Glutamate is the most abundant excitatory transmitter in the central nervous system, and the transporters responsible for its reuptake play critical roles in both normal and pathological brain processes. We have isolated three glutamate transporter cDNAs from human motor cortex by expressed them in Xenopus oocytes and a human kidney cell line. We propose to study their structural and functional properties in these model systems using a combination of electrophysiological and biochemical approaches.
Aim 1 is to study the transporters' biophysical properties in oocytes using two-electrode voltage clamp, cut-open vaseline gap recording, and excised patch recordings. A second approach will involve whole cell recording of HEK 293 cells stably transfected with human glutamate transporters. The time-, voltage-, temperature-, and concentration-dependence of both influx and efflux mediated by the transporters will be examined, and the requirements for ion co-substrates will be defined. Radiolabel flux measurements will made to define the electrochemical gradient coupling and stoichiometry.
Aim 2 is to characterize the pharmacology of the three transporter isoforms. Classically described glutamate transport antagonists and novel analogs including pyrrolidine dicarboxylate derivatives related to kainate will be screened for the ability to act as substrates for transport or to antagonize glutamate transport. Antagonists will be tested for their ability to discriminate between the transporter isoforms and tested for selectivity by assaying their actions at NMDA and Non-NMDA inotropic receptor subtypes. Using both pharmacological and biophysical approaches, the effects of selectively modulating transporter function on glutamatergic synaptic transmission in cultured neurons will be investigated.
Aim 3 involves structure-function studies aimed at defining the external site for glutamate binding to the transporter. Chimeric transporters will be constructed from kainate sensitive and insensitive isoforms to map domains involved in binding this competitive antagonist and to target individual residues involved in binding for site-directed mutagenesis. The quaternary structure of the transporter will be probed by coexpressing pharmacologically distinct isoforms in oocytes in order to test for their ability to assemble into functionally discrete multimers. The results of these studies will be of significance in neurobiology and medicine because of the insights they will provide into the molecular mechanisms underlying glutamate transporter function. A clear understanding of molecular biophysics of glutamate transporters will help define the transporters' roles under normal conditions as well as in pathological processes such as stroke.
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