In the mammalian brain the extracellular concentration of the excitatory neurotransmitter glutamate is, among other factors, controlled by transporters, which actively take up glutamate into glial cells and neurons. The broad aim of this proposal is to investigate the molecular mechanisms by which these transporters accomplish this glutamate uptake. Recombinant glutamate transporters will be expressed in HEK293 cells and Xenopus oocytes, and currents that originate from electrogenic glutamate transport will be recorded using the patch clamp technique. Currents will be induced by glutamate concentration jumps generated within 100 mus, allowing the resolution of rapid transporter reaction steps in time. The hypotheses that guide this research are: Both inward and outward glutamate transport occur via a multistep electrogenic mechanism; charged amino acids in the transmembrane segments contribute to the function and electrogenicity of the transporter; translocation of glutamate across the membrane requires molecular movement of the transport protein. Experiments with the following specific aims will test these hypotheses: (1) To determine the reaction steps associated with outward glutamate transport, by investigating the sequence and voltage dependence of intracellular Na+ and glutamate binding. (2) To determine the contributions of conserved charged amino acids to transporter function and electrogenicity, by investigating transporters with specifically charge-neutralized amino acids. (3) To characterize molecular movement of the transporter machinery by determining which parts of the transporter move and at what steps during a complete transport cycle they move. Movement of the transporter will be detected by the movement of charges attached to the transporter at specific sites. Understanding the molecular mechanism of glutamate transport will give insight into the involvement of glutamate transporters in acute and slowly progressing neurodegenerative diseases, such as stroke, amyotrophic lateral sclerosis, and Alzheimer's disease. ? ? ?
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