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  • PKC s effect on quantal size may involve regulation of

    2021-11-24

    PKC’s effect on quantal size may involve regulation of endocytotic protein components responsible for mediating kiss-and-run exocytosis. There is a growing body of evidence to suggest that canonical endocytotic proteins—dynamin, amphiphysin, syndapin, and others—may play a role in modulating fusion pore expansion or collapse via a kiss-and-run mechanism [127], [145], [146], [147], [148], [149]. Dynamin and other endocytotic proteins are known to be modulated by PKC activity [150], [151]. By targeting these proteins, PKC could be modulating fusion pore dynamics or closure and thus affecting the amount of insulin release. This casr has not been fully explored and future work with insulin granules are an ideal system to determine if kiss-and-run or sub-quantal cargo release could play an important role in physiological insulin levels. PKC may also modulate fusion pore expansion and kinetics through its control of exocytotic protein factors. Barclay et al. suggest munc18 phosphorylation has a direct effect on fusion pore kinetics, though munc18 itself is unlikely to have direct effects on the membrane [117]. Instead munc18 could be either allowing more trans-SNARE complex assembly or enabling more efficient zippering of SNAREs [152], [153]. In addition to modulating the amount of SNARE complex formation, munc18 could directly affect syntaxin-mediated regulation of fusion pore diameter and behavior [154]. Munc18 also appears to help regulate the selection of larger diameter vesicles for docking and fusion, and vesicle size directly contributes to fusion pore size [154]. Recent work has implicated synaptotagmin in fusion pore kinetic control [127], though it is not clear how its phosphorylation might affect this putative function. Obviously, lipids play a central role in fusion pore structure and dynamics [155], and Xue et al. invoke a lipid-mediated mechanism to account for PKC effects on fusion pore dynamics [144]. PKC activates phospholipase D, an enzyme which produces phosphatidic acid via the cleavage of phosphatidylcholine in the membrane. Phosphatidic acid has a small headgroup and high spontaneous negative curvature that would help to accommodate the high membrane curvatures at the nascent fusion pore. Overall, it seems possible PKC could exert an effect at the nascent fusion pore itself, though the mechanism of this remains unclear and requires further study, particularly with respect to insulin granules.
    Bringing it all together PKC also potentiates insulin release by enhancing the calcium sensitivity of the exocytotic machinery, but it remains unclear how this is achieved. No known modifications of exocytotic proteins directly increases their calcium affinity, which would be the most straightforward mechanism to enhance the calcium sensitivity of exocytosis. One mechanism to achieve calcium sensitization could involve isoform specific phosphorylation of munc18 or synaptotagmin. As reserve vesicles translocate to the plasma membrane, PKC may help to determine which vesicles successfully dock. Different isoforms of both munc18 and synaptotagmin display distinct calcium affinities that could be used to modulate exocytotic calcium sensitivity [124], [125]. Such a model is consistent with observations that PKC acts to increase the size of the highly calcium-sensitive vesicle pool [156], [157], [158]. Several studies have shown newcomer vesicles are more likely to fuse and are more calcium sensitive, which is consistent with this model [159], [160]. Another possible mechanism to enhance the calcium sensitivity of exocytosis is to increase the size of the RRP [52]. Phosphorylation of all three of the exocytotic proteins we discussed—munc18, synaptotagmin, and SNAP25—could enhance vesicle docking by either providing more docking sites or increasing the affinity of the docking machinery for vesicles. A larger pool of docked vesicles with a homogeneous calcium sensitivity for each vesicle could yield a larger exocytotic response to a given calcium stimulus [52].