Excitatory amino acid transporters (EAATs) are assembled as oligomers consisting of three subunits, each of which appears to transport glutamate independently from the neighboring subunits. EAATs do not only sustain a secondary-active glutamate transport, but also function as anion channel. Here we report that inter-subunit interactions are necessary for activating the pore- mediated anion conduction. We expressed a neuronal isoform, rEAAT4, heterologously in Xenopus oocytes and mammalian cells and measured glutamate fluxes and anion currents under various concentrations of Na+ and glutamate. EAAT4 anion channels are active in the absence of both substrates, and increasing concentrations activate EAAT4 anion currents with a sigmoidal concentration dependence. Since only one glutamate is co-transported per uptake cycle, the cooperativity between glutamate binding sites must arise from an interaction between different carrier domains. Two point mutations, G464S and Q467S, alter the dissociation constants and Hill coefficient of the substrate dependence of anion currents, as well as the time- and voltage dependence anion channel gating. The concentration dependence of glutamate uptake was not affected by these mutations, suggesting an independent operation of these two transport processes. Our results demonstrate glutamate carriers cooperatively interacting during the anion channel activation.