The central hypothesis of this proposal is that GAD65 and GAD67 catalyze the synthesis of distinct pools of GABA-one vesicular and the other cytosolic. According to our working model, membrane-associated GAD65 produces vesicular GABA and cytosolic GAD67 produces cytosolic GABA. Whereas vesicular GABA, released by exocytosis, is likely to be particularly important in point to point signaling via synapses, cytosolic GABA, released via plasma-membrane transporters, may serve a longer-range paracrine role-inhibiting neuronal activity in a limited region of the nervous system. To address this hypothesis, we will undertake three sets of studies of genetically modified cells in culture, as well as of primary cell cultures from wild-type, GAD65- and GAD67 deficient mice. These experiments are organized around three Specific Aims: (1) to identify the determinants of the distinctive intracellular distributions of GAD65 and GAD67 (2) to test the distinct roles of the two GADs in vesicular and nonvesicular GABA release; and (3) to examine the functional significance of changes in the ratio of GAD65 to GAD67 and of apoGAD65 to -holoGAD65. An important long-term goal of this project period is the development of presynaptic pharmacological interventions in GABA signaling. Specifically, the establishment of cell culture model systems to study GABA dynamics will also enable us to use these systems as a rapid and high-throughput screen for drugs that selectively affect one or the other branch of the GABA release system. Drugs identified in such screens will have major importance as candidates for the treatment of epilepsy, movement disorders such as Parkinson's Disease, and ischemic or traumatic injury to the nervous system.
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