To determine whether the hydrophobic pocket in GIRK2 mediates alcohol activation, we examined the effects of the side-chain volume using an amino acid with a small (Ala) or large (Trp) side-chain (Fig. 3a,b). Six mutants did not express basal K+ currents (< −1 pApF−1) (Fig. 3b). In mutant channels engineered with an extracellular hemagglutinin (HA)-tag, we investigated whether the lack of basal current was due to a trafficking defect using confocal microscopy. Four mutants, HA-GIRK2-Y58W, HA-GIRK2-Y58A, HA-GIRK2-L342W and HA-GIRK2-Y349A, expressed on the plasma membrane but did not conduct currents (Fig. 3b; Supplementary Fig. S2). Mutations at GIRK2-I244 impaired expression on the membrane surface (Fig. 3b). These findings suggest the hydrophobic pocket in GIRK2 is important for channel gating and/or assembly in the absence of alcohol. Four other mutants, GIRK2-L257A, GIRK2-L257W, GIRK2-L342A and GIRK2-Y349W, produced functional channels that were activated by EtOH (Fig. 3c). However, GIRK2-L257W displayed significantly smaller EtOH-activated currents (Fig. 3c), suggesting that Leu at position 257 in the βD-βE ribbon is a key residue that is required for alcohol-dependent activation of GIRK2 channels.
