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 of concentrations (25, 125, 250 mM) for 1-PrOH or MPD (Fig. 5b), indicating a significant impairment in alcohol sensitivity. In addition to the change in alcohol response, mutations at L257 also reduced the m2R-mediated currents (Figs. 4c-e and 5a), indicating that L257 is involved in both alcohol-mediated and Gβγ-mediated activation (see Discussion). Taken together, these results demonstrate that increasing the side-chain volume at L257 leads to a progressive loss in alcohol-mediated activation (Fig. 5a and inset). A switch in alcohol activation occurred with an increase in volume from wild-type Leu (101 Å3) to Tyr (133 Å3) or Trp (168 Å3). In addition, bulky substitutions at L257 affected larger alcohols (MPD) more than smaller alcohols (EtOH), suggesting the molecular volume of the pocket is an important determinant of alcohol specificity.
