In the brain, the neurotransmitter glutamate is removed from the synaptic cleft by (Na(+) + K(+))-coupled transporters by an electrogenic process. Moreover, these transporters mediate a sodium- and glutamate-dependent uncoupled chloride conductance. In contrast to the wild type, the uptake of radiolabeled substrate by the I421C mutant is inhibited by the membrane-impermeant [2-(trimethylammonium)ethyl]methanethiosulfonate and also by other sulfhydryl reagents. In the wild-type and the unmodified mutant, substrate-induced currents are inwardly rectifying and reflect the sum of the coupled electrogenic flux and the anion conductance. Remarkably, the I421C mutant modified by sulfhydryl reagents exhibits currents that are non-rectifying and reverse at the equilibrium potential for chloride. Strikingly, almost 10-fold higher concentrations of d-aspartate are required to activate the currents in the modified mutant as compared with untreated I421C. Under conditions in which only the coupled currents are observed, the modified mutant does not exhibit any currents. However, when the uncoupled current is dominant, sulfhydryl reagents cause >4-fold stimulation of this current. Thus, the modification of the cysteine introduced at position 421 impacts the coupled but not the uncoupled fluxes. Although both fluxes are activated by substrate, they behave as independent processes that are in dynamic equilibrium.