Gamma-aminobutyric acid type A (GABA/A) receptors are the main effectors of neuronal inhibition in the mammalian central nervous system and are responsible for transducing the neuroactive properties of benzodiazepines (BZDs). BZDs are a clinically important class of drugs with potent anxiolytic, sedative-hypnotic, myorelaxant, and anti-epileptic effects. Although both alpha and gamma subunits are thought to contribute to the formation of the BZD binding pocket, a detailed structural map of the binding site does not exist.. Using site-directed mutagenesis and the substituted cysteine accessibility method, I will identify novel residues important for BZD binding and will map the secondary structure of two potential binding site domains in the alpha (alpha1P96-S106) and gamma subunits (gamma2 Y72-783). Additionally, comparisons of the rates of modification for a variety of sulfhydryl-specific reagents and residue mutagenesis will yield information about the local chemical environment in which BZDs bind. Lastly, I will identify the structural changes in the binding site that underlie BZD modulation of the receptor by examining changes in the accessibility of substituted cysteines in the presence of GABAA receptor activators. Allosteric changes in the BZD site will also be examined by destabilizing backbone structure of residues implicated in conformation movements using unnatural amino acid substitution. These studies represent a step towards understanding the structural determinants of BZD modulation of the GABA/Z receptor, and will provide the most detailed map of the BZD binding domain to date.
