The GABAA receptor is a hetero-oligomeric membrane protein that hyperpolarizes neuronal membranes by opening an intrinsic Cl- channel gated by the neurotransmitter GABA. The principle function of GABAA receptors is inhibitory and aberrant GABAergic transmission has been associated with the etiology of several neurological and psychological disorders including epilepsy, myotonia, panic disorders and chronic drug dependence. Numerous clinically efficacious drugs enhance central nervous system inhibition by acting at their associated recognition sites and allosterically potentiating GABA-gated Cl- currents. These include the barbiturates, steroids, volatile anesthetics and benzodiazepines (BZDs). While it has been known for some time that BZDs exert their therapeutic action by modulating the GABAA receptor, the molecular mechanism by which this occurs is still somewhat controversial. The proposal will use a combination of molecular biological and electrophysiological techniques to obtain structural and functional information regarding the actions of BZDs at GABAA receptors. Residues critical for the actions of several BZD ligands will be identified in the alpha1 (a1) subunit (for alpha-beta-gamma receptors, a-b-g receptors) and the gamma2 (g2) subunit (beta-gamma receptors, b-g receptors) using site- directed mutagenesis and recombinant expression studies. Whole-cell electrophysiological recording techniques will be used to evaluate mechanisms by which these mutations impair the sensitivities of the recombinant receptors to BZDs and the actions of BZDs on the kinetic properties (such as agonist sensitivity and desensitization) of the GABAA receptor. These whole-cell and single-channel data will then be used to test and adapt working hypotheses that BZDs exert their effects by modulating receptor activation at steps subsequent to GABA binding. The design of more efficacious BZDs that can target the many different GABAA receptor subtypes that have been identified in the brain ultimately depend on understanding the structural requirements and precise mechanisms of action of this important class of compounds. Collectively, results from the proposed experiments should contribute to that effort.
