(A) Current-Voltage relationships of HEK 293 cells expressing murine SLC26A9 and (B) human SLC26A6. Data were recorded in the whole-cell configuration in symmetric (150 mM) Cl- concentrations and asymmetric conditions with equimolar (150 mM) concentrations of intracellular Cl- and extracellular HCO3-. Values show mean of independent experiments (SLC26A9 Cl-/Cl- n=6, Cl-/ HCO3-, n=6; SLC26A6 Cl-/Cl- n=6, Cl-/ HCO3-, n=6). Dashed lines in (B) correspond to SLC26A9 data displayed in (A). (C) Protein expression at the surface of HEK cells determined by surface biotinylation. Ratio of biotinylated (right) over total protein (left) as quantified from a Western blot against a myc-tag fused to the C-terminus of the respective constructs. (D) Uncoupled Cl- transport mediated by either the modified murine construct SLC26A9T or SLC26A6 reconstituted into proteoliposomes, as monitored by the fluorescence change of the pH gradient-sensitive fluorophore ACMA. Traces show mean of seven and three replicates from two independent reconstitutions for SLC26A9T and SLC26A6. (E) Coupled Cl-/HCO3- exchange monitored by the time- and concentration-dependent quenching of the fluorophore lucigenin trapped inside proteoliposomes containing SLC26A6. Traces show mean of six independent experiments from two reconstitutions. (F) Cl- concentration dependence of transport. Initial velocities were obtained from individual measurements displayed in (E), the solid line shows a fit to the Michaelis Menten equation with an apparent Km of 16 mM. (G) Coupled Cl-/HCO3- exchange monitored by the time- and concentration-dependent luminescence increase of the HCO3-selective probe [Eu.L1+] trapped inside proteoliposomes containing SLC26A6. Traces show mean of five independent experiments from three reconstitutions. ‘neg.’ refers to mock liposomes. (E, G), Hashtag indicates addition of the assayed anion. (H) Electrogenic oxalate uptake followed by the fluorescence change of the pH gradient sensitive fluorophore ACMA. Traces show mean quenching of ACMA fluorescence in a time- and concentration-dependent manner for SLC26A6 proteoliposomes with outside oxalate concentrations of 9.4 mM (n=3), 37.5 mM (n=5), 75 mM (n=6), 150 mM (n=8, all from two independent reconstitutions). Neg. refers to mock liposomes assayed upon addition of 75 mM oxalate as defined in Figure 1—figure supplement 1G. (D, H), Asterisk indicates addition of the H+ ionophore CCCP, which allows counterion movement and electrogenic Cl– transport to proceed. (A, B, D–H), errors are s.e.m. (D, E, G, H) Scheme of the respective assay is shown left.