The accessibility of a novel reentrant loop of the glutamate transporter GLT-1 is restricted by its substrate.
The excitatory neurotransmitter glutamate is removed from the synaptic cleft by several related sodium- and potassium-coupled transporters. They thereby restrict the neurotoxicity of this transmitter. Based on the accessibility of single cysteines to the large sulfhydryl reagent 3-N-maleimidyl(propionyl)biocytin, we have proposed a topological model for the astroglial glutamate transporter GLT-1 (Grunewald, M., Bendahan, A. and Kanner, B. I. (1998) Neuron 21, 623-632). Because of several unexpected observations, we have investigated the topological disposition of 19 cysteine residues engineered into a loop proposed to be intracellular. We have probed the accessibility of these cysteines to small and large sulfhydryl reagents. The impermeant hydrophilic sulfhydryl reagent [(2-trimethylammonium)ethyl] methanethiosulfonate inhibits transport activity only at two of these positions, weakly at G365C and potently at A364C. Glutamate and its nontransportable analogue dihydrokainate markedly protect A364C transporters against this impermeant reagent. Using a biotinylated maleimide, we found that, among the 14 mutants tested with it, only A364C is accessible to it from the extracellular side. This, together with our previous observations, indicates that the loop-including amino acid residues 354, 359, 373, and 379-is largely intracellular, but a short region of it forms a reentrant pore-loop-like structure, the accessibility of which is dependent on the conformation of the transporter.
Grunewald
M
M
Department of Biochemistry, Hadassah Medical School, The Hebrew University, P. O. Box 12272, Jerusalem 91120, Israel.
Kanner
B I
BI
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D001692:Biological Transport; D018698:Glutamic Acid / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D007202:Indicators and Reagents; D018698:Glutamic Acid; D000409:Alanine