Author

Paul J. Focke

Date

December 2009

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Neuroscience

Institution

Oregon Health & Science University

Abstract

The remarkable ability to maintain low levels of glutamate in the extracellular space following signaling events is achieved mainly by excitatory amino acid transporters (EAATs) located in the plasma membranes of both glial cells and neurons. Abnormal glutamate transporter function is implicated in Parkinson’s disease, Alzheimer’s disease, cerebral ischemia, epilepsy, and amyotrophic lateral sclerosis, underscoring the importance of understanding how these transporters function. Our research is centered on elucidating the structural and functional properties of glutamate transporters to reveal novel (i.e. smallmolecule) approaches for treating these neuropathological conditions. To this end, we have developed a technique of using double site-directed spin labeling (DSDSL) electron paramagnetic spectroscopy (EPR) on a bacterial glutamate transporter homolog from Pyrococcus horikoshii, GltPh. Briefly, this technique provides the ability to make distance measurements in the ~8-25 Å range between two spin labels, based on the extent of the dipolar interactions between them. This thus allows us to monitor conformational changes in GltPh in response to events such as ion, substrate, and inhibitor-binding. In our current work, we have utilized this technique specifically to examine regions of the GltPh transporter proposed to contribute to the extracellular gating mechanism. Our research validates the conclusions from previous electrophysiological studies and crystal structures, as well as provides new insight into previously undescribed conformational changes in the extracellular gate of GltPh that occur in response to the binding of ions to the apo-state of the transporter.

Identifier

doi:10.6083/M4DR2SF7

School

School of Medicine

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