Broad Long-Term Objectives: Initiate an interdisciplinary reseach endeavor, dedicated to the measurement of the gating mechanism of the Glycine Receptor (GlyR), and the correlation of structure with function.
Specific Aims : In this research we will determine: 1) distances, side-chain dynamics, and depths of immersion constraints to site-specifically map the resting state of the GlyR. (2) distances, side-chain dynamics, and depths of immersion constraints to site-specifically map a desensitized state of the GlyR. (3) distance constraints to site-specifically map an open state of the GlyR . The ESR data will provide site-specific details of molecular movements to determine the identities, directions, and amplitudes of domain movements that generate ion-flux in the GlyR. We will also be able to determine the details of how ligand binding in the extracellular domain leads to channel opening in the transmembrane domain. Method: Functional GlyR mutants with cysteines at selected sites will be generated and spin-labeled. Electron spin resonance will provide intersubunit and intrasubunit proximities, and local structural constaints in each allosteric state of the GlyR. These structural constraints, coupled with molecular modeling will allow us to develop a detailed picture of molecular movements that open and desensitize the GlyR. Health Relatedness: Determination of the gating mechanism of the GlyR will lead to an understanding of the molecular basis for many nuerological diseases, such as pathogenesis of spasticity, loss of motor control associated with amyotrophic lateral sclerosis and Parkinsonism, inherited congenital myoclonus, and familial startle disease (hyperekplexia). It will also enhance our understanding of mechanisms of action of many drugs that interact with the GlyR, such as anesthetics, alcohols, and various narcotics and therapeutic agents used for the treatment of a wide range of neurologic disorders.
