Cholesterol enantiomer (ent-cholesterol) consistently fails to inhibit BK channels. Representative single-channel recordings obtained after cbv1 incorporation into control (A), ent-cholesterol- (B), and cholesterol-containing (C) POPE/POPS (3:1 wt/wt) bilayers. Ent-cholesterol and cholesterol were introduced at 33 mol% into the lipid mixture. For A–C, channel openings are shown as upward deflections; arrows on the top trace of each panel indicate the baseline. Channel activity (Po) was calculated as described in the legend to Fig. 2. (D) Scatter graph showing individual Po values in control and in the presence of ent-cholesterol or natural cholesterol in the lipid mix. Each data point corresponds to one independent bilayer preparation. (E) Averaged Po values underscoring that ent-cholesterol fails to reduce cbv1 Po from control values while cholesterol remains effective under identical recording conditions. C and D: **, significantly different from control (P < 0.01).