Figure 3.

Reversible and sequential binding of two Ag⁺ ions to a single cysteine residue. (A) Current recordings at different free Ag⁺ ion concentrations, [Ag⁺]_free (Ag⁺ was added as AgNO₃ to the cis compartment). Each current level is labeled on the right: P_C = unoccupied pore (level 0); P_CS-Ag = one Ag⁺ ion-bound (level 1); P_CS⁺(Ag)₂ = two Ag⁺ ions-bound (level 2). All-points amplitude histograms are shown on the left. Conditions: 2 M KNO₃, 10 mM MOPS, 10 mM EDTA, pH 7.4, at 22°C and –50 mV. Under these conditions, P_C has an open pore current of –96 ± 2 pA (n = 5). (B) Dwell-time histograms for P_C, P_CS-Ag, and P_CS⁺(Ag)₂ in the presence of 66 nM [Ag⁺]_free. Each histogram was fitted to a single-exponential function using Clampfit (Molecular Devices) to determine the mean dwell time $\left(\overline{\tau }\right)$. (C) Plots of the measured rates of each transition versus [Ag⁺]_free. Each data point (mean ± SD) was obtained from three experiments: level 0→level 1 (blue solid rhombus), level 1→0 (blue open rhombus), level 1→2 (red solid triangle), level 2→1 (red open triangle). The determination of rate constants from these plots is described in the Materials and Methods section. (D) Kinetic scheme describing the sequential association of two Ag⁺ ions with the cysteine side chain in P_C. Direct transitions between P_C and P_CS⁺(Ag)₂ are not observed. This scheme was used in QuB (see Materials and Methods) for the determination of rate constants.