, 2009), possibly due to
the effect of additional regulatory mechanisms—for example, it is possible that Syt1 may become inhibited by phosphorylation or that Syt7 may be constitutively activated by phosphorylation or by alternative splicing ( Sugita et al., 2001). The simple model of Syt1 and Syt7 function that we propose accounts for all of the available observations but raises new questions. What precisely are the mechanisms mediating the functions of Syt1 and Syt7 in Ca2+ triggering of evoked release and in clamping minirelease? SNARE and phospholipid binding by Syt1 and Syt7 are probably involved (Perin et al., 1990, Bennett et al., 1992, Li et al., 1995, Chapman et al., 1995 and Sugita et al., 2002), but the precise nature of the protein selleck chemical complexes that mediate these functions remains to be established. In particular, the clamping function of Syt1 and of Syt7 probably operates upstream of Ca2+ influx and thus implies that Syt1 and Syt7 perform Ca2+-independent selleck compound actions in release. A second question regards the identity of the Ca2+ sensor for the increased
minirelease in Syt1 KO synapses that is not mediated by Syt7. This sensor has a lower apparent Ca2+ cooperativity than the Ca2+ sensor for evoked release (Xu et al., 2009) and is also clamped by complexin (Yang et al., 2010), suggesting that it is not a synaptotagmin. It is tempting to speculate that this Ca2+ sensor may
catalyze SNARE complex assembly and could be involved in Munc13 function upstream of regular Ca2+ triggering by Syt1 and Syt7 (Augustin et al., 1999 and Varoqueaux et al., 2002), but this possibility has not been tested. Furthermore, not all asynchronous release is blocked in Syt1/Syt7 double-deficient neurons (see Figures 2, 3, and 6). The residual asynchronous release may be mediated by remaining Syt1 or Syt7 protein or by other synaptotagmin isoforms. Alternatively, 3-mercaptopyruvate sulfurtransferase the remaining asynchronous release may reflect an additional, qualitatively different release process that may be driven by the same molecular mechanisms as those that lead to the increased minirelease in Syt1 KO synapses. Finally, our data do not rule out additional functions for Syt1 and Syt7. For example, Syt1 and Syt7 could additionally contribute to vesicle priming and/or regulate the repriming rate of synaptic vesicles. However, the phenotypes we observed cannot be explained solely by a potential function of Syt7 in priming or in regulating repriming. Specifically, impairments in priming or in the Ca2+-dependent regulation of repriming could not account for the sustained increased minifrequency in Syt1/Syt7 double-deficient neurons (Figure 4), for the decrease in isolated single responses in these neurons (Figure 3) and for the selective loss of asynchronous responses in Syt7 KO neurons still expressing Syt1 (Figure 7).