GLUTAMATE-101 IS CRITICAL FOR THE FUNCTION OF THE SODIUM AND CHLORIDE-COUPLED GABA TRANSPORTER GAT-1

We have investigated the possible role of selected negatively-charged amino acids of the sodium and chloride-coupled GABA transporter GAT-1 on sodium binding, These residues located adjacent to putative transme mbrane domains and which are conserved throughout the large superfamil y of neurotransmitter transporters were changed by site-directed mutag enesis. The functional consequences were that one of the residues, glu tamate-101, was critical for transport. Its replacement by aspartate l eft only 1% of the activity, and no activity could be detected when it was replaced by other residues. Expression levels and targeting to th e plasma membrane of the mutant transporters appeared normal. Transien t sodium currents were not observed in the mutants, and increased sodi um concentrations did not affect the percentage of wild type transport of the E101D mutant. It is concluded that residue glutamate-101 is cr itical for one or more of the conformational changes of GAT-1 during i ts transport cycle.