Gamma-aminobutyric acid (GABA) is released from presynpatic terminals and binds to postsynaptic GABA receptors. The binding of GABA on the extracellular amino terminal domain leads to a structural change at the pore, buried deep within the membrane. This structural change at the pore permits the flux of chloride down its concentration gradient, thereby inhibiting the postsynaptic neuron. The general aim of this project is to understand the mechanism by which this binding of GABA leads to the gating of the pore. For these studies we use recombinant receptors expressed in oocytes and, where necessary, other exogenous expression systems. We have assembled a variety of techniques that will allow us to probe the activation mechanism from both a structural and functional vantage point. The first aim will use mutagenesis, cysteine accessibility, and electrophysiological techniques to identify the key domains that play a role in activation. This will set the stage for the next two aims that will investigate the dynamics of the activation process. For example, the second aim will use site-specific fluorescence labeling in combination with two-electrode voltage clamp to identify functionally-relevant molecular rearrangements. The third aim employs a novel approach in which charged amino acids are substituted at select positions to identify parts of the receptor within the membrane field that move during activation. GABA is the major inhibitory neurotransmitter in the mammalian brain. Dysfunctions in GABA-mediated inhibition have been implicated in the etiology of a variety of brain disorders. Furthermore, many clinicallyprescribed compounds (barbiturates, benzodiazepines, neurosteroids) exert their therapeutic effects by modulating the function of GABA receptors. Crucial to understanding the molecular basis of these diseases and drugs is a detailed understanding of the molecular mechanism of activation. The results from this proposal should facilitate this endeavor.