Panel A shows an alignment of protein sequences from LRRC8A and LRRC8C, highlighting differences in the second transmembrane domain. Panel B illustrates schematic images of heteromeric and chimeric LRRC8 channels, with regions from 8A shown in red and regions from 8C shown in blue. Panels C to H display representative whole cell current traces for various channels under different conditions, with and without the application of DCPIB. Panel I presents a bar graph showing the percentage inhibition by DCPIB at positive 100 millivolts for the indicated channels, with data points representing means and standard errors.
Identification of essential regions for DCPIB inhibition in chimeras of 8A and 8C. (A) Alignment of the first extracellular loop (EL1) and the second TM domain (TM2) of LRRC8A and LRRC8C. The differences in TM2 are highlighted in yellow. Asterisks denote identical aminio acid residues between the two aligned sequences. (B) Schematic images of heteromeric and chimeric LRRC8 channels. Red and blue indicate regions of 8A and 8C, respectively. (C–H) Representative whole-cell currents of the indicated channels recorded from a cell dialyzed with normal or low ionic strength pipette solution plus cell swelling with hypotonic buffer (left panel), after bath application of 10 μM DCPIB (right panel). The scale is the same for all traces. (I) Inhibition by 10 μM DCPIB at +100 mV in the indicated channels. All cells were exposed to a hypotonic buffer to activate the current. After full activation, 10 μM DCPIB was continuously applied until a new steady state was reached (3–4 min). Data are means ± SEM (8A/8C, n = 5; 8A, n = 5; 8A 26 mM CsCl, n = 4; 8A-8C(TM2) 26 mM CsCl, n = 5; 8A-8C(EL1), n = 4; 8A-8C(EL1+TM2), n = 4). **P = 0.0065; ****P < 0.0001.