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Structure of an open K_ATP channel reveals tandem PIP₂ binding sites mediating the Kir6.2 and SUR1 regulatory interface.

ABSTRACT: 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_ATP channel opening is stimulated by PIP₂ and inhibited by ATP. Mutations that increase channel opening by PIP₂ reduce ATP inhibition and cause neonatal diabetes. Although considerable evidence has implicated a role for PIP₂ in K_ATP channel function, previously solved open-channel structures have lacked bound PIP₂, and mechanisms by which PIP₂ regulates K_ATP channels remain unresolved. Here, we report the cryoEM structure of a K_ATP channel harboring the neonatal diabetes mutation Kir6.2-Q52R, in the open conformation, bound to amphipathic molecules consistent with natural C18:0/C20:4 long-chain PI(4,5)P₂ at two adjacent binding sites between SUR1 and Kir6.2. The canonical PIP₂ binding site is conserved among PIP₂-gated Kir channels. The non-canonical PIP₂ binding site forms at 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 PIP₂ binding and gating, explain the antagonistic regulation of K_ATP channels by PIP₂ and ATP, and provide a putative mechanism by which Kir6.2-Q52R stabilizes an open channel to cause neonatal diabetes.

SUBMITTER: Driggers CM

PROVIDER: S-EPMC10954709 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Structure of an open KATP channel reveals tandem PIP2 binding sites mediating the Kir6.2 and SUR1 regulatory interface.

Driggers Camden M CM Kuo Yi-Ying YY Zhu Phillip P ElSheikh Assmaa A Shyng Show-Ling SL

Nature communications 20240320 1

ATP-sensitive potassium (KATP) channels, composed of four pore-lining Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1) subunits, control insulin secretion in pancreatic β-cells. KATP channel opening is stimulated by PIP2 and inhibited by ATP. Mutations that increase channel opening by PIP2 reduce ATP inhibition and cause neonatal diabetes. Although considerable evidence has implicated a role for PIP2 in KATP chan ...[more]

PMID: 38509107

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Project description:Background: KATP channels have diverse roles, including regulation of insulin secretion and blood flow, and protection against biological stress responses and are excellent therapeutic targets. Different subclasses of KATP channels exist in various tissue types due to the unique assemblies of specific pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against the various KATP channel subclasses. Methods and Results: We used 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures of channels in both the open and closed states to identify a potential agonist binding pocket in a functionally critical area of the channel. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds yielded 15 top-ranked "hits", which were tested for activity against KATP channels using patch clamping and thallium (Tl+) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl+ fluxes. One of them (CL-705G) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC50 of 9 µM and 11 μM, respectively). Remarkably, compound CL-705G had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1, or Kir3.1/Kir3.4 channels, or Na+ currents of TE671 medulloblastoma cells. CL-705G activated Kir6.2Δ36 in the presence of SUR2A, but not when expressed by itself. CL-705G activated Kir6.2/SUR2A channels even after PIP2 depletion. The compound has cardioprotective effects in a cellular model of pharmacological preconditioning. It also partially rescued activity of the gating-defective Kir6.2-R301C mutant that is associated with congenital hyperinsulinism. Conclusion: CL-705G is a new Kir6.2 opener with little cross-reactivity with other channels tested, including the structurally similar Kir6.1. This, to our knowledge, is the first Kir-specific channel opener.

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Structure of an open K_ATP channel reveals tandem PIP₂ binding sites mediating the Kir6.2 and SUR1 regulatory interface.

Publications

Structure of an open K<sub>ATP</sub> channel reveals tandem PIP<sub>2</sub> binding sites mediating the Kir6.2 and SUR1 regulatory interface.

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