Year of Award

2008

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Neuroscience

Department or School/College

Department of Biomedical and Pharmaceutical Sciences

Committee Chair

Michael P. Kavanaugh

Commitee Members

Richard J. Bridges, John M. Gerdes, J. B. Alexander Ross, J. Stephen Lodmell

Keywords

biophysics, electrophysiology, hEAATs, Neuronal glutamate transporter

Abstract

Glutamate transporters have a homotrimeric subunit structure with a large central water-filled cavity that extends partially into the plane of the lipid bilayer (Yernool et al., 2004). In addition to uptake of glutamate, the transporters also mediate a chloride conductance that is gated by Na+ and glutamate. Our data indicate that glutamate binding sites, transport pathways, and chloride channels reside in individual subunits in the trimer and function independently and that the anion channel is gated by alkali cations binding from either side of the membrane. We also investigated conformational changes during glutamate binding by incorporating a fluorescent probe into a site near the postulated external gate (HP2) of a mutant transporter that can bind but not transport L-Glu. Fluorescence changes were observed upon ligand binding that strongly depended on the number of subunits labeled; this suggests quenched fluorophore dimers form at the center of the trimer that are subject to HP2 loop closure upon substrate binding. This supports a model of gate motion that is also consistent with recent x-ray structural data (Boudker et al., 2007). Finally, we propose that the large aqueous central cavity in the trimeric complex (Yernool et al., 2004) may function to restrict the diffusion of molecules near the three ligand binding sites, resulting in an increase in the probability of rebinding.

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© Copyright 2008 Gregory Patrick Leary