The available clamping data also raise additional questions. It is striking that the clamping function of Syt1 requires the wild-type sequences selleck chemicals llc of both its C2A and its C2B domain Ca2+-binding sites (Shin et al., 2009 and Lee et al., 2013). Clamping itself cannot depend on Ca2+ binding to these domains because such Ca2+ binding triggers exocytosis (Fernández-Chacón et al., 2001, Rhee et al., 2005, Pang et al., 2006a and Xu et al., 2009). It is unlikely

that the Ca2+-binding sites of the C2 domains are partially occupied in nerve terminals at rest given the high cooperativity of Ca2+ binding to C2 domains (Davletov and Südhof, 1993 and Kohout et al., 2002). The most parsimonious interpretation is that prior to Ca2+ binding, the sequences of the C2 domain Ca2+-binding sites interact with an unidentified target in a Ca2+-independent Selleckchem Dolutegravir manner, such that this interaction clamps mini release but is severed by Ca2+ binding (Figure 2). Apart from prompting the question of the nature of this target, this interpretation implies that contrary to current models, Syt1 acts on the release process

upstream of Ca2+ triggering, before the last millisecond in the lifetime of a vesicle, during the stage during which the fusion machinery is set up to prepare for the demise of the vesicle and the popping of its fusion pore (Figure 2). Future experiments will have to explore the nature of this activity. Complexin is a universal cofactor for synaptotagmin in all Ca2+-triggered fusion reactions that have been examined (e.g., see Reim et al., 2001, Tang et al., 2006, Cai et al., 2008, Jorquera et al., 2012 and Cao et al., 2013). Three distinct changes caused by the loss of function of complexin have been defined: a decrease in Ca2+ triggering of release, an increase in spontaneous mini release, and a decrease in the size of the RRP. In two of these activities—the Ca2+ triggering of release and the clamping of mini release—complexin

performs analogous roles to Syt1 and Syt2 but with considerably smaller effect sizes. How does a small molecule like complexin, composed of only ∼130 residues, act to activate and clamp synaptic vesicles for synaptotagmin action? Atomic structures revealed that complexin, those when bound to assembled SNARE complexes, contains two short α helices flanked by flexible sequences (Chen et al., 2002). The central, more C-terminal α helix is bound to the SNARE complex and is essential for all complexin functions (Maximov et al., 2009). The accessory, more N-terminal α helix is required only for the clamping but not for the activating function of complexin (Yang et al., 2010). The flexible N-terminal sequence of complexin, conversely, mediates only the activating but not the clamping function of complexin (Xue et al., 2007 and Maximov et al., 2009).