Synergistic activation of G protein-gated inwardly rectifying potassium channels by cholesterol and PI(4,5)P₂

G-protein gated inwardly rectifying potassium (GIRK or Kir3) channels play a major role in the control of the heart rate, and require the membrane phospholipid phosphatidylinositol-bis-phosphate (PI(4,5)P₂) for activation. Recently, we have shown that the activity of the native heterotetrameric Kir3.1/Kir3.4 channel that underlies atrial K_ACh currents was enhanced by cholesterol. Similarly, the activities of both the Kir3.4 homomer and its active pore mutant Kir3.4* (Kir3.4_S143T) were also enhanced by cholesterol. Here we employ planar lipid bilayers to investigate the crosstalk between PI(4,5)P₂ and cholesterol, and demonstrate that these two lipids act synergistically to activate Kir3.4* currents. Further studies using the Xenopus oocytes heterologous expression system suggest that PI(4,5)P₂ and cholesterol act via distinct binding sites. Whereas PI(4,5)P₂ binds to the cytosolic domain of the channel, the putative binding region of cholesterol is located at the center of the transmembrane domain overlapping the central glycine hinge region of the channel. Together, our data suggest that changes in the levels of two key membrane lipids - cholesterol and PI(4,5)P₂ - could act in concert to provide fine-tuning of Kir3 channel function.