Information processing in the central nervous system depends on the rapid and precise release of amino acid neurotransmitters gamma- aminobutyric acid (GABA) and glutamate. Although much is known about the molecular basis of the postsynaptic response to GABA and glutamate, the presynaptic mechanisms involved in release are less well understood. Since classical neurotransmitters are synthesized in the cytoplasm, they require transport into synaptic vesicles for exocytotic release. Previous studies have demonstrated the energy - dependent uptake of GABA and glutamate into synaptic vesicles, but the proteins responsible for these activities remain unknown. The long-term objectives of this proposal are to determine the molecular basis for vesicular transport of amino acid transmitters and its role in synaptic transmission. Genetic and pharmacological analysis has implicated the C. elegans gene unc-47 in the release of GABA. WE have recently demonstrated that a vertebrate homologue of unc-47 encodes vesicular GABA transport. The amino acid sequence of this rat vesicular GABA transporter (VGAT) shows no similarity to previously identified neurotransmitter transporters, indicating that it defines a novel family of proteins involved in the vesicular transport of amino acid neurotransmitters that may also include the vesicular transporter for glutamate. We have now isolated a cDNA with sequence homology to unc-47 and VGAT. Preliminary characterization shows restricted expression of this putative rat vesicular transporter 2 (VT2) in the brain and the kidney by Northern analysis and in glutamatergic neurons in the brain by in situ hybridization. To assess the role of the predicted protein in vesicular glutamate transport, we propose the following specific aims: 1) generate antibodies to VT2 and determine its tissue distribution and subcellular localization; 2) characterize the biochemical function of VT2 expressed in mammalian cell lines; 3)isolate cDNAs related to VGAT and VT2; and 4)target the disruption of the VT2 gene in mice. The crucial role of neurotransmitter packaging for regulated exocytotic release indicates that the results of these studies will have relevance for normal synaptic function, behavior and human neuropsychiatric disease.
