STRUCTURAL DETERMINANTS OF GABA RECEPTOR FUNCTION

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DESCRIPTION: Molecular cloning studies have revealed numerous mammalian neuronal GABA-A receptor subunits and have helped explain the diversity of receptors found in vitro. Certain subunit combinations or mutations result in different GABA affinity, efficacy or pharmacology of resultant receptors; however little is known about what microscopic mechanisms underlie these observations or how these receptors function under synaptic-like conditions of transient GABA exposure. The hypothesis is: Receptor composition and specific molecular determinants alter events or processes involved in microscopic kinetics of GABA binding, channel gating and receptor deactivation and allosteric modulation that are relevant to the synapse. In an effort to study the role of subunit composition and molecular determinants for GABA receptor function, three specific aims are proposed: #1) Determine functional consequences of subunit composition on affinity, gating and pharmacological modulation of expressed receptors; #2) Evaluate specific amino acids involved in binding, gating and modulation of expressed GABA receptors; and #3) Correlate GABA-A receptor subunit mRNA and with electrophysiological properties of receptors in individual neurons. Ultrafast ligand exchange (100 su) and electrophysiological techniques will be used to study excised patches of defined subunit combinations expressed in HEK 293 cells. The investigators will directly resolve processes involved in receptor activation and gating and also study receptor function and pharmacology that better recapitulates agonist activity in the synapse. To further correlate information on genetic expression with receptor function, single cell RT-PCR techniques will qualitatively determine mRNA present in living neurons and kinetic fingerprints will be obtained with neuronal receptor system responses evoked by <1 ms GABA applications. This project will improve our understanding of the molecular correlates of GABA receptor structure and function which can result in better therapeutic intervention in epilepsy, anxiety and anesthesia.