Structural Determinants of Pip2 Regulation of Inward Rectifier KATP Channels

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP2) activates KATP 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 KATP 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.

Novel Kv7.1-Phosphatidylinositol 4,5-Bisphosphate Interaction Sites Uncovered by Charge Neutralization Scanning

“…Residues outside of the C terminus have been detected before, for instance in the N terminus of K ir channels (42,46,48) and at the end of S4 in K v 7.1 (50). Thus, it is possible that all basic residues in a certain distance to the membrane mediate interaction with PIP 2 without forming a specific binding site.…”

Interplay between PIP 3 and calmodulin regulation of olfactory cyclic nucleotide-gated channels

“…Binding of PIP 2 to both K ir 1 and K ir 6 channels promotes channel activation by stabilizing the open state (32)(33)(34), and PIP 3 has been proposed to activate both epithelial sodium channels and TRPC6 channels by direct binding (35,36). Like PIP 2 -binding regions identified in other ion channels (37)(38)(39), the stretch of amino acids between residues 61-90 of CNGA2 contains multiple basic residues that may be important for the interaction with negatively charged phospholipids. Similar to PIP 2 inhibition of TRPV1 channels (40), PIP 3 inhibited activation of olfactory CNG channels.…”

Neuroprotective role of ATP-sensitive potassium channels in cerebral ischemia

“…Cellular regulation of K ATP channels K ATP channel activity is regulated by phosphatidylinositol 4,5-biphosphate (PIP2) via interaction with the cytoplasmic domain that is close to the ATP binding site of Kir6.2 (including residues R54, R176, R177, and R206) [28,67,68] . PIP2 decreases the ATP sensitivity of the channels by preventing the channel from closing, thus stabilizing the open state.…”