FIGURE 1.

Mutation of Thr¹⁰¹ to alanine interferes with the ability of Na⁺ to induce anion current. A, illustration of the transport cycle. B, multiple sequence alignment of EAATs and GltPh shows that Thr¹⁰¹ (red, arrow) is conserved within the glutamate transporter family. C, typical leak anion currents induced by Na⁺ application to EAAC1_WT in the absence of external substrate (left panel, black), nontransfected control cells (left panel, red), and EAAC1_T101A (middle and right panels). The gray bars indicate the length of Na⁺ application. The extracellular solution contained MES as the anion and Na⁺/NMG⁺. The intracellular solution contained 140 mm SCN⁻ and 10 mm glutamate (see inset in C). D, [Na⁺] dependence of specific leak anion currents (red circles, WT; black circles, T101A; black triangles, T101S, control responses were subtracted; open square, control response in the absence of SCN⁻). The lines represent fits to the Michaelis-Menten equation (WT, T101S) or a sum of two Michaelis-Menten equations (T101A). E, K_Na⁺ values determined from the fits. F, voltage dependence of the 60 mm Na⁺-induced apparent outward current (T101A, open circles) indicates that it is mediated by inhibition of a tonic inward anion conductance. For comparison, inhibition of the tonic leak anion conductance of EAAC1_WT by TBOA as a function of the voltage is shown as the solid circles.