STRUCTURE OF NA+ DEPENDENT GLUTAMATE TRANSPORTERS

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High affinity, sodium-dependent glutamate transporters are responsible for maintaining glutamate concentrations below neurotoxic levels and have been implicated in the neuropathological changes associated with ALS, acute ischemia and stroke. Elucidating the structural features underlying the mechanism of transport will provide insight on how the carriers contribute to disease and neurodegeneration and may suggest possible therapeutic interventions. The proposed studies aim to further resolve the transmembrane topology and to begin to elucidate the structural correlates of substrate and inhibitor binding and chloride ion permeation. First, the membrane orientation of a FLAG epitope, inserted into hydrophilic loops in the protein, will be determined using indirect immunofluorescence microscopy in permeabilized and nonpermeabilized transfected COS cells. Second, single cysteine residues will be placed in these domains and their membrane orientation determined in Xenopus oocytes using membrane-permeant and impermeant sulfhydryl-reactive compounds. Finally, the Substituted Cysteine Accessibility Method (SCAM) will be used to examine whether residues located in putative TMD 8 interact with substrates and inhibitors or are involved in the permeation of chloride ions. These studies will focus on a highly conserved domain which has already been implicated in substrate and inhibitor binding. Twenty-four consecutive residues will be replaced by cysteine in a previously-generated functional cysteineless transporter and the transport properties and currents associated with each of the mutants will be assessed in oocytes. Sulfhydryl-reactive MTS reagents will be used to identify residues that contribute to the interactions of substrates in inhibitors and ions with the carrier.