Purified GIRK2 channels, containing amino acids 52 - 380, were reconstituted into liposomes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (POPG) at a 3:1 ratio15. PE:PG lipids were found previously to support both Gβγ and Na+-dependent activation15. We first examined the requirement of PIP2 for channel activation12, 14. PIP2 is found in the plasma membrane at concentrations up to 1%33 and has been shown previously to be required for GIRK activity12, 14, 34. We measured the relative K+ flux through GIRK2 channels reconstituted in either the absence or presence of 1% brain PIP2, under conditions that saturate the cytoplasmic Na+ binding site in GIRK235, 36. GIRK2-containing liposomes containing brain PIP2 exhibited a robust quenching of fluorescence upon addition of CCCP (‘GIRK2 /PIP2’, Fig. 1b). Subsequent addition of a small molecular inhibitor of GIRK2 channels, MTS-HE (100 μM)37, abruptly slowed the rate of quenching, indicating closure of GIRK2 channels (‘GIRK2/PIP2 + HE’, Fig. 1b). Similarly, pre-incubating GIRK2-containing liposomes with the K channel inhibitor BaCl2 (2 mM) prevented the CCCP-dependent quenching (data not shown). In the absence of PIP2, however, there was no significant
