Purified GIRK2 channels were reconstituted into lipid vesicles as described previously15, 34. Briefly, a lipid mixture containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (POPG), and L-α-phosphatidylinositol-4,5-bisphosphate (Brain, PI(4,5)P2 (Porcine)) at mass ratios of 3:1:0.04 (POPE:POPG:PIP2) or 3:1 (POPE:POPG) was prepared, reconstituted in vesicle buffer (20 mM K-HEPES, pH 7.4; 150 mM KCl; 0.5 mM EDTA containing 35 mM CHAPS) and incubated with protein in detergent at a 1:200 protein: lipid ratio unless otherwise indicated. Where indicated, cholesterol (Ovine wool) was added to vesicles at a mole percentage of 1%, 5% or 10%. Cholesterol could not be incorporated into liposomes at concentrations higher than 10% as noted previously25. Detergent was removed through sequential addition of Bio-beads SM-2 (Bio-rad). All phospholipids, cholesterol, and Brain PIP2 were purchased from Avanti Polar Lipids, Inc. Soluble PIP2 (diC8-PIP2) was purchased from Echelon Biosciences.
