Structural determinants of PIP2 regulation of inward rectifier K-ATP channels

Phosphatidylinositol 4,5-bisphosphate (PIP2) activates K-ATP and other inwa rd rectifier (Kir) channels. To determine residues important for PIP2 regul ation, we have systematically mutated each positive charge in the COOH term inus of Kir6.2 to alanine. The effects of these mutations on channel functi on were examined using Rb-86 efflux assays on intact cells and inside-out p atch-clamp methods. Both methods identify essentially the same basic residu es in two narrow regions (176-222 and 301-314) in the COOH terminus that ar e important for the maintenance of channel function and interaction with PI P2. Only one residue (R201A) simultaneously affected ATP and PIP2 sensitivi ty, which is consistent with the notion that these ligands, while functiona lly competitive, are unlikely to bind to identical sites. Strikingly, none of 13 basic residues in the terminal portion (residues 315-390) of the COOH terminus affected channel function when neutralized. The data help to defi ne the structural requirements for PIP2 sensitivity of K-ATP, channels. Mor eover, the regions and residues defined in this study parallel those uncove red in recent studies of PIP2 sensitivity in other inward rectifier channel s, indicating a common structural basis for PIP2 regulation.