Direct evidence for two ASCT2 Na⁺-binding sites. (A) Original anion current recordings upon the application of 10 mM alanine (timing indicated by the bars) at [Na⁺] of 0.2 mM (left), 2 mM (middle), and 140 mM (right). The membrane potential was 0 mV, and the pipette solution contained 140 mM NaSCN and 10 mM alanine. The external anion was Mes⁻. Na⁺ was replaced by NMG⁺. (B) [Na⁺] dependence of the maximum alanine-induced current, I_max, at saturating [alanine] (V = 0 mV, normalized to the current at 140 mM Na⁺). The solid line represents a fit of Eq. 8 (see Appendix), assuming a two-site binding model. The fitted parameters are: K_Na1 = 0.1 mM, K_Na1 = 2.8 mM, and r = 1.9. (C) Determination of the K_m of ASCT2 for alanine at different [Na⁺]. The solid lines represent fits to a Michaelis–Menten-type equation. (D) Dependence of the K_m for alanine on [Na⁺]. The solid red line represents a fit using the equation K_m = (1 + K_Na1/[Na⁺])K_S/(1 + [Na⁺]/K_Na2) (Eq. 9; see Appendix), assuming a two-site binding model (K_Na1 = 0.1 mM, K_Na2 = 8.5 mM, and K_S = 7.5 mM; K_Na1 was fixed in the fitting procedure). The dashed blue line represents a model in which two [Na⁺] bind before the amino acid (Eq. 10; see Appendix). The black solid line represents a model in which only alanine binding and one subsequent Na⁺-binding step were included (K_m = K_S/(1 + [Na⁺]/K_Na2), K_Na2 = 6.5 mM, and K_S = 9 mM). (E) Minimal sequential mechanism for Na⁺ and amino acid (AA) binding to the transporter, T. The bars illustrate the anion-conducting states.