Key residue Unlocks Dual PIP ₂ -Dependent and Independent Gating in G Protein-Gated Inwardly Rectifying Potassium Channels

G protein-gated inwardly rectifying potassium (GIRK) channels mediate membrane hyperpolarization in response to G protein-coupled receptor activation and are critical for regulating neuronal excitability. The membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP ₂ ) is essential for regulating a large family of ion channels, and disruptions in PIP ₂ interactions contribute to some neurological diseases. Structural analyses have identified key residues in PIP ₂ -mediated gating of the GIRK2 channel. Notably, Arginine-92 (R92), a highly conserved basic residue at the membrane interface in GIRK2, interacts with PIP ₂ as well as cholesteryl hemisuccinate (CHS), a potentiator of GIRK2. Here, we used a combination of electrophysiological assays, fluorescent K ⁺ flux measurements, cryo-electron microscopy, and molecular dynamics simulations, and find that mutations at R92 (Y, F, and Q) not only alter PIP ₂ sensitivity but can also reveal a novel gating mechanism that is independent of PIP ₂ . These findings indicate that R92 plays a crucial role in modulating GIRK2 channel gating, offering new insights into developing potential therapeutic targets for treating neurological disorders linked to GIRK channel dysfunction.