Mouse GIRK2 channels were expressed in Pichia pastoris, purified, and reconstituted into liposomes of defined compositions (Fig. 1a), following a protocol described previously15, 32. GIRK2 channels express efficiently in Pichia pastoris and retain their activation by Gβγ subunits15. To study K+ flux through GIRK2 channels, we used a fluorescence-based K+ flux assay15 (Fig. 1b) with a plate-reader equipped with a fluidic system to add compounds acutely. In GIRK2-containing liposomes loaded with K+ and incubated with the membrane permeable pH-sensitive ACMA dye, the addition of the proton ionophore CCCP results in quenching of the ACMA dye if GIRK channels are open, i.e., H+ enter via CCCP if K+ can exit the proteoliposome (Fig. 1b). Thus the decrease in fluorescence, i.e., quenching, provides a measure of the relative K+ flux that correlates directly with GIRK2 channel activity.
