The purpose of this proposal is to continue molecular and genetic analysis of acetylcholine synthesis and packaging in the soil nematode Caenorhabditis elegans. These studies focus on three genes and proteins: cha-1 is the structural gene for choline acetyltransferase (ChAT), the enzyme which synthesizes the neurotransmitter acetylcholine; unc-17 is the gene which encodes the synaptic vesicle acetylcholine transporter (VAChT); and cho-1 encodes the plasma membrane choline high-affinity transporter (CHT). Two of these genes, cha-1 and unc-17, are tightly linked and define the """"""""cholinergic gene locus"""""""". C. elegans is being used for these studies because of its simple nervous system, its ease of genetic and molecular analysis, and the availability of DNA-mediated transformation techniques.
The specific aims i nclude development of a standardized sensitive assay system for quantifying cholinergic-mediated behavior, analysis of multiple cha-1 transcripts and multiple ChAT isoforms to determine their roles in ChAT function, isolation of additional cho-1 alleles and extragenic suppressors of cho-1, determining the role of phosphorylation in localization and function of the three cholinergic proteins, and (in collaboration with two other laboratories) structure-function studies of nematode and mammalian ChAT and VAChT. The results of these studies will elucidate the mechanisms used within nerve terminals to coordinate neurotransmitter supply and demand. Although this is basic research, it is clearly relevant to health, since alterations in acetylcholine metabolism and/or cholinergic function have been identified in many neurological and psychiatric disorders, including congenital myasthenic syndromes, Alzheimers disease, and depression.
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