Cl⁻-driven H⁺ pumping in E203 mutants. Uphill movements of H⁺ driven by Cl⁻ gradients were examined in WT CLC-ec1 and the indicated Glu_in mutants. (A) Raw recordings of pH of the liposome suspension, with upward deflection indicating proton uptake against a pH gradient. Transport was triggered by the addition of Vln (wedge) and reversed by FCCP (circle). (B) Summary of proton uptake measured after 30 s, with black or gray bars indicating coupled or uncoupled mutants, respectively, as determined from direct coupling measurements (Fig. 2). Each bar represents mean ± SEM of approximately three to eight determinations.