Neurotransmitter is released from presynaptic nerve terminals by the fusion of synaptic vesicles with the plasma membrane. Synaptic vesicle exocytosis requires the formation of SNARE complexes, comprised of synaptobrevin, SNAP-25 and syntaxin. In solution, syntaxin adopts a closed configuration that is incompatible with SNARE complex formation. UNC-18, a neuronal protein implicated in vesicle exocytosis, binds to syntaxin in the closed configuration. Loss-of-function mutations in
unc-18 nearly eliminate vesicle exocytosis. A model based on these data proposed that UNC-18 bound closed syntaxin, promoted the opening of syntaxin, and thereby promoted the formation of the SNARE complex and hence exocytosis. A prediction of this model is that if one could eliminate the syntaxin - UNC-18 interaction then neurotransmission would be blocked. To test this model we engineered mutations into syntaxin that reduce UNC-18 binding, and mutations into UNC-18 that reduce syntaxin binding. We have tested the consequence of these mutations on synaptic vesicle exocytosis. Scanning alanine replacement experiments of the mammalian syntaxin homolog identified two residues, L165/E166, that when mutated to alanine render syntaxin incapable of binding UNC-18. We made the corresponding mutations into C. elegans syntaxin, L166A/E167A. Pulldown experiments demonstrate that this mutant form of syntaxin does not bind UNC-18 from worm lysates. We expressed this mutant form of syntaxin in a null background,
unc-64(
js115) . Mutant syntaxin that does not bind UNC-18 is capable of rescuing the lethality and locomotory phenotypes associated with the syntaxin null, suggesting that the mutant protein functions in vivo and that UNC-18 function is not via syntaxin. In fact, such worms are hypersensitive to Aldicarb, suggesting that UNC-18 interactions with syntaxin inhibit release. Studies of the UNC-18 homolog in Drosophila , ROP, identified a mutation, R50C, that decreases the affinity of ROP for syntaxin without affecting other known UNC-18 interactions. We made the corresponding mutation in UNC-18, R39C, and expressed the protein in
unc-18(
e234) animals. Expression of R39C UNC-18 rescues the paralysis associated with
e234 suggesting the mutant protein functions in vivo and that the interaction of UNC-18 with syntaxin is not relevant to the UNC-18 role in facilitation of exocytosis. However, animals expressing the mutant UNC-18 are hypersensitive to the effects of Aldicarb, suggesting that more acetylcholine is released in these animals. Thus, UNC-18 binding to syntaxin appears to inhibit rather than facilitate vesicle exocytosis. Finally, if UNC-18 is required to promote the open configuration of syntaxin for SNARE complex formation then a constitutively open form of syntaxin should bypass the requirement for UNC-18. The L165/E166 mutations which reduce UNC-18 binding also renders syntaxin in a constitutively open configuration. To test if the open form of syntaxin can bypass the requirement of UNC-18 we made doubles with
unc-18(
e234) . Expression of open syntaxin in the
unc-18 mutant fails to suppress the Unc-18 phenotype. This suggests that the facilitory role of UNC-18 is not to promote the open configuration of syntaxin for SNARE complex formation. Consistent with this result our preliminary ultrastructural analysis of
unc-18 mutant synapses suggests that the facilitory role of UNC-18 is in synaptic vesicle docking, which precedes SNARE complex formation. Together, these data suggest that UNC-18 has dual roles in synaptic transmission: a facilitory role in vesicle docking that precedes SNARE complex formation, and an inhibitory role via the SNARE protein syntaxin. We are currently testing in vivo whether the inhibitory role of UNC-18 is via inhibition of SNARE complex formation.