The long-term goal of our work is to define the role of GABA transporters, which are a critical element of the GABAergic system that maintains brain excitability within normal limits. Many neuroscientists view GABA transporters simply as scavengers of GABA that has been released by vesicular fusion. However, new data suggest that the behavior of GABA transporters is much more complex, and that they play an active role in neuronal inhibition that goes far beyond simply reuptake of GABA. For example, among the neurotransmitter transporters they have a particularly low threshold for reversal, and when they reverse they release GABA into the extracellular fluid. Even when they don't reverse they play an important role in regulation of the amount of tonic inhibition, a newly discovered form of GABA signaling due to continuous activation of high affinity extrasynaptic GABAA receptors. Thus, accumulating evidence indicates that GABA transporters are not just GABA vacuum cleaners, but play a much more dynamic role in control of brain excitability. We have proposed the novel hypothesis that during neuronal firing the increase in membrane potential and rise in intracellular [Na+] leads to GABA transporter reversal, an increase in extracellular [GABA], and more tonic inhibition. We have further proposed that GABA transporters are one of the major determinants of extracellular [GABA] at rest, by virtue of the fact that they will only transport GABA into cells until they reach their equilibrium, and under normal conditions this equilibrium is reached when extracellular [GABA] is still relatively high. Thus, by establishing the """"""""floor level"""""""" of extracellular GABA, they are responsible for maintaining a minimum amount of tonic inhibition. Here we plan experiments that test these hypotheses by: 1) Directly measuring how easily GAT1 and GAT3 reverse, using a novel, highly sensitive functional assay of transporter reversal;2) Determining whether neurons can release GABA during action potentials via GAT1 reversal, 3) Measuring intracellular and extracellular [GABA] in response to treatment with the anticonvulsant vigabatrin, which selectively enhances tonic inhibition;4) Determining the relative importance of GAT1 reversal compared to other forms of nonvesicular GABA release, and;5) Defining the mechanism of a GAT1- independent nonvesicular form of GABA release that appears to come from glia. Loss of normal GABAergic inhibition can lead to seizures, and enhancement of inhibition may limit excitotoxicity during ischemia. Thus, the work proposed here will lead to better insight into normal synaptic physiology and control of inhibition during pathophysiological conditions such as epilepsy and strokes. The anticipated results may lead to new treatments for neurological disease aimed at enhancing nonvesicular GABA release and targeting the newly discovered form of tonic inhibition.
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