A critical residue mediates proper assembly and gating of GIRK2 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 (PIP2) is essential for regulating the large family of inward rectifiers, and disruptions in PIP2 interactions contribute to some neurological diseases. Structural analyses have identified arginine-92 (R92) in GIRK2 as a key amino acid interacting with PIP2 as well as the potentiator cholesteryl hemisuccinate (CHS). Using electrophysiological assays and fluorescent K+ flux measurements, we show that substitutions at R92 (F, Y, or Q) disrupt PIP2 regulation, as well as G protein and alcohol activation. Cryo-EM structures of R92F and R92Q show an unexpected change in the orientation of the slide helix that leads to a "domain swap" between adjacent subunits in the cytoplasmic domain, producing a unique arrangement of the alcohol-binding pocket and G protein-interacting domain. These findings indicate that R92 plays a crucial role in how GIRK2 channel subunits assemble for physiological gating, and likely extend to gating of most inward rectifiers due to the high conservation of arginine in that location.