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In addition to G proteins, ethanol can activate G protein-gated inwardly rectifying K (GIRK) channels. The mechanism underlying GIRK channel activation by alcohol is not well understood. Based on a crystal structure of a related IRK1 channel which contains the alcohol (2-methyl,2-4-pentanediol- MPD) bound to a cytoplasmic hydrophobic pocket, we used structure-based mutagenesis and patch-clamp electrophysiology to investigate the role of the homologous alcohol pocket in GIRK2 channels. In HEK293T cells transfected with GIRK2 cDNA, both ethanol and MPD activated GIRK2 channels. Replacing a conserved Leucine (L257) in this pocket with a bulkier Tyrosine or Tryptophan led to significant attenuation or loss of alcohol-dependent activation of GIRK2 channels, suggesting these larger hydrophobic side-chains filled the pocket. Based on structure and functional evidence, we conclude that this hydrophobic pocket is the site for alcohol activation of GIRK channels. We hypothesized that tethering a hydrophobic group near the pocket might mimic alcohol mediated activation of the channel. To test this idea, we introduced a S246C mutation in a Cysteine-less GIRK2 channel and examined the effect of bath applied MTS-Benzene. Application of 10 micromolar MTS-Benzene dramatically increased the size of basal GIRK currents by 336+66% n=5. This rapid activation was reversed by application of reducing agent DTT (10 mM), indicating a disulfide bond had formed. In addition to the change in basal current, MTS modification of S246C channel altered the rank order for alcohol activation -with significantly less activation by the larger alcohol MPD. These results suggest that attachment of a bulky hydrophobic amino acid near the hydrophobic alcohol-binding pocket can produce sustained activation of the channel by associating with the activation site. These experiments provide a launching point to study molecular events at this hydrophobic pocket that lead to activation of GIRK channels.

Alcohols can activate G protein-gated inwardly rectifying K (GIRK) channels but the molecular mechanism is not well understood. To investigate the possibility of a physical alcohol pocket located in the cytoplasmic domain of GIRK channels, we used a crystal structure of related IRK1 channel containing a bound alcohol (2-methyl, 2-4-pentanediol - MPD) and structure-based mutagenesis of GIRK2 and GIRK4 channels combined with patch-clamp electrophysiology. In transiently transfected HEK293 cells, both wild-type GIRK2 and GIRK4 channels were activated by 100 mM ethanol, MPD and 1-Propanol. Replacing a conserved Leucine (GIRK2-L257 / GIRK4-L252) in the betaD-betaE region of the cytoplasmic domain with bulkier Tyrosine or Tryptophan led to significant attenuation or loss of alcohol-dependent activation for both GIRK2 and GIRK4 channels. Constitutively open channels, such as IRK1 and GIRK2-PIP2 (engineered to bind PIP2 with high affinity), on the other hand, were inhibited by ethanol, 1-propanol, 1-butanol and MPD. Mutating the homologous Leucine in IRK1 (L257) or in GIRK2-PIP2 channels did not alter the sensitivity to inhibition by these alcohols, suggesting a second site in the channel is involved in inhibition by alcohols. Consistent with this, mutagenesis of the extracellular pore-helix of GIRK4 and GIRK2-PIP2 channels reduced the sensitivity to alcohol-mediated inhibition. Interestingly, mutation of the conserved Leucine (L257/L252) in the betaD-betaE domain also disrupted G protein-dependent activation, suggesting a common mechanism of activation by alcohols and G-proteins. Using our data and an analysis of high-resolution structures of inwardly rectifying K channels, we propose a novel model for alcohol activation of GIRK channels that is mediated by the cytoplasmic hydrophobic alcohol pocket.