Na⁺ binding to the empty transporter is electrogenic, but alanine binding is electroneutral. (A) Currents in response to voltage jumps (top panel indicates voltage protocol) at 0.5 mM of extracellular Na⁺ (left) and 140 mM Na⁺ (right) (counterion was Mes⁻). The intracellular solution contained 120 mM NaMes/10 mM alanine. (B) Voltage dependence of the charge movement of the on-response (solid line represent fit to a Boltzmann equation). (C) Voltage dependence of the K_m of ASCT2 for alanine. The intracellular solution contained 120 mM NaSCN/10 mM alanine, and the extracellular solution contained 140 mM NaMes. (D) Predicted valences computed by PB analysis for alanine binding as well as for the binding of Na⁺ to three hypothesized binding sites. The inset shows an illustration of the alanine-binding reaction used for calculation of the valence.