Heterogeneous substrate binding in WT Glt _Ph . (a–c) Aspartate binding isotherms derived from the ITC experiments performed at 15°C in the presence of 500 mM Na-gluconate (red circles; a); NaCl (green squares; b); or NaNO₃ (blue triangles; c). (d and e) Aspartate binding isotherms in 500 mM Na-gluconate at 25°C (d) or 35°C (e). Experiments in a–e were performed at least twice on independently prepared protein samples, producing similar results. All data were fitted to the two-state model (black lines); however, the binding parameters were not uniquely determined (see Fig. S2 for further information). Insets show the thermal power with the corresponding scales. (f) Aspartate transport of Glt_Ph (C321A/N378C) was measured using the single-transporter FRET-based assay in NaNO₃ (blue) or NaCl (green). Transport was initiated by perfusing surface-immobilized proteoliposomes with buffer containing 200 mM sodium salt, 1 μM valinomycin, and 1 μM L-Asp. Data are shown as fractions of total observable transport over time, fitted to triexponential functions (black lines). The fitted parameters are in Fig. S4 b. Data are means and SE from three independent experiments.