Trans-SNARE complexes catalyze fast synaptic vesicle fusion and bind complexin, but the function of complexin binding to SNARE complexes remains unclear. Here we show that in neuronal synapses, complexin simultaneously suppressed spontaneous fusion and activated fast Ca 2+ -evoked fusion. The dual function of complexin required SNARE binding, and additionally involved distinct N-terminal sequences of complexin that localize to the point where trans-SNARE complexes insert into the fusing membranes, suggesting that complexin controls the force that trans-SNARE complexes apply onto the fusing membranes. Consistent with this hypothesis, a mutation in the membrane insertion sequence of the v-SNARE synaptobrevin/VAMP phenocopied the complexin loss-of-function state without impairing complexin-binding to SNARE complexes. Thus, complexin probably activates and clamps the force-transfer from assembled trans-SNARE complexes onto fusing membranes.Synaptic vesicle fusion is driven by assembly of trans-SNARE complexes (or SNAREpins) from syntaxin-1 and SNAP-25 on the plasma membrane, and synaptobrevin/ VAMP on the vesicle membrane [1][2][3]. Ca 2+ then triggers fast synchronous synaptic vesicle fusion by binding to the Ca 2+ -sensor synaptotagmin [4][5][6]. Besides SNARE proteins and synaptotagmin, fast Ca 2+ -triggered fusion requires complexin [7]. Complexin is composed of short N-and C-terminal sequences and two central α-helices. Complexin binds to SNARE complexes via its central α-helix, which inserts in an anti-parallel orientation into a groove formed by synaptobrevin/VAMP and syntaxin-1 [8,9]. Although multiple approaches have revealed an essential role of complexin in synaptic fusion [7,[10][11][12][13][14][15], the nature of this role remains unclear. In vertebrate autapses, deletion of complexin selectively impairs fast synchronous neurotransmitter release without changing asynchronous or spontaneous release [7,10]. In in vitro fusion assays, conversely, addition of complexin causes a general block of SNARE-dependent fusion, indicating that complexin is a SNARE clamp [11][12][13][14]. In Drosophila neuromuscular synapses, deletion of complexin produces a >20-fold increase in spontaneous release but only a small decrease in evoked release [15]. Thus, the role of complexin in fusion is unclear. Moreover, even the importance of complexin SNARE- [16,17]. Here we addressed these questions with two complementary approaches -RNAi-dependent knockdown of complexin with rescue, and replacing wild-type synaptobrevin with specific mutants using synaptobrevin knockout (KO) mice [18].We knocked down complexin expression in cultured cortical neurons using an shRNA that targets both complexin-1 and -2, the only complexin isoforms significantly expressed in these neurons [19]. For this purpose, we used lentiviruses that simultaneously synthesize the complexin shRNA and either GFP, wild-type complexin-1, or 4M-mutant complexin-1 that is unable to bind to SNARE complexes (19,20). Lentivirus expressing GFP without the shRNA ...