Liposomes were diluted 1:20 into flux buffer (20 mM Na-HEPES, pH 7.4; 150 mM NaCl; 0.5 mM EDTA) containing 5 μM of the H+ sensitive dye 9-Amino-6-chloro-2-methoxyacridine (ACMA). Fluorescence was measured using a Flexstation 3 microplate reader (Molecular Devices) with the following parameters: 410 nm excitation, 480 nm emission, 455 nm cutoff, medium PMT sensitivity, and sampling at 2 seconds. After a stable baseline fluorescence (150 s) was obtained, the H+ ionophore m-chlorophenyl hydrazone (CCCP) was automatically added (1 μM), then a second addition consisting of different compounds or vehicle was added 150 s later, followed 900 s later by a third addition with the K+ ionophore Valinomycin (100 nM), to determine the maximal K+ flux. GIRK2 channels are likely arranged in both orientations in the liposomes. However, we expect the channels oriented inside-out to support high K+ flux because of high Na+ in the flux buffer and high K+ in the liposome14, 35, 36.
