neurons to show that the C-terminal sequence of complexin-1 is not required for its synaptotagmin-activating function but is essential for its priming and clamping functions. Wild-type complexin-3 did not clamp exocytosis but nevertheless fully primed and activated exocytosis. Strikingly, exchanging the complexin-1 C terminus for the complexin-3 C terminus abrogated clamping, whereas exchanging the complexin-3 C terminus for the complexin-1 C terminus enabled clamping. Analysis of point mutations in the complexin-1 C terminus identified two single amino-acid substitutions that impaired clamping without altering the activation function of complexin-1. Examination of release induced by stimulus trains revealed that clamping-deficient C-terminal complexin mutants produced a modest relative increase in delayed release. Overall, our results show that the relatively large C-terminal complexin-1 sequence acts in priming and clamping synaptic exocytosis and demonstrate that the clamping function is not conserved in complexin-3, presumably because of its distinct C-terminal sequences.