Characterization of cholesterol-binding mutants of MEC-2. (A) Alignment of selected PHB family members. MEC-2 and UNC-24 are coexpressed in vivo. MEC-2 is also represented schematically on the right. In yellow, P134, required for cholesterol binding. In green, C140 and C174 required for palmitoylation (Huber et al., 2006). (B) I-V relationship of channels lacking MEC-2, containing MEC-2 double mutant, and MEC-2 triple mutant. MEC-2(P134S) single mutant currents are essentially indistinguishable from the triple mutant and are omitted for clarity. (C) Average currents recorded from MEC-2 mutant-expressing cells (top). Bars are mean ± SEM. Sample size is indicated in parentheses below each bar. †, P < 0.005, compared with the absence of MEC-2; *, P < 10−6 compared with wild-type MEC-2. Western blot of MEC-2 isoforms (bottom). For clarity, lanes corresponding to each isoform are aligned with the data in A. All lanes were from the same blot (with identical contrast manipulation). Similar results were obtained in a total of three independent experiments. (D) Single-channel activity from an outside-out patch of a cell expressing triple mutant MEC-2. These data were used to measure single-channel conductance. Solid lines indicate zero, one or both channels open; all-points histogram is shown on the right.
