Characteristics of LRRC8 channels. (A) Cartoon showing key structural features of a LRRC8 protein. LRRC8 proteins comprise four predicted transmembrane domains (TMDs), a large EL1 connecting TMDs 1 and 2, an IL connecting TMDs 2 and 3, and a C-terminal LRR domain. (B) Current–voltage relationships of currents in Lrrc8^−/− HCT116 cells transfected with GFP cDNA. Cell volume–sensitive anion currents are undetectable. Values are means ± SEM (n = 7). (C) Time course of swelling-induced activation and shrinkage-induced inactivation of whole-cell Cl⁻ currents in Lrrc8^−/− HCT116 cells coexpressing LRRC8A and LRRC8C. Cells were swollen by exposure to a 250-mOsm bath solution and then shrunken by returning to hypertonic 400-mOsm bath. Values are means ± SEM (n = 7). (D) Current–voltage relationships (top) of basal current, peak swelling-induced current, and stable current observed after shrinking swollen cells in a hypertonic bath. Values are means ± SEM (n = 7). Representative traces (bottom) of swelling-activated 8A + 8C current. (E) Top: Current–voltage relationships of currents in LRRC8A-expressing cells. LRRC8A expression induces a small, outwardly rectifying current that is insensitive to cell volume changes. Values are means ± SEM (n = 5–6). Bottom: Fluorescent and brightfield images of a cell expressing LRRC8A with a C-terminal GFP tag. Bar, 5 μm. Protein appears to be localized primarily in or near the plasma membrane. (F) Top: Current–voltage relationships of currents in LRRC8C-expressing cells. Currents were not significantly different (P > 0.07) from those in GFP-expressing Lrrc8^−/− HCT116 cells (see B). Values are means ± SEM (n = 7). Bottom: Fluorescent and brightfield images of a cell expressing LRRC8C with a C-terminal GFP tag. Bar, 5 μm. Protein appears to be localized primarily to the cytoplasm.