FIGURE 2.

Mutations of Asp-467 in the proposed Na1 site affect substrate binding and transport. A, current-voltage relationship elicited by 300 μm l-serine in Cl⁻ containing buffer (open squares) and NO₃⁻ containing buffer (closed squares), at pH 7.5 in wild type ASCT1. Sample currents in response to 100-ms voltage jumps from −30 to +60 mV (top panel depicts protocol) at 1 mm l-serine and 96 mm NaNO₃ are shown for ASCT1 (B) and D467T (C) (lower panels). Imemb refers to the membrane potential, and V refers to the applied voltage. Concentration-response curves are shown for l-serine (D) and Na⁺ (E) in ASCT1 (closed triangles), D467N (circles), D467S (closed squares), D467T (diamonds), and D467A (open squares) at +60 mV. l-Serine concentrations were varied in a NO₃⁻ based buffer with 96 mm NaNO₃. Na⁺ titrations were performed with 1 mm l-serine and NMDG⁺ as the substitute cation and leak currents were subtracted from baseline measurements. F, l-[³H]serine uptake into oocytes expressing wild type and mutant ASCT1 transporters. Oocytes were incubated in Cl⁻ containing buffer with 10 μm l-[³H]serine at room temperature, pH 7.5, for 10 min. Values presented are mean ± S.E., see Table 1 for n values.