Panel A shows structural views of the 8C 8A(IL1 25) channel, highlighting the positions of Y126 and L136 within the transmembrane domains and pore region. The views include labeled transmembrane domains TM1, TM2, TM3, and TM4, with structural orientations shown from different perspectives. Panel B shows representative whole cell current traces for the Y126F, L136K, and L136E mutants. Current scale bars are shown in picoamperes, and time scale bars are shown in milliseconds. Panel C shows concentration response curves for 8C 8A(IL1²⁵), Y126F, L136E, and L136K. The x-axis represents DCPIB concentration in micromolar on a logarithmic scale, and the y-axis represents percentage inhibition. Panel D shows relative current over time during application of DCPIB followed by washout for 8C 8A(IL1 25) and the L136K mutant. The x-axis represents time in minutes, and the y-axis represents relative current. Panel E shows relative current over time for the L136E mutant at positive 100 millivolts and negative 100 millivolts. Different DCPIB concentrations are indicated from 0.1 to 10 micromolar. The x-axis represents time in minutes, and the y-axis represents relative current. Error bars are shown throughout the graphs.
Analysis of 8C-8A(IL1 25 ) TM2 mutations on DCPIB sensitivity. (A) Side (left) and top (right) views of the 8C-8A(IL125) TMD (PDB ID: 8DXN) along with the solvent-accessible permeation pathway (gray surface) calculated using HOLE (Smart et al., 1996). (B) Representative whole-cell currents of the indicated mutants recorded from a cell dialyzed with normal pipette solution and cell swelling with hypotonic buffer. WT current is shown in Fig. 3 A. (C) CRC data recorded at +100 mV for the indicated mutants. Fit of WT CRC data (black line) from Fig. 3 C is shown for comparison (8C-8A(IL125) n = 6, mutants n = 5 each). (D) Time course of drug washout in L136K. WT data (gray line) from Fig. 3 E is shown for comparison (8C-8A(IL125) n = 4, L136K n = 4). After full activation, 10 μM DCPIB was applied for 7 min and then washed out for 5 min by continuous perfusion at 1 ml/min. (E) Time course of additional DCPIB-dependent activation of swelling-induced in L136E mutant (n = 5). Triangles and numbers indicate the timings and concentrations of DCPIB application, respectively. Data are the mean ± SEM.