Figure 5.

ZPT activates VRAC currents under isotonic conditions. A: time course of whole cell Cl⁻ currents from HCT116 cells. Currents were measured using a ramp protocol from −100 mV to +100 mV. Cells are exposed to an isotonic bath (300 mOsm) containing DMSO (vehicle) (n = 4) or 30 μM ZPT (n = 4) at time = 0 min. B: summary of current density of DMSO-treated and ZPT-treated cells from −100 mV and +100 mV at time = 3 min. Data were analyzed by two-way ANOVA and Sidak’s multiple-comparisons test, ****P < 0.0001. C: whole cell currents from HCT116 cells were measured using a step protocol from −120 mV to +120 mV, with 20-mV steps. Cells were exposed to an isotonic bath (300 mOsm) containing DMSO (vehicle) or 30 μM ZPT. D: I–V plot from currents recorded in C [DMSO (n = 3); ZPT (n = 5)]. E: representative time course of ZPT-activated and DCPIB-inhibitable whole cell Cl⁻ currents from HCT116 cells (n = 4). Currents were measured using a ramp protocol from −100 mV to +100 mV. Cells were exposed to a hypotonic bath (250 mOsm) containing 30 μM ZPT at time = 0. Solution exchange with a hypotonic bath containing 30 μM ZPT and 10 μM DCPIB occurred once steady state was reached (∼time = 4 min). F: summary of steady-state current density from E. Data were analyzed by two-way ANOVA and Sidak’s multiple-comparisons test, ***P < 0.001, ****P < 0.0001. G: images from a ZPT-treated cell at time = −1 min, +3 min, and +5 min. H: time course of cell volume measurements from HCT116 cells. Cells were exposed to an isotonic bath (300 mOsm) containing DMSO (n = 3) or 30 µM ZPT (n = 3) at time = 0 min. Data were analyzed by two-way ANOVA and Sidak’s multiple-comparisons test, not significant. DCPIB, 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid; HCT, human colorectal carcinoma; n.s., not significant; VRAC, volume-regulated anion channels; ZPT, zinc pyrithione.