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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 channel function, previously solved open-channel structures have lacked bound PIP2, and mechanisms by which PIP2 regulates KATP channels remain unresolved. Here, we report the cryoEM structure of a KATP 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 KATP channels by PIP2 and ATP, and provide a putative mechanism by which Kir6.2-Q52R stabilizes an open channel to cause neonatal diabetes.KATP channels regulate insulin secretion and are activated by PIP2. Here, the authors show PIP2 binds between SUR1 and Kir6.2 to open the channel, and a neonatal diabetes mutation stabilizes KATP channels in a PIP2-bound open conformation. [ABSTRACT FROM AUTHOR]

Gated by intracellular ATP and ADP, ATP-sensitive potassium (KATP) channels couple cell energetics with membrane excitability in many cell types, enabling them to control a wide range of physiological processes based on metabolic demands. The KATP channel is a complex of four potassium channel subunits from the Kir channel family, Kir6.1 or Kir6.2, and four sulfonylurea receptor subunits, SUR1, SUR2A, or SUR2B, from the ATP-binding cassette (ABC) transporter family. Dysfunction of KATP channels underlies several human diseases. The importance of these channels in human health and disease has made them attractive drug targets. How the channel subunits interact with one another and how the ligands interact with the channel to regulate channel activity have been long-standing questions in the field. In the past 5 yr, a steady stream of high-resolution KATP channel structures has been published using single-particle cryo-electron microscopy (cryo-EM). Here, we review the advances these structures bring to our understanding of channel regulation by physiological and pharmacological ligands. [ABSTRACT FROM AUTHOR]

Vascular tone is dependent on smooth muscle KATP channels comprising pore-forming Kir6.1 and regulatory SUR2B subunits, in which mutations cause Cantú syndrome. Unique among KATP isoforms, they lack spontaneous activity and require Mg-nucleotides for activation. Structural mechanisms underlying these properties are unknown. Here, we determined cryogenic electron microscopy structures of vascular KATP channels bound to inhibitory ATP and glibenclamide, which differ informatively from similarly determined pancreatic KATP channel isoform (Kir6.2/SUR1). Unlike SUR1, SUR2B subunits adopt distinct rotational “propeller” and “quatrefoil” geometries surrounding their Kir6.1 core. The glutamate/aspartate-rich linker connecting the two halves of the SUR-ABC core is observed in a quatrefoil-like conformation. Molecular dynamics simulations reveal MgADP-dependent dynamic tripartite interactions between this linker, SUR2B, and Kir6.1. The structures captured implicate a progression of intermediate states between MgADP-free inactivated, and MgADP-bound activated conformations wherein the glutamate/aspartate-rich linker participates as mobile autoinhibitory domain, suggesting a conformational pathway toward KATP channel activation. [ABSTRACT FROM AUTHOR]

ATP-sensitive potassium (K ATP ) channels function as metabolic sensors that link cell membrane excitability to the cellular energy status by controlling potassium ion (K + ) flow across the cell membrane according to intracellular ATP and ADP concentrations. As such, K ATP channels influence a broad spectrum of physiological processes, including insulin secretion and cardiovascular functions. K ATP channels are hetero-octamers, consisting of four inward rectifier potassium channel subunits, Kir6.1 or Kir6.2, and four sulfonylurea receptors (SURs), SUR1, SUR2A, or SUR2B. Different Kir6 and SUR isoforms assemble into K ATP channel subtypes with distinct tissue distributions and physiological functions. Mutations in the genes encoding K ATP channel subunits underlie various human diseases. Targeted treatment for these diseases requires subtype-specific K ATP channel modulators. Rubidium ions (Rb + ) also pass through K ATP channels, and Rb + efflux assays can be used to assess K ATP channel function and activity. Flame atomic absorption spectroscopy (Flame-AAS) combined with microsampling can measure Rb + in small volume, which provides an efficient tool to screen for compounds that alter K ATP channel activity in Rb + efflux assays. In this chapter, we describe a detailed protocol for Rb + efflux assays designed to identify new K ATP channel modulators with potential therapeutic utilities., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Gated by intracellular ATP and ADP, ATP-sensitive potassium (KATP) channels couple cell energetics with membrane excitability in many cell types, enabling them to control a wide range of physiological processes based on metabolic demands. The KATP channel is a complex of four potassium channel subunits from the Kir channel family, Kir6.1 or Kir6.2, and four sulfonylurea receptor subunits, SUR1, SUR2A, or SUR2B, from the ATP-binding cassette (ABC) transporter family. Dysfunction of KATP channels underlies several human diseases. The importance of these channels in human health and disease has made them attractive drug targets. How the channel subunits interact with one another and how the ligands interact with the channel to regulate channel activity have been long-standing questions in the field. In the past 5 yr, a steady stream of high-resolution KATP channel structures has been published using single-particle cryo-electron microscopy (cryo-EM). Here, we review the advances these structures bring to our understanding of channel regulation by physiological and pharmacological ligands., (© 2022 Driggers and Shyng.)

Regulation of pancreatic K ATP channels involves orchestrated interactions of their subunits, Kir6.2 and SUR1, and ligands. Previously we reported K ATP channel cryo-EM structures in the presence and absence of pharmacological inhibitors and ATP, focusing on the mechanisms by which inhibitors act as pharmacological chaperones of K ATP channels (Martin et al., 2019). Here we analyzed the same cryo-EM datasets with a focus on channel conformational dynamics to elucidate structural correlates pertinent to ligand interactions and channel gating. We found pharmacological inhibitors and ATP enrich a channel conformation in which the Kir6.2 cytoplasmic domain is closely associated with the transmembrane domain, while depleting one where the Kir6.2 cytoplasmic domain is extended away into the cytoplasm. This conformational change remodels a network of intra- and inter-subunit interactions as well as the ATP and PIP 2 binding pockets. The structures resolved key contacts between the distal N-terminus of Kir6.2 and SUR1's ABC module involving residues implicated in channel function and showed a SUR1 residue, K134, participates in PIP 2 binding. Molecular dynamics simulations revealed two Kir6.2 residues, K39 and R54, that mediate both ATP and PIP 2 binding, suggesting a mechanism for competitive gating by ATP and PIP 2 ., Competing Interests: Competing interests The authors declare that they have no competing financial or non-financial interests with the contents of this article., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

ATP-sensitive potassium (K ATP ) channels are multimeric protein complexes made of four inward rectifying potassium channel (Kir6.x) subunits and four ABC protein sulfonylurea receptor (SURx) subunits. Kir6.x subunits form the potassium ion conducting pore of the channel, and SURx functions to regulate Kir6.x. Kir6.x and SURx are uniquely dependent on each other for expression and function. In pancreatic β-cells, channels comprising SUR1 and Kir6.2 mediate glucose-stimulated insulin secretion and are the targets of antidiabetic sulfonylureas. Mutations in genes encoding SUR1 or Kir6.2 are linked to insulin secretion disorders, with loss- or gain-of-function mutations causing congenital hyperinsulinism or neonatal diabetes mellitus, respectively. Defects in the K ATP channel in other tissues underlie human diseases of the cardiovascular and nervous systems. Key to understanding how channels are regulated by physiological and pharmacological ligands and how mutations disrupt channel assembly or gating to cause disease is the ability to observe structural changes associated with subunit interactions and ligand binding. While recent advances in the structural method of single-particle cryo-electron microscopy (cryoEM) offers direct visualization of channel structures, success of obtaining high-resolution structures is dependent on highly concentrated, homogeneous K ATP channel particles. In this chapter, we describe a method for expressing K ATP channels in mammalian cell culture, solubilizing the channel in detergent micelles and purifying K ATP channels using an affinity tag to the SURx subunit for cryoEM structural studies., (Copyright © 2021 Elsevier Inc. All rights reserved.)