NBI 27914 hydrochloride australia Evidence suggests that pho

Evidence suggests that phosphorylation increases synaptotagmin affinity for the SNARE complex [15], [128]. It is, however, unclear how this might affect release. Synaptotagmin has been identified as part of the minimal vesicle docking machinery in chromaffin NBI 27914 hydrochloride australia [129]. Thus, phosphorylation of synaptotagmin could modulate docking and because vesicle fusogenicity is heterogeneous, docking of different vesicle populations could lead to changes in apparent calcium sensitivity of exocytosis. For example, phosphorylation of synaptotagmin-7 by PKC would enhance its affinity for the SNARE complex and consequently enhance docking of synaptotagmin-7-labeled vesicles with the plasma membrane. This could lead to a change in the apparent calcium sensitivity of exocytosis, as synaptotagmin-7 has a higher calcium affinity than synaptotagmin-1 [127]. Consistent with a scenario under which different synaptotagmin isoforms are relevant PKC targets in different cellular systems, Nagy et al. showed in chromaffin cells that overexpression of synaptotagmin-1 phosphomutants and phosphomimetics did not appear to alter exocytosis, whereas in another system, hippocampal neurons, phosphomimetics of synaptotagmin-1 regulate exocytosis [73], [130]. Synaptotagmin isoform-specific phosphorylation has yet to be demonstrated, but it represents an exciting hypothesis for the molecular mechanisms underlying the sensitization of the exocytotic machinery by PKC. Finally, SNAP-25 appears to be a PKC substrate in vivo, though the role of this modification in insulin secretion is still controversial. The most compelling evidence for physiologically relevant phosphorylation-dependent control of SNAP-25 is that knock-in mice expressing a SNAP-25 mutant that cannot be phosphorylated display neurological defects including anxiety and epilepsy [131], [132]. SNAP-25 phosphorylation was first observed in vitro and was quickly shown to occur in response to PMA treatment in PC12 cells, correlating with enhanced exocytosis [109], [133]. The primary site for PKC-mediated phosphorylation of SNAP-25 is Ser187 [133]. Further studies in insulin-secreting, neuroendocrine, and neuronal systems have been at odds over whether PKC-mediated phosphorylation of SNAP25 directly affects or is only correlated with exocytosis [134], [135], [136], [137], [138], [139]. It is currently thought that phosphorylation of SNAP-25 increases its interaction with syntaxin and that this increases the population of the highly-calcium sensitive pool of vesicles [139], [140]. More recent work suggests that endogenous SNAP-25 may have complicated earlier studies on phosphomimetic SNAP-25 mutants [138], [139]. Despite phenotypic effects in neurons of model organisms, SNAP-25’s physiological role in insulin secretion remains controversial. In addition to the exocytotic proteins discussed above, PKC could affect insulin release by modulating the amount of cargo released at single vesicles (Fig. 1B3–4). More complete release of insulin from single vesicles could enhance the overall amount of insulin released without changing the total number of fusion events per cell. Early evidence for a role for PKC modulating fusion pore behavior in neuromuscular junctions came from the observation that staurosporine, a PKC inhibitor, affected fusion pore behavior and vesicle collapse into the plasma membrane [141]. In neurons, PKC was shown to affect exocytosis after calcium binding to synaptotagmin, suggesting that fusion pore regulation could play a role [142]. In chromaffin cells, amperometry has been used to examine the kinetics of the fusion pore at very high time resolution. PKC activation appears to accelerate fusion pore expansion [117], [143], [144], however, some studies have also shown that PKC tends to decrease the average quantal size of fusion events [117], [143]. Quantal size could be linked to the probability of kiss-and-run exocytosis, which may be a function of the stimulation protocol, making it challenging to interpret these findings with respect to physiological glucose stimulation.