Structural Determinants of Pip2 Regulation of Inward Rectifier KATP Channels

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP₂) activates K_ATP and other inward rectifier (Kir) channels. To determine residues important for PIP₂ regulation, we have systematically mutated each positive charge in the COOH terminus of Kir6.2 to alanine. The effects of these mutations on channel function were examined using ⁸⁶Rb efflux assays on intact cells and inside-out patch-clamp methods. Both methods identify essentially the same basic residues in two narrow regions (176–222 and 301–314) in the COOH terminus that are important for the maintenance of channel function and interaction with PIP₂. Only one residue (R201A) simultaneously affected ATP and PIP₂ sensitivity, which is consistent with the notion that these ligands, while functionally 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 define the structural requirements for PIP₂ sensitivity of K_ATP channels. Moreover, the regions and residues defined in this study parallel those uncovered in recent studies of PIP₂ sensitivity in other inward rectifier channels, indicating a common structural basis for PIP₂ regulation.