Ligand-gated ion channels (LGICs) are integral membrane proteins that mediate fast signal transduction at chemical synapses. GABAA receptors belong to a major family of LGICs, called Cys-loop receptors, which allow rapid diffusion of Cl- through a central pore upon binding of the neurotransmitter GABA. The conducting channel is formed from a pentameric assembly of identical or homologous subunits giving rise to multiple receptor subtypes with a diverse array of pharmacological properties. Specifically, binding of commonly prescribed benzodiazepines, such as diazepam, enhances the observed Cl- conductance of receptors composed of 1/2/3 subunit isoforms. However, the lack of high resolution structural information prevents a detailed analysis of the molecular mechanism that couples GABA binding to positive allosteric modulation by diazepam. This proposal outlines the use of proven strategies to determine a high resolution crystal structure of the 11/22/32 receptor in complex with GABA and diazepam by x-ray diffraction methods. The highly sensitive technique of fluorescence detection size exclusion chromatography will be employed to rapidly screen for optimized gene constructs and conditions to isolate a functional GABAA receptor. Then optimized constructs will be expressed in Sf9 insect cells to obtain mg quantities of receptor for crystallization trials. Co-crystallization experiments with GABA and diazepam will be used to identify the ligand binding sites and conformational changes in the structure of the 11/22/32 receptor. The relationship of these conformational changes to the functional properties of the receptor will be assessed by a combination of mutagenesis, ligand binding assays and electrophysiological experiments. The results of this work will substantially contribute to the understanding of GABAA receptor structure and the allosteric effects of ligand binding on channel behavior.
The dysfunction of GABAA receptor activity has been linked to a variety of neurological disorders, such as epilepsy, which can be treated with potent receptor modulators like diazepam. However, the broad interaction spectrum of diazepam (and other benzodiazepines) with multiple GABAA receptor subtypes leads to undesired side effects including amnesia. The results of this application will significantly increase the knowledge of the therapeutic value of diazepam-GABAA receptor interactions and will inform rational drug design strategies for the development of receptor subtype-specific drugs with reduced side effects.