Reduction and reformation of disulfides. (A) Structure of 4,4′-(azodicarbonyl)-bis-[1,1-dimethylpiperazinium, diiodide] (QPD). (B) Reduction and reoxidation of the disulfide between G18C and L199C (left) and R20C and R201C (right). The intact cells were surface biotinylated, reduced with DTT, and oxidized for 6 min with 40 μM QPD. The extent of cross-linking was estimated from each pair of lanes. (C) Functional effects of reduction and reoxidation: G-V curve in outside-out macropatches from cells expressing the mutant α G18C-L199C (left) and R20C- R201C (right). The cells were treated with 20 mM DTT, pH 8.0, for 20 min, before pulling the patch. After first G-V curve, the patch was exposed to 40 μM QPD and the effects monitored with 1-s ramps (−100 to +100 mV) from a holding potential of −80 mV, with 5-s intervals at −80 mV. When the reaction was complete, a second G-V curve was determined. The G-V curve from a different cell expressing the same double-Cys mutant that was neither reduced nor reoxidized is shown as a dashed line. The pipet contained 10 μM Ca²⁺. (D) The mean change in V₅₀ induced by QPD acting on cells expressing the double-Cys mutants, R20C-R201C and G18C-L199C, and each of the single-Cys mutants, from records such as in C. The means, standard errors, and n are shown.