Assmaa ElSheikh

ATP-sensitive potassium (K _ATP ) channels composed of Kir6.2 and sulfonylurea receptor 1 (SUR1) couple glucose metabolism with insulin secretion in pancreatic β-cells and are vital to glucose homeostasis. Loss-of-function mutations in SUR1 and Kir6.2, encoded by ABCC8 and KCNJ11 , respectively are the commonest causes of severe persistent hypoglycemia in infants and children seen in the rare disease congenital hyperinsulinism (HI). The N-terminal transmembrane domain, TMD0, and the linker immediately C-terminal to TMD0, L0, of SUR1 (TMD0/L0) forms direct contact with Kir6.2 in K _ATP channels. Mutations in SUR1-TMD0 often impair K _ATP channel trafficking to the plasma membrane, causing severe disease unresponsive to treatment by the K _ATP activator diazoxide; however, surface expression and function of many such mutant channels can be rescued by reversible K _ATP inhibitor pharmacochaperones. Here, we identified seven new SUR1 missense mutations in TMD0/L0 from HI patients unresponsive to diazoxide and investigated their effects on K _ATP channel expression, function, and response to pharmacochaperones. All seven mutations, N32K, Y124F, P133R, W143R, L171P, G228D, and Y230C, reduced channel function in Rb ⁺ efflux assays. Further characterization by immunoblotting, immunostaining and electrophysiology revealed that Y124F primarily causes defective channel gating, while the others impair channel trafficking to different extents. The trafficking mutations showed varied response to surface expression and function rescue by the reversible K _ATP inhibitor pharmacochaperones, tolbutamide and Aekatperone. The study underscores the critical role of SUR1-TMD0/L0 in K _ATP expression and gating. It further highlights the importance of detailed biochemical and functional studies of mutant channels in understanding their pathogenic roles and response to potential pharmacological therapies.

Pancreatic KATP channel trafficking defects underlie congenital hyperinsulinism (CHI) cases unresponsive to the KATP channel opener diazoxide, the mainstay medical therapy for CHI. Current clinically used KATP channel inhibitors have been shown to act as pharmacochaperones and restore surface expression of trafficking mutants; however, their therapeutic utility for KATP trafficking impaired CHI is hindered by high-affinity binding, which limits functional recovery of rescued channels. Recent structural studies of KATP channels employing cryo-electron microscopy (cryoEM) have revealed a promiscuous pocket where several known KATP pharmacochaperones bind. The structural knowledge provides a framework for discovering KATP channel pharmacochaperones with desired reversible inhibitory effects to permit functional recovery of rescued channels. Using an AI-based virtual screening technology AtomNet® followed by functional validation, we identified a novel compound, termed Aekatperone, which exhibits chaperoning effects on KATP channel trafficking mutations. Aekatperone reversibly inhibits KATP channel activity with a half-maximal inhibitory concentration (IC50) ∼ 9 μM. Mutant channels rescued to the cell surface by Aekatperone showed functional recovery upon washout of the compound. CryoEM structure of KATP bound to Aekatperone revealed distinct binding features compared to known high affinity inhibitor pharmacochaperones. Our findings unveil a KATP pharmacochaperone enabling functional recovery of rescued channels as a promising therapeutic for CHI caused by KATP trafficking defects.

Pancreatic K _ATP channel trafficking defects underlie congenital hyperinsulinism (CHI) cases unresponsive to the K _ATP channel opener diazoxide, the mainstay medical therapy for CHI. Current clinically used K _ATP channel inhibitors have been shown to act as pharmacochaperones and restore surface expression of trafficking mutants; however, their therapeutic utility for K _ATP trafficking impaired CHI is hindered by high-affinity binding, which limits functional recovery of rescued channels. Recent structural studies of K _ATP channels employing cryo-electron microscopy (cryoEM) have revealed a promiscuous pocket where several known K _ATP pharmacochaperones bind. The structural knowledge provides a framework for discovering K _ATP channel pharmacochaperones with desired reversible inhibitory effects to permit functional recovery of rescued channels. Using an AI-based virtual screening technology AtomNet® followed by functional validation, we identified a novel compound, termed Aekatperone, which exhibits chaperoning effects on K _ATP channel trafficking mutations. Aekatperone reversibly inhibits K _ATP channel activity with a half-maximal inhibitory concentration (IC ₅₀ ) ∼ 9 μM. Mutant channels rescued to the cell surface by Aekatperone showed functional recovery upon washout of the compound. CryoEM structure of K _ATP bound to Aekatperone revealed distinct binding features compared to known high affinity inhibitor pharmacochaperones. Our findings unveil a K _ATP pharmacochaperone enabling functional recovery of rescued channels as a promising therapeutic for CHI caused by K _ATP trafficking defects.

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.

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.

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