GABA/A receptors are the major inhibitory neurotransmitter receptors in the mammalian CNS and are the site of action of benzodiazepines (BZDs). GABA analogues and BZDs are used in the treatment of a variety of neurological and psychiatric disorders, but detailed molecular structures of the GABA and BZD binding sites are unknown. The long-term goal of our research program is to understand the function of the GABA/A receptor in terms of its molecular structure. As a first step, we propose to identify and locate in the receptor structure the amino acid residues that form the GABA and BZD binding sites and determine the secondary protein structures which contribute to forming these sites. We are utilizing the substituted cysteine accessibility method in combination with mutagenesis, affinity labeling and cross-linking experiments to achieve these aims. The combination of these approaches is quite powerful and can provide details about the molecular structure of these binding sites which are not obtainable by mutagenesis and photoaffinity labeling techniques alone. If the structure of the GABA and BZD binding sites were known in detail, it is possible that whole new classes of site-specific compounds would be discovered and existing compounds could be modified to exploit the physicochemical features of the binding site to yield higher affinity, more selective drugs. The substituted cysteine accessibility method is a combination of single, consecutive cysteine- substitutions by site-directed mutagenesis of wild-type amino acid residues, heterologous functional expression of the mutants, and the probing of the substituted cysteines with sulfhydryl-specific (SH-) reagents. If a cysteine-substituted residue is part of a binding site, reaction with a SH-reagent will alter binding irreversibly, and site-selective ligands will protect the engineered cysteine from covalent modification. Direct detection of a modified cysteine can be accomplished by using the SH- reagent, MTSEA-Biotin, in combination with avidin-bead precipitation and Western blotting techniques. By examining the pattern of accessibility of consecutively engineered cysteines to reaction with SH-reagents, the secondary structure of these residues can be determined; e.g. alpha-helix or beta-strand. Elaborations of this approach allow us to determine the charge-selectivity of the binding sites and to determine the distance between pairs of engineered cysteines. Thus, detailed structural maps of the GABA and BZD binding sites can be obtained, and the 3-dimensional structure of the sites can be potentially modeled. In the absence of an X- ray crystal structure, this biochemical approach will provide, at a molecular level, the most detailed structural picture of the GABA/A receptor available.
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