Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. Dysfunction in GABA-mediated inhibition are implicated in the etiology of a variety of brain disorders and are key target for many clinically-prescribed, neuroactive compounds. Crucial to understanding the mechanisms of these diseases and therapeutic drugs is a molecular understanding of the structure/function relationship of the GABA receptor. Although much has been learned during the previous decade, there are still major gaps in our understanding of GABA receptor activation. This project uses a combination of molecular biological, ligand binding, and electrophysiological techniques to gain insight into the activation mechanism of the GABA receptor from both a structural and functional vantage point. The first specific aim will take advantage of a modular GABA receptor subunit we have constructed that contains multiple unique restriction sites at key junctures throughout the cDNA. We will use this modular subunit to move segments between different GABA and non-GABA subunits in an effort to transfer, and hence localize, key domains involved in activation. We will also assemble tandem GABA receptor subunit constructs with both two and three rho subunits connected. This will enable us to examine the function of GABA receptors with mutations at defined subunits with the pentamer and allow us to elucidate intersubunit interactions. In addition, we have recently developed a new technique that allows one to perform [3H]-ligand binding and electrophysiological studies in the same individual oocytes expressing recombinant GABA receptors. This enables a direct correlation between ligand binding and receptor activation in the same set of functional receptors. In the second specific aim, we will use this dual ligand binding/electrophysiology approach to directly determine the affinity and efficacy of several GABA receptor agonists and complement this with a single channel analysis to further delineate the specific activation mechanism(s) of different agonists. Finally, we will examine the inter-relationships between receptor activation (opening), deactivation (closing) and desensitization, using a combination of single oocyte binding in conjunction with whole-cell and single channel recordings. We expect the proposed studies to provide the most comprehensive picture to date for the activation mechanism of this receptor-operated ion channel. The working hypothesis derived from these experiments should provide a framework for understanding the molecular mechanism by which GABA-modulatory compounds, as well as other factors, shape synaptic inhibition in the central nervous system.
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