Mutational analysis of ethanol interactions with G-protein-coupled inwardly rectifying potassium channels

Abstract

G-protein-coupled inwardly rectifying potassium (GIRK) channels play a major role in regulating inhibitory responses in the nervous system. Previously, we showed that function of GIRK channels, but not that of the related inwardly rectifying potassium (IRK) channels, was enhanced by intoxicating concentrations of ethanol in native and recombinant systems. In addition, by using chimeras constructed between GIRK2 and IRK1 channels and truncation mutants of GIRK2 channel, a region of 43 amino acids in the carboxyl (C) terminus of GIRK2 channels was shown to be important for enhancement by ethanol. In the present study, we attempted to define the critical amino acids for ethanol actions within this 43 amino acid region. We made GIRK2 channel mutants with single or multiple amino acids changed to those corresponding to IRK1 channel by using site-directed mutagenesis and expressed them in Xenopus oocytes. Each of the amino acid residues not identical between GIRK2 and IRK1 channels was systematically changed. Ethanol (100 mM) enhanced function of wild type GIRK2 channel by 38%, as observed previously. However, none of the mutants showed any significant difference from wild type, indicating that none of the mutations tested were sufficient to abolish the enhancement by ethanol.