Glutamate transporters located in the brain maintain low synaptic concentrations of the neurotransmitter by coupling its flux to that of sodium and other cations. In the binding pocket of the archeal homologue Glt Ph , a conserved methionine residue has been implicated in the binding of the benzyl moiety of the nontransportable substrate analogue threo--benzyloxyaspartate. To determine whether the corresponding methionine residue of the neuronal glutamate transporter EAAC1, Met-367, fulfills a similar role, M367L, M367C, and M367S mutants were expressed in HeLa cells and Xenopus laevis oocytes to monitor radioactive transport and transport currents, respectively. The apparent affinity of the Met-367 mutants for D-aspartate and L-glutamate, but not for L-aspartate, was 10 -20-fold reduced as compared with wild type. Unlike wild type, the magnitude of I max was different for each of the three substrates. D-Glutamate, which is also a transportable substrate of EAAC1, did not elicit any detectable response with M367C and M367S but acted as a nontransportable substrate analogue in M367L. In the mutants, substrates inhibited the anion conductance as opposed to the stimulation observed with wild type. Remarkably, the apparent affinity of the blocker D,L-threo--benzyloxyaspartate in the mutants was similar to that of wild type EAAC1. Our results are consistent with the idea that the side chain of Met-367 fulfills a steric role in the positioning of the substrate in the binding pocket in a step subsequent to its initial binding.Glutamate transporters keep the synaptic concentrations of the neurotransmitter below neurotoxic levels and are key elements in the termination of the synaptic actions of the neurotransmitter. Glutamate transport is an electrogenic process (1, 2) consisting of two sequential translocation steps: 1) cotransport of the neurotransmitter with three sodium ions and a proton (3, 4) and 2) the countertransport of one potassium ion (5-7). Glutamate transporters mediate two distinct types of substrate-induced steady-state current: an inward-rectifying or "coupled" current, reflecting electrogenic sodium-coupled glutamate translocation, and an "uncoupled" sodium-dependent current, which is carried by chloride ions and further activated by substrates of the transporter (8 -10). Substrate analogues, such as D,L-threo--benzyloxyaspartate (TBOA), 2 can bind to the transporters, but their extra bulk prevents them from being transported. These analogues are expected to "lock" the transporter in an outward-facing conformation and not only are competitive inhibitors of the two types of substrate-induced current but also inhibit the basal sodium-dependent anion conductance (11,12). Moreover, when the membrane voltage is suddenly changed in the absence of substrate, sodium-dependent transient currents are observed (13). These transient currents presumably represent a charge-moving conformational change following binding/debinding of sodium. The addition of a transportable substrate enables transport and thereby co...