Neurotransmitters are loaded into synaptic vesicles by protein transporters located in the membrane of these vesicles. These proteins were first identified as biochemical activities in cell extracts, and now the cDNAs for all the classical neurotransmitter transporters, except glutamate, have been isolated. The Edwards lab has recently identified another putative vesicular transporter cDNA (VT2) related to the GABA transporter cDNA sequence. From in situ analysis this mRNA is expressed in the brain in a pattern consistent with a function in excitatory neurotransmission. For this proposal, antibodies will be produced to the predicted VT2 protein sequence, and the cellular and subcellular expression pattern of the VT2 protein will be examined. This information will be used to determine the function of the protein using a heterologous expression system and standard uptake assays. In addition, VT2 and VGAT function will be analyzed in the presence of a fodrin protein fragment, an inhibitor of endogenous vesicular glutamate and GABA transport. I will also determine whether VT2 is co-expressed with other vesicular neurotransmitter transporters in the same cells and, if so, in the same synaptic vesicles. These studies will help us to understand how neurotransmitter packaging influences synaptic transmission.
