Direct and specific activation of human inward rectifier K⁺ channels by membrane phosphatidylinositol 4,5-bisphosphate

Many ion channels are modulated by phosphatidylinositol 4,5-bisphosphate (PIP₂), but studies examining the PIP₂ dependence of channel activity have been limited to cell expression systems, which present difficulties for controlling membrane composition. We have characterized the PIP₂ dependence of purified human Kir2.1 and Kir2.2 activity using ⁸⁶Rb⁺ flux and patch clamp assays in liposomes of defined composition. We definitively show that these channels are directly activated by PIP₂ and that PIP₂ is absolutely required in the membrane for channel activity. The results provide the first quantitative description of the dependence of eukaryotic Kir channel function on PIP₂ levels in the membrane; Kir2.1 shows measureable activity in as little as 0.01% PIP ₂, and open probability increases to ∼0.4 at 1% PIP₂. Activation of Kir2.1 by phosphatidylinositol phosphates is also highly selective for PIP₂; PI, PI(4)P, and PI(5)P do not activate channels, and PI(3,4,5)P₃ causes minimal activity. The PIP₂ dependence of eukaryotic Kir activity is almost exactly opposite that of KirBac1.1, which shows marked inhibition by PIP₂. This raises the interesting hypothesis that PIP₂ activation of eukaryotic channels reflects an evolutionary adaptation of the channel to the appearance of PIP₂ in the eukaryotic cell membrane.