MPD is bound to a hydrophobic pocket in the crystal structure of IRK1, suggesting that MPD might inhibit IRK1 channels like other alcohols7,8. Bath applying 100 mM MPD inhibited nearly 50% the basal inwardly rectifying K+ current through IRK1 channels (Fig. 7a). The MPD inhibition was dose dependent and had an IC50 of 104 ± 23 mM and a Hill coefficient of 0.93 ± 0.02 (n=8) (Fig. 7b). We next investigated whether Trp substitutions in the hydrophobic alcohol-binding pocket of IRK1 altered alcohol-dependent inhibition (Fig. 7c). IRK1-F47W, IRK1-L232W, IRK1-L245W IRK1-L330W but not IRK1-Y337W produced significant basal K+ current (data not shown). Like wild-type IRK1, MPD inhibited the basal currents of mutant channels in a dose-dependent manner. In fact, the IC50 for MPD inhibition was indistinguishable among the different IRK1 mutants (Fig. 7d). Furthermore, IRK1-L245W mutation did not alter inhibition by EtOH, 1-PrOH or 1-BuOH (data not shown). Thus, mutations at the hydrophobic pocket of IRK1 channels do not appear to alter the sensitivity to inhibition by alcohols.
