Figure 9.

Perturbations to the conserved salt-bridge network alter metal selectivity and increase K_m for Mn²⁺. (A) Average initial Ca²⁺ transport rates ± SEM (n ≥ 4) at −120 mV with pH 7.0 on both sides of the membrane and 750 µM Ca²⁺. (B) Schematic of mutation locations. M230A, or R353A, 15 Å away; both increased Ca²⁺ transport. D56 and N59 mutations eliminated Ca²⁺ uptake, and other mutants were similar to WT. (C and D) Dose–response curves of Cd²⁺ (C) or Mn²⁺ (D) transport at −150 mV with pH 7.0 on both sides of the membrane for a subset of mutants that reduce or eliminate ΔΨ dependence, ΔpH stimulation, or metal-dependent proton transport. Data are averages ± SEM (n ≥ 2). The resulting transport kinetic values (middle) show significant overlap for Cd²⁺ transport but wider separation for the physiological substrate Mn²⁺. M230A is the only mutation that impaired Cd²⁺ transport more severely than Mn²⁺ transport. Errors in K_m and V_max encompass the uncertainty of the fit to the data. See also Fig. S10 for representative time traces of the data in A.