Figure 2. Na⁺ ions control on- and off-rates of L-asp binding.

(a) The values of k_obs for L-asp binding as a function of Na⁺ concentration. The binding rates were measured as a function of Na⁺ concentrations upon additions of L-asp to P11W^IFS to the following final concentrations in mM: 0.01 (blue), 0.025 (red), 0.05 (green), 0.1 (purple), 0.25 (orange), 0.5 (black), 1 (brown) and 2 (dark blue). (b) The dissociation rates of L-asp as a function of Na⁺ concentration in the media measured for P11W^IFS (solid squares) and P11W^IFS-M311A (open squares). The lines through data for P11W^IFS are the fits to Equation 2 with Hill coefficients n fixed to 1 (red), 2 (green) and 3 (blue). The fitted values of k_off,asp and K_D,Na were, respectively: 0.5 ± 0.2 s⁻¹, 40 ± 20 µM; 0.2 ± 0.02 s⁻¹, 130 ± 20 µM; 0.2 ± 0.01 s⁻¹, 150 ± 13 µM. (c) Schematic representation of the mechanism in Equation 2 exemplified for two rapidly equilibrating cooperatively binding Na⁺ ions. (d) Comparison of the rate of L-asp uptake (open circles) and exchange (solid circles) in proteoliposomes containing wild type Glt_Ph. Background uptake was measured in the absence of the external NaCl and subtracted from the data. All experiments were performed in triplicate and means and standard errors are shown.

Figure 2—figure supplement 1. Na⁺ dependence of L-asp binding kinetics in low (a) and high (b) L-asp.

In the presence of high concentrations of L-asp the dependence of k_obs on Na⁺ concentration is linear. However, at low L-asp concentrations, the dependence is non-linear, and can be approximated by a quadratic function.