GABAA receptors (GABAARs) are the major sites for fast synaptic inhibition in the brain. The long-term goal of this project is to understand the neuronal regulation of GABAAR density and subcellular distribution. The investigators have previously demonstrated that acute exposure of cortical neurons to GABA or benzodiazepine agonists induces the transfer of surface GABAARs into a labile intracellular pool. In order to examine the underlying mechanisms and to evaluate their role in agonist-evoked GABAAR downregulation, three specific objectives are developed in this proposal. (1) To test the hypothesis that acute exposure of cortical neurons to benzodiazepines induces the sequestration of GABAAR subunits. Exoplasmic regions of GABAAR polypeptides on living neurons will be labeled with impermeant cleavable reagents. Following acute exposure to agonists, sequestered receptors will be recovered by stripping the surface label and doubly immunoprecipitating cell extracts with antibodies against GABAAR alpha1, beta2, and beta4 subunits. (2) To test the hypothesis that interactions of GABAARs with coated-pit proteins are evoked by agonists. The agonist-induced interactions of GABAAR subunits with inositol polyphosphate binding proteins as well as proteins identified by yeast two-hybrid screening will be examined by co-precipitation. (3) To test the hypothesis that GABAARs are degraded by distinct agonist-dependent and agonist-independent pathways. GABAAR subunits will be labeled internally by incorporation of 35S-Met/Cys and externally by impermeant non-cleavable reagents. The effect of agonists and protease inhibitors on the turnover rates will be determined. It is suggested that this project will provide new insights into pathways which modulate synaptic function. By means of these regulatory mechanisms, cell-cell communication and drug-cell interaction can produce persistent changes in neuronal excitability. Furthermore, these are likely to represent molecular mechanisms which establish tolerance and habituation to benzodiazepines, barbiturates, and alcohol.