GIRK2 channels reconstituted in cholesterol-containing liposomes (5%) lacking brain PIP2 exhibited no basal K+ flux, indicating that PIP2 is required for cholesterol activation, similar to alcohol activation. We hypothesized that cholesterol may enhance GIRK2 activity by altering the sensitivity to PIP2. To test this, we measured the rate of K+ flux in GIRK2-containing liposomes containing 5% cholesterol that were directly exposed to different concentrations of diC8-PIP2 (Fig. 5a). The dose-response curve for diC8-PIP2 in the presence of cholesterol shifted to lower concentrations, compared to that without cholesterol (Fig. 5b). The EC50 for PIP2 activation of GIRK2 in the presence of cholesterol was 12.2 ± 2.5 μM (n = 6), compared to an EC50 of 25.1 ± 3.3 μM without cholesterol. Thus, the apparent affinity for PIP2 increases nearly 2-fold in the presence of cholesterol. In GIRK2 proteoliposomes containing both brain PIP2 (1%) and 5% cholesterol, acute application of neomycin decreased the rate of K+ flux, with an IC50 of 15.0 ± 4.6 μM (Fig. 5c,d). The IC50 for neomycin inhibition is indistinguishable from that measured in the absence of cholesterol
