In previous studies, extracellular application of alcohol enhanced GIRK channel activity, both with heterologously expressed channels, or with natively expressed channels in cerebellar neurons16, 17. Alcohol activation was determined to be insensitive to pertussis toxin (PTX) or antibodies to the G protein β1 subunit, suggesting that G proteins were not involved17. Further, deletion studies16, the identification of an alcohol pocket in x-ray crystallographic structures18, 51 and alcohol-tagging studies19 all indicated that alcohol activation was likely mediated by the direct interaction of alcohol with the channel. However, the involvement of endogenous proteins, e.g. Gβγ subunits, could not be ruled out with heterologous expression systems, such as oocytes or HEK293 cells, or native cell systems, i.e., neurons. In addition, a component of ethanol activation could include an alteration in the lipid bilayer fluidity21, since this was not directly tested. By reconstituting purified GIRK2 channels in liposomes, controlling the components of the bilayer, and adding alcohol in isolation of any other proteins, we could overcome these previous limitations and demonstrate that intoxicating concentrations of ethanol, i.e., >20 mM, directly activate GIRK2 channels in
