Regulation of synaptic vesicle fusion by protein kinase C

Activation of protein kinase C (PKC) by phorbol esters stimulates secretion of neurotransmitters from most presynaptic terminals. This action of PKC may be even more general, because secretion from many non-neuronal cells is also stimulated by these agents. Despite recent advances in our understanding of the molecular mechanisms underlying transmitter release, the mode of action of PKC in nerve terminals remains unclear. PKC activation might enhance the presynaptic Ca2+ signal that triggers release, for example by modulating ion channels to increase Ca2+ influx or by decreasing Ca2+ buffering or removal. Alternatively, PKC could act independently of Ca2+ entry, by increasing the number of release sites or releasable vesicles, or by making individual vesicles more sensitive to entering Ca2+. Distinguishing among these possible mechanisms has proven difficult, in part because the electrophysiological measurements typically used for such studies provide only indirect information about Ca2+ signalling and vesicle trafficking. The report by Yawo in this issue of The Journal of Physiology (Yawo, 1999) provides clear new insight into this long-standing question. Extracellular application of phorbol esters increased transmission at the classical giant synapse of the chick ciliary ganglion. Although this was due to a stimulation of transmitter release, direct optical measurements from the giant presynaptic terminal showed that phorbol ester treatment had no effect on presynaptic Ca2+ influx, or Ca2+ buffering and removal. These observations unambiguously establish the important point that the target of phorbol esters lies downstream of the presynaptic Ca2+ signal that triggers release. The work of Yawo (1999) also provides new clues about the mechanisms by which phorbol esters stimulate neurotransmitter release. Phorbol esters activate PKC by binding to the regulatory C1-domain found in most PKC isoforms. However, at least one additional presynaptic target of phorbol esters, the Munc-13 protein family, has recently been identified (Betz et al. 1998). The drug bisindolylmaleimide (BIS) can be used to distinguish between these two target proteins. BIS is a potent and selective inhibitor of the binding of ATP to PKC, yet should have no effect on Munc-13 (which apparently does not bind ATP). Yawo (1999) found that BIS eliminated the action of phorbol esters on transmitter release, suggesting that phorbol esters act via PKC at the chick synapse. In contrast, at the rat calyx of Held synapse, blockade of presynaptic phorbol ester effects requires both inhibition of PKC and disruption of the binding of Munc-13 to the vesicle protein Doc2 (T. Hori & T. Takahashi, personal communication). It therefore seems likely that both PKC and Munc-13 are targets of phorbol esters in certain presynaptic terminals. A molecular understanding of how phorbol esters modulate transmitter release will require further identification of the physiological substrates and binding interactions of PKC and Munc-13. PKC phosphorylates several cytoskeletal proteins in vitro, suggesting that phorbol esters could enhance release by recruiting additional releasable vesicles. This is consistent with previous indications that PKC activators ‘mobilize’ vesicles in Aplysia presynaptic terminals (Ghirardi et al. 1992) and increase the size of the readily releasable pool of vesicles in hippocampal neurons (Stevens & Sullivan, 1998). However, the work of Yawo (1999) indicates that this is unlikely to be the case at the chick synapse. His quantitative analysis of the kinetics of synaptic depression indicated that phorbol esters did not change the size of a readily releasable pool of synaptic vesicles, the rate of vesicle pool refilling, or the number of release sites. Thus, phorbol esters may have diverse actions in different presynaptic terminals, perhaps due to the differential distribution of PKC and Munc-13 isoforms. Yawo (1999) observed that phorbol esters cause a leftward shift in the relationship between extracellular Ca2+ and transmitter release, suggesting that phorbol esters increase the Ca2+ sensitivity of exocytosis without affecting the number of releasable vesicles or Ca2+-binding sites. This implies that phorbol esters are affecting the priming or fusion of synaptic vesicles, and among the many proteins implicated in these two processes are several possible downstream targets of phorbol esters. The SNARE complex proteins that appear to mediate synaptic vesicle priming and fusion reactions serve as PKC substrates. For example, phosphorylation of SNAP-25 by PKC decreases its interaction with syntaxin (Shimazaki et al. 1996), which might enhance Ca2+-dependent exocytosis by accelerating dissociation of the SNARE complex. Further, PKC-dependent phosphorylation of n-Sec1 (also called Munc-18) inhibits its interaction with syntaxin, which might enhance exocytosis by increasing the amount of syntaxin available to form SNARE complexes (Fujita et al. 1996). Synaptotagmin I, a Ca2+-binding protein thought to serve as a Ca2+ receptor for transmitter release, is a substrate for PKC both in vitro and in vivo (S. Hilfiker, V. A. Pieribone, C. Nordstedt, P. Greengard & A. J. Czernik, unpublished observations). PKC might enhance exocytosis by modulating the Ca2+ sensitivity of synaptotagmin, by enhancing its binding to either Ca2+ or to the SNARE complex. Finally, the phorbol ester-mediated enhancement of exocytosis might be mediated through effects on Munc-13 proteins. These proteins interact with syntaxin, as well as with the vesicle protein Doc2, a putative Ca2+-dependent regulator of neurotransmitter release (Betz et al. 1998); phorbol ester binding to Munc-13 could then affect transmitter release by altering the availability of syntaxin, or by affecting the Ca2+ sensitivity of Doc2. Thus, a phorbol ester-mediated change in the affinity and/or availability of SNARE proteins may make more vesicles available for fusion by increasing the priming of vesicles within the releasable pool, followed by an increase in the efficiency by which Ca2+-binding proteins such as synaptotagmin or Doc2 transmit the Ca2+ signal to the SNARE-based fusion machinery. In summary, the work of Yawo (1999) very elegantly emphasizes that PKC enhances the sensitivity of neurotransmitter release to entering Ca2+. Continued investigation of the molecular mechanism(s) underlying this physiological response should further clarify our understanding of synaptic vesicle fusion and its control by modulatory signalling pathways.