West Coast Worm Meeting Abstracts - Caenorhabditis elegans ...

ELECTROPHYSIOLOGICAL ANALYSIS OF UNC-18
MUTANTS.
J. E. Richmond, R. Weimer, W. S. Davis, E. M. Jorgensen
University of Utah, Salt Lake City, Utah 84112
West Coast Worm Meeting 2000
UNC-18 is a C. elegans neuron-specific protein belonging to a conserved protein family implicated in
vesicle fusion. UNC-18 interacts with the SNARE protein syntaxin, which is believed to mediate fusion of
synaptic vesicles with the plasma membrane. Mutations in unc-18 result in severe locomotory defects and
acetylcholine accumulation, implying a critical role for UNC-18 in synaptic transmission. Three mutants
were examined, unc-18(e81 and e234) which have premature stop codons and unc-18(b403) which
disrupts binding to syntaxin. The mutant phenotype was not caused by developmental abnormalities since
GFP-expression patterns revealed motor neurons had normal morphology and exhibited wild-type
distributions of synapses. We have used electrophysiological techniques to analyze synaptic function in
these unc-18 mutants. Evoked release at the C. elegans neuromuscular junction was reduced to 5% of
wild-type amplitudes (from 1800pA to 100pA) in all three mutants. A similar reduction in endogenous
synaptic event frequency (from 44Hz to 5 Hz) was observed. The mutant synaptic event amplitudes were
unaffected demonstrating that the reduction in the evoked response was not due to a defect in vesicle
neurotransmitter filling or in post-synaptic receptor sensitivity. Preliminary EM data showed an
accumulation of vesicles at the neuromuscular synapses of unc-18 mutants consistent with a defect in
exocytosis as opposed to defects in vesicle biogenesis or retrieval. In the absence of external Ca2+, the
rates of spontaneous fusion events were also reduced, suggesting that either the docking or fusion
competence of unc-18 mutants is defective. Given these data, how might UNC-18 function in exocytosis?
UNC-18 forms a heterodimer with syntaxin, which is mutually exclusive of SNARE complex formation.
Several studies suggest that the interaction of UNC-18 and syntaxin is an essential step in regulated
release, and that this interaction may be required to promote a conformational change in syntaxin which
facilitates SNARE complex formation. To test this hypothesis, we have engineered a double mutation in
syntaxin, which, in vitro eliminates UNC-18 binding, and induces the syntaxin open configuration. We are
currently examining the effects of this mutation in wild-type, unc-64 (syntaxin) and unc-18 mutants.
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