Show-Ling Shyng

Vascular smooth muscle K _ATP channels, composed of the pore-forming Kir6.1 and regulatory SUR2B subunits, control vascular tone, dysfunction of which causes systemic disease. Vascular K _ATP is regulated by Mg-nucleotides, but the underlying structural mechanism has remained elusive. Here, we determined cryoEM structures of these channels in the presence of MgATP and MgADP. Two key structures captured, one showing the SUR2B-nucleotide binding domains (NBDs) separated and one showing the SUR2B-NBDs dimerized, reveal conformation-specific organization of intrinsically disordered regions (IDRs) found in both Kir6.1 and SUR2B. In the NBD-separated conformation, the Kir6.1-N terminal IDR (KNt) sits within the central cleft of SUR2B’s ABC-core. In the NBD-dimerized conformation, KNt is excluded from the central cleft and instead forms contacts with an ED domain comprising 15 consecutive glutamate and aspartate residues within a SUR2B IDR, the N1-T2 linker connecting NBD1 (N1) to transmembrane domain 2 (T2). Moreover, within the N1-T2 linker a regulatory helix seen between the two NBDs in the NBD-separated conformation moves to outside the dimerized NBDs, interacting with the C-terminal residues unique to SUR2B, in the NBD-dimerized conformation. MD simulations further reveal that transient but frequent interactions mediated by the IDRs may facilitate Mg-nucleotide dependent conformational switch in vascular K _ATP channels.

Glucokinase and ATP-sensitive potassium (KATP) channels in pancreatic β-cells control insulin secretion in response to glucose stimulation to maintain glucose homeostasis. It is well established that loss-of-function (LOF) variants in GCK, which encodes glucokinase, are diabetogenic, whereas LOF variants in KATP channel genes lead to congenital hyperinsulinism (HI) and hypoglycemia. However, how the co-occurrence of GCK and KATP channel variants manifests in glycemic phenotypes is unknown. This study presents a multiplex pedigree with a heterozygous GCK deletion along with a heterozygous ABCC8 variant that results in the E1209K missense variant in the regulatory subunit of the KATP channel sulfonylurea receptor 1 (SUR1). The three-generation pedigree exhibits a complex spectrum of dysglycemia depending on genotype and age at referral. Individuals harboring the heterozygous GCK deletion alone present with maturity-onset diabetes; those harboring the heterozygous ABCC8 E1209K variant alone exhibit HI resulting from the LOF of KATP channels, with some developing diabetes later in life; and those harboring both GCK and ABCC8 variants escape HI but not diabetes. This unique pedigree offers insights into the complex interplay between LOF genetic variants of glucokinase and KATP channels in age-dependent dysglycemia. Article Highlights: Genetic variants causing loss of function (LOF) of glucokinase or ATP-sensitive potassium (KATP) channels underlie diabetes or congenital hyperinsulinism, respectively, but how the co-occurrence of such variants affects glucose control is unknown. This study presents genotypes and clinical phenotypes in a pedigree with LOF variants in both GCK and the pancreatic KATP channel. Heterozygous glucokinase deletion masked infantile hyperinsulinemia and hypoglycemia caused by a heterozygous LOF KATP channel variant. Carriers of the LOF KATP variant exhibited a shift from hypoglycemia to diabetes, as has been reported previously in some carriers of KATP LOF variants.

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 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 (IC₅₀) ~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.

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, 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.

K_ATP channels are ligand-gated potassium channels that couple cellular energetics with membrane potential to regulate cell activity. Each channel is an eight subunit complex comprising four central pore-forming Kir6 inward rectifier potassium channel subunits surrounded by four regulatory subunits known as the sulfonylurea receptor, SUR, which confer homeostatic metabolic control of K_ATP gating. SUR is an ATP binding cassette (ABC) protein family homolog that lacks membrane transport activity but is essential for K_ATP expression and function. For more than four decades, understanding the structure-function relationship of Kir6 and SUR has remained a central objective of clinical significance. Here, we review progress in correlating the wealth of functional data in the literature with recent K_ATP cryoEM structures.