Glutamate transporters mediate electrogenic glutamate transport and evoke glutamate-gated chloride conductance. To identify amino acid residues involved in substrate binding and chloride conductance, conserved charged amino acid residues in a small region (region A, residues 345-356) of the human glutamate transport, EAAC1, were mutated (E345A, E346A, D351A, R342H, K352H and R356D). In addition, the "serine rich" motif (SSSS 331-334), which is conserved in the glutamate transporter isoforms GLAST1, EAAT4 and EAAT5, but not in GLT1, was mutated to the corresponding sequence in GLT1 (ASSA). Mutants were expressed in Xenopus oocytes and analyzed by voltage-clamp studies and isotope flux measurements. The L-glutamate and L-cysteine affinities of mutants E345A, E346A, D351A were slightly decreased but were increased for D-aspartate. Mutants K352H, R356D and ASSA (331-334) exhibited significantly increased affinities for all three substrates. The largest increase was observed for R356D and ASSA (331-334) (about 5- and 2-fold, respectively). The latter mutants also exhibited significant increases in chloride conductance, and the reversal potentials (Vr) were dramatically altered. At 500μM extracellular glutamate concentration, Vr of wild-type EAAC1 was +35 mV, that of ASSA (331-334) +5mV and that of R356D +8mV. These data indicate that substrate binding and opening of the chloride pathway are related processes, and that the "serine rich" motif and the charged amino acid residues in region A play crucial roles in these processes.