Gamma-aminobutyric acid type A receptors (GABAARs) mediate synaptic inhibition in the brain and the actions of several clinically important drugs, including benzodiazepines, barbiturates, ethanol and anesthetics. Several mutations in the receptor are linked to inherited forms of epilepsy. The long-term goal of our research program is to understand the function and pharmacological modulation of the GABAAR in terms of its molecular structure. Work during the current project period significantly advanced our understanding of the structure and dynamics of the GABA and benzodiazepine (BZD) binding sites. Experiments proposed herein build on this information to advance our understanding, on a structural level, of how GABA binding triggers channel gating and how BZD binding is coupled to receptor modulation. We propose to test the following hypotheses: 1) that intra- and inter-subunit salt bridges relay GABA binding site movements in the extracellular domain to gating movements in the transmembrane channel domain by connecting rigid-body protein blocks, 2) that residues in Loop 2 are involved in GABAAR activation and desensitization, 3) that residues in Loop 9 at non-binding site interfaces comprise a `hinge'element important for coupling GABA binding to gating, and 4) that BZD binding modulates GABAAR function by triggering movements in the extracellular domain that are transduced to the transmembrane helices via residues in Loop 2, Loop 7, Loop 9, pre-M1 and M2-M3 regions of the 11 and 32 subunits. The approach combines site-directed mutagenesis, disulfide crosslinking, mutant cycle analysis, substituted cysteine accessibility method, patch-clamping and kinetic analysis. The experiments will be interpreted with the aid of recently elucidated atomic-level structures to gain a deeper understanding of the molecular mechanisms underlying the function of GABAARs and related receptors. The central role played by GABAARs in brain function make this basic research directly relevant to human health. The relevance of the research proposed herein extends beyond the GABAA receptor itself. GABAA receptors are members of a family of receptors that function as ligand-gated ion channels, and their activity regulates information flow throughout the brain. Defects in these channels lead to a wide variety of diseases, and they are the targets of a large number of clinically used drugs. Improvements in our understanding of how these channels work at a molecular level will improve our ability to predict the actions of drugs that act on these channels, to design safer and more effective drugs, to develop better therapeutic strategies, and to understand the etiology of disease-causing mutations. The research proposed here will increase our understanding of how one type of ion channel, the GABAA receptor, functions in health and disease and will establish testable hypotheses for elucidating how other related ligand-gated ion channels function.
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