Strikingly, mutant Doc2B not only rescued minirelease at all Ca2+ concentrations, but even slightly enhanced it (Figure 4D) and reversed the small increase in apparent Ca2+ affinity observed in the DR KD neurons (Figure 4E). Thus, mutant Doc2B is fully active in this functional assay. Spontaneous minirelease probably mediates important information transfer and may be mechanistically distinct from

evoked release (Sara et al., 2005, Fredj and Burrone, 2009, Stacey and Durand, 2000 and Sutton et al., 2006). Most spontaneous release is Ca2+ dependent, and controlled by at least selleck two different Ca2+ sensors: a low-affinity, high-cooperativity Ca2+ sensor in wild-type synapses and a high-affinity, low-cooperativity Ca2+ sensor in synaptotagmin- or complexin-deficient synapses (Sun et al., 2007, Xu et al.,

2009 and Yang et al., 2010). For wild-type synapses, two Ca2+ sensors for spontaneous release were proposed: synaptotagmins (Xu et al., 2009) and Doc2A and Doc2B (Groffen et al., 2010). No candidate Ca2+ sensor exists for minirelease in synaptotagmin-deficient synapses, although this Ca2+ sensor may be the same as that for asynchronous release, analogous to the proposed role of synaptotagmin as a Ca2+ sensor for both spontaneous and synchronous release in wild-type selleck chemicals llc synapses. Both synaptotagmin and Doc2 are attractive Ca2+ sensor candidates for spontaneous release based on their biochemical properties, but only for synaptotagmin is there evidence linking changes in Ca2+-binding affinity to changes in spontaneous release (Xu et al., 2009). Here, we have examined the potential role of Doc2 proteins as Ca2+ sensors in spontaneous release and their relation to asynchronous release. In doing so, we strove

to avoid potential problems caused by the expression of four closely related isoforms of Doc2 proteins that could produce functional redundancy and developed an approach that allowed simultaneous KD of four different targets with a rescue control (Figures 1A and 1B). Our data confirm KO studies showing that much Doc2 proteins are essential for normal minirelease—in fact, the degree of impairment in spontaneous release we observed with a 75% KD of all four isoforms (Figure 1 and Figure S1) is strikingly similar to that described for the Doc2A and Doc2B double KO (Groffen et al., 2010). We show that in DR KD synapses, the apparent Ca2+ dependence of minirelease exhibits a small but significant increase (Figure 1), but that otherwise no change in Ca2+ triggering of either spontaneous or evoked release is detected (Figure 2). Moreover, our results indicate that the DR KD does not alter synchronous or asynchronous evoked release and—importantly—does not impair the enhanced spontaneous release detected in Syt1 KO synapses (Figure 2). This latter result confirms the notion that spontaneous release events in Syt1 KO and wild-type neurons are qualitatively different, consistent with their distinct Ca2+ dependence (Xu et al., 2009).