Structure of an open K_ATPchannel reveals tandem PIP₂binding sites mediating the Kir6.2 and SUR1 regulatory interface

ATP-sensitive potassium (K_ATP) channels, composed of four pore-lining Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1) subunits, control insulin secretion in pancreatic β-cells. K_ATPchannel opening is stimulated by PIP₂and inhibited by ATP. Mutations that increase channel opening by PIP₂reduce ATP inhibition and cause neonatal diabetes. Although considerable evidence has implicated a role for PIP₂in K_ATPchannel function, previously solved open-channel structures have lacked bound PIP₂, and mechanisms by which PIP₂regulates K_ATPchannels remain unresolved. Here, we report cryoEM structure of a K_ATPchannel harboring the neonatal diabetes mutation Kir6.2-Q52R, bound to natural C18:0/C20:4 long-chain PI(4,5)P₂in open conformation. The structure reveals two adjacent PIP₂molecules between SUR1 and Kir6.2. The first PIP₂binding site is conserved among PIP₂-gated Kir channels. The second site forms uniquely in K_ATPat the interface of Kir6.2 and SUR1. Functional studies demonstrate both binding sites determine channel activity. Kir6.2 pore opening is associated with a twist of the Kir6.2 cytoplasmic domain and a rotation of the N-terminal transmembrane domain of SUR1, which widens the inhibitory ATP binding pocket to disfavor ATP binding. The open conformation is particularly stabilized by the Kir6.2-Q52R residue through cation-π bonding with SUR1 - W51. Together, these results uncover the cooperation between SUR1 and Kir6.2 in PIP₂binding and gating, explain the antagonistic regulation of K_ATPchannels by PIP₂and ATP, and provide the mechanism by which Kir6.2-Q52R stabilizes an open channel to cause neonatal diabetes.