While investigating alcohol modulation of GIRK4*, we discovered that 1-BuOH activated GIRK4*, in contrast to inhibition of GIRK4 wild-type (Supplementary Fig. S3) or GIRK1/4 heterotetramers7,8. GIRK4 and GIRK4* differ by a point mutation (S143T) in the pore-helix27 suggesting that S143T may regulate sensitivity to alcohol inhibition. To assess whether this site is generally involved in alcohol-mediated inhibition of GIRK channels, we introduced a Thr at the equivalent Ser (S148T) in GIRK2-PIP2 channels. As predicted, GIRK2-PIP2--S148T significantly shifted the IC50 for MPD-dependent inhibition compared to GIRK2-PIP2 (Fig. 7f). Taken together, these experiments implicate amino acids in the pore-helix in regulating the extent of alcohol-dependent inhibition and support the conclusion that the cytoplasmic alcohol-binding pocket mediates alcohol-dependent activation, but not inhibition, of GIRK channels.
