Benzodiazepines (BDZs) exert anxiolytic and anti-epileptic effects in the central nervous system (CNS) by allosteric modulation of the GABAA receptor Cl- current (IGABA). Because of the widespread clinical utility of these drugs, understanding the mechanism(s) by which BDZs alter ion channel function is an important pharmacological inquiry. To identify the structural determinants underlying BDZ modulation of kinetic transitions of channel activation, one must first determine the effects of BDZs on identified, wild-type GABAA receptors. This will be approached using rapid drug application techniques and single-channel recordings to test which of the microscopic kinetic rate constants are altered by modulatory BDZs in a1 B2g2 receptors. We will make use of mutations in the GABA binding site, the pore region, and the extracellular loop to tease apart the contributions of BDZs to binding/unbinding and gating/desensitization of IGABA. We will also make use of tethered tandem subunits to functionally separate the contributions of each of the two GABA binding sites to these processes. The studies outlined in this application will help to establish the functional mechanisms of action for clinically relevant drugs on a major CNS variant of the GABAA receptor, and aid in establishing testable hypotheses regarding structure-function relationships in this and other ligand-gated ion channels.
