In the IRK1-MPD structure, L245, which is homologous to L257, is positioned at the base of the pocket, and interacts intimately with MPD. The decrease in EtOH-activated current of GIRK2-L257W raised the possibility that amino acids with bulky side-chains might generally interfere with alcohol activation. We systematically evaluated the effect of substituting twelve different amino acids of increasing molecular side-chain volume in GIRK2-L257. Of the twelve, five expressed (> −1 pApF−1) and could be investigated further for possible changes in alcohol-mediated activation (Fig. 4a). The magnitude and rank order (1-PrOH > MPD > EtOH) for alcohol activation with smaller molecular volume substitutions, such as Ala, Cys and Met, were indistinguishable from wild-type Leu in GIRK2 channels (Figs. 4b,c and 5a). On the other hand, GIRK2-L257Y reduced 1-PrOH and MPD but not EtOH activation while GIRK2-L257W affected EtOH, 1-PrOH and MPD dependent activation (Figs. 4d,e and 5a). Interestingly, 100 mM MPD no longer activated and now inhibited the basal currents for GIRK2-L257W (Figs. 4e and 5a). For GIRK2-L257Y and GIRK2-L257W, the decrease in alcohol activation was observed at a full range
