GABAA 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 the molecular structures of the GABA and BZD binding sites are unknown. The long-term goal of our researach program is to understand the function of the GABAA 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. An innovative approach, the substituted cysteine accessibility method, will be used to probe systematically the entire surface of the GABA and BZD binding site pockets of the GABAA receptor. This method can provide detailed molecular information about the structure of binding sites beyond that usually obtained using traitional mutagenesis or affinity labeling techniques. If the structure of the GABA and BZD binding sites were known in molecular detail, it is possible that whole neew 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-substitution by site directed mutagenesis of wild-type amino acid residues, heterologous functional expression of the mutants, and the probing of the substituted cysteines with sulfhydry1-specific reagents. These reagents are small, charged, hydrophilic, and lipophobic, and thus reactonly at the water-accessible surface of the receptor. If binding to a cysteine substitution mutnt is irreversibly blocked by the sulfhydry1-specific reagents, and if this blockade of binding can be prevented by site-specific ligands, we would infer that the side chain of the corresponding wild-type residue lines the binding site pocket. The pattern of accessibility of consecutive engineered cysteines to reaction with the sulfhydry1-specific reagents reflects the secondary structure of these residues;e.g. alpha helix or beta sheet. Inmutants with abnormal function and binding, the accessibility of neighboring residues to reaction with the sulfhydry1-specific reagents will allow us to deduce the secondary structure of the region containing this residue,and thus determine whether or not the residue is exposedin the binding site. Thus, all residues which structurally form the binding site can be successfully mapped, and the 3-dimensional structure of the site can be potentially modeled. In the absence of an X-ray crystal structure, this biochemical aproach will provide, at a molecular level, the most detailed structural picture of the GABAA receptor yet available.
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