IRK1 channels are constitutively open, producing a large basal K+ current. We speculated that alcohols might therefore inhibit GIRK channels engineered to be constitutively open. We introduced a high affinity PIP2 site shown previously to produce a large basal current28,29 (GIRK2-PIP2). In contrast to wild-type GIRK2, GIRK2-PIP2 exhibited large basal currents as expected (−530 ± 197 pApF−1, n=5). Application of 100 mM MPD inhibited the basal K+ current of GIRK2-PIP2 (Fig. 7e). Similar to IRK1, we hypothesized that mutating L257 to Trp in GIRK2-PIP2 would have no effect in alcohol-mediated inhibition. Accordingly, GIRK2-PIP2-L257W produced large Ba++-sensitive currents (−363 ± 182 pApF−1, n=6) that were inhibited by MPD, similar to GIRK2-PIP2 channels (Fig. 7f). We conclude that the hydrophobic alcohol-binding pocket in IRK1 or GIRK2 is not involved in alcohol-dependent inhibition. Furthermore, these results show that constitutively open inwardly rectifying K+ channels are not activated further by alcohols.
