Motor function in C. elegans is controlled primarily by the excitatory neurotransmitter acetylcholine. Choline aceyltransferase (ChAT) encoded by the
cha-1 gene, synthesizes acetylcholine which is transported into synaptic vesicles by the vesicular acetylcholine transporter (vAChT) encoded by
unc-17 (Alfonso, A. et al. Science 261, 1993). Function of these genes is essential for synaptic transmission as null mutations in either gene is lethal. Mutants with residual
unc-17 gene function are uncoordinated, slow growing, small, and are resistant to cholinesterase inhibitors. Acetylcholine is only released into the synaptic cleft after vesicles packaged with the transmitter fuse with the pre-synaptic terminal. To study the role of vAChT in regulating neurotransmitter release in C. elegans ,
unc-17 was overexpressed in animals. Transgenic animals exhibited hypersensitivity to the cholinesterase inhibitor aldicarb. This suggests acetylcholine release into the synaptic cleft was increased and that neurotransmission can be modulated by changes in the level of expression of transporter. Suppressors of the locomotory defect of non null mutant
unc-17 (
e245) may elucidate transport function, isolate components that interact with vesicular transport and may participate in the regulation of transmitter release. The mutation in
unc-17 (
e245) converts a glycine to an arginine in transmembrane domain nine of vAChT (Alfonso, A. et al. Science 261, 1993). We have identified an intragenic suppressor of
unc-17 (
e245),
unc-17(
mg301), that changes this arginine to lysine. Extragenic expression of
unc-17(
mg301) rescues the locomotory defect of
unc-17 (
e245). This suggests that the arginine interferes with gene function but that glycine at that position is not essential even though this glycine is conserved in vertebrate acetylcholine and serotonin transporters. For example, the pH at this position in the membrane may be key.
sup-8 (
e1563) suppresses the movement defects of
unc-17(
e245) but not other
unc-17 alleles. We found that
sup-8 corresponds to a dominant allele of synaptobrevin (
snb-1).
sup-8(
e1563) is an isoleucine to aspartate substitution in the single transmembrane domain of synaptobrevin.
unc-17 (
e245) animals expressing the
snb-1 (
e1563) from a transgene in a
snb-1(+) chromosomal background show wild-type movement and normal aldicarb sensitivity. Thus,
snb-1 (
e1563) is a dominant suppressor of
unc-17(
e245), restoring normal movement and sensitivity to aldicarb.
snb-1(1563) animals exhibit no phenotype; for example they are not aldicarb sensitive, suggesting that this
snb-1 allele does not facilitate synaptic signaling. The location of the transporter and the synaptobrevin mutations in the transmembrane domains suggests a possible interaction between neurotransmitter packaging and vesicle fusion which we are trying to show by engineering mutations in these transmembrane segments, as well as biochemically.