Abstract

Recent work has established membrane phospholipids such as phosphatidylinositol-4,5-bisphosphate (PIP₂) as potent regulators of K_ATP channels controlling open probability and ATP sensitivity. We here investigated the effects of phospholipids on the pharmacological properties of cardiac type K_ATP(Kir6.2/SUR2A) channels. In excised membrane patches K_ATPchannels showed considerable variability in sensitivity to glibenclamide and ATP. Application of the phosphatidylinositol phosphates (PIPs) phosphatidylinositiol-4-phosphate, PIP₂, and phosphatidylinositol-3,4,5-trisphosphate reduced sensitivity to ATP and glibenclamide closely resembling the native variability. Insertion of the patch back into the oocyte (patch-cramming) restored high ATP and glibenclamide sensitivity, indicating reversible modulation of K_ATP channels via endogenous PIPs-degrading enzymes. Thus, the observed variability seemed to result from differences in the membrane phospholipid content. PIP₂ also diminished activation of K_ATPchannels by the K⁺ channel openers (KCOs) cromakalim and P1075. The properties mediated by the sulphonylurea receptor (sensitivity to sulfonylureas and KCOs) seemed to be modulated by PIPs via a different mechanism than ATP inhibition mediated by the Kir6.2 subunits. First, polycations abolished the effect of PIP₂on ATP inhibition consistent with an electrostatic mechanism but only weakly affected glibenclamide inhibition and activation by KCOs. Second, PIP₂ had clearly distinct effects on the concentration-response curves for ATP and glibenclamide. However, PIPs seemed to mediate the different effects via the Kir6.2 subunits because a mutation in Kir6.2 (R176A) attenuated simultaneously the effects of PIP₂ on ATP and glibenclamide inhibition. Finally, experiments with various lipids revealed structural features necessary to modulate K_ATP channel properties and an artificial lipid (dioleoylglycerol-succinyl-nitriloacetic acid) that mimicked the effects of PIPs on K_ATP channels.