Biology subjects

Shyng, S.-L.

Publications and source records attributed to Shyng, S.-L..

10 recordsLinked to original sources

bioRxiv2026

MgATP/MgADP-dependent conformational dynamics and intrinsically disordered regions of vascular KATP channels revealed by cryoEM

Vascular smooth muscle KATP channels, composed of the pore-forming Kir6.1 and regulatory SUR2B subunits, control vascular tone, dysfunction of which causes systemic disease. Vascular KATP 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 the ABC-core of SUR2B. 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 KATP channels.

biochemistry↗

bioRxiv2025

Identification and Rescue of Congenital Hyperinsulinism-Associated ABCC8 Mutations that Impair KATP Channel Trafficking

ATP-sensitive potassium (KATP) channels composed of Kir6.2 and sulfonylurea receptor 1 (SUR1) couple glucose metabolism with insulin secretion in pancreatic {beta}-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 KATP channels. Mutations in SUR1-TMD0 often impair KATP channel trafficking to the plasma membrane, causing severe disease unresponsive to treatment by the KATP activator diazoxide; however, surface expression and function of many such mutant channels can be rescued by reversible KATP inhibitor pharmacochaperones. Here, we identified seven new SUR1 missense mutations in TMD0/L0 from HI patients unresponsive to diazoxide and investigated their effects on KATP 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 KATP inhibitor pharmacochaperones, tolbutamide and Aekatperone. The study underscores the critical role of SUR1-TMD0/L0 in KATP 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.

biochemistry↗

bioRxiv2024

AI-Based Discovery and CryoEM Structural Elucidation of a KATP Channel Pharmacochaperone

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(R) 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 M. 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.

biochemistry↗

bioRxiv2023

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

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 {beta}-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 cryoEM structure of a KATP channel harboring the neonatal diabetes mutation Kir6.2-Q52R, bound to natural C18:0/C20:4 long-chain PI(4,5)P2 in open conformation. The structure reveals two adjacent PIP2 molecules between SUR1 and Kir6.2. The first PIP2 binding site is conserved among PIP2-gated Kir channels. The second site forms uniquely in KATP 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-{pi} 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 the mechanism by which Kir6.2-Q52R stabilizes an open channel to cause neonatal diabetes.

biochemistry↗

bioRxiv2022

Ligand-mediated structural dynamics of a mammalian pancreatic KATP channel

Regulation of pancreatic KATP channels involves orchestrated interactions of channel subunits, Kir6.2 and SUR1, and their ligands. How ligand interactions affect channel conformations and activity is not well understood. To elucidate the structural correlates pertinent to ligand interactions and channel gating, we compared cryo-EM structures of channels in the presence and absence of pharmacological inhibitors and ATP, focusing on channel conformational dynamics. We found pharmacological inhibitors and ATP enrich a channel conformation in which the Kir6.2 cytoplasmic domain is closely associated with the transmembrane domain relative to one where the Kir6.2 cytoplasmic domain is extended away into the cytoplasm. This conformation change remodels a network of intra and inter-subunit interactions as well as both the ATP and PIP2 binding pockets. The structures resolved key contacts between the distal N-terminus of Kir6.2 and SUR1s ABC module involving residues implicated in channel function. A SUR1 residue, K134, is identified to directly contribute to the PIP2 binding pocket. Molecular dynamics simulations revealed two Kir6.2 residues, K39 and R54, that mediate both ATP and PIP2 binding, suggesting a mechanism for competitive gating by ATP and PIP2.

biochemistry↗

bioRxiv2021

Vascular KATP channel structural dynamics reveal regulatory mechanism by Mg-nucleotides

Vascular tone is dependent on smooth muscle KATP channels comprising pore-forming Kir6.1 and regulatory SUR2B subunits, in which mutations cause Cantu 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 the first cryoEM 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 previously unseen ED-rich linker connecting the two halves of the SUR-ABC core is observed in a quatrefoil-like conformation. MD 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 ED-rich linker participates as mobile autoinhibitory domain, suggesting a conformational pathway toward KATP channel activation.

biochemistry↗

bioRxiv2020

AKAP79/150 coordinates leptin-induced PKA activation to regulate KATP channel trafficking in pancreatic β-cells

The adipocyte hormone leptin regulates glucose homeostasis both centrally and peripherally. A key peripheral target is the pancreatic {beta}-cell, which secretes insulin upon glucose stimulation. Leptin suppresses glucose-stimulated insulin secretion by promoting trafficking of KATP channels to the {beta}-cell surface, which increases K+ conductance and causes {beta}-cell hyperpolarization. Here we investigate the signaling mechanism underlying leptin-induced KATP channel translocation with a focus on protein kinase A (PKA). Using FRET-based PKA activity reporters, we show that leptin increases PKA activity at the cell membrane via a signaling pathway involving NMDA receptors, CaMKK{beta} and AMPK. Genetic knockdown and rescue experiments reveal that leptin activation of PKA requires tethering of PKA to the membrane-targeted PKA-anchoring protein AKAP79/150. Interestingly, disrupting protein phosphatase 2B (PP2B) anchoring to AKAP79/150, known to elevate basal PKA signaling, increases surface KATP channels. Our findings uncover a novel role of AKAP79/150 in coordinating leptin and PKA signaling to regulate {beta}-cell function.

cell biology↗

bioRxiv2019

Mechanism of pharmacochaperoning in KATP channels revealed by cryo-EM

ATP-sensitive potassium (KATP) channels composed of a pore-forming Kir6.2 potassium channel and a regulatory ABC transporter sulfonylurea receptor 1 (SUR1) regulate insulin secretion in pancreatic {beta}-cells to maintain glucose homeostasis. Mutations that impair channel folding or assembly prevent cell surface expression and cause congenital hyperinsulinism. Structurally diverse KATP inhibitors have been shown to act as pharmacochaperones to correct mutant channel expression, but the mechanism is unknown. Here, we compare cryoEM structures of KATP channels bound to pharmacochaperones glibenclamide, repaglinide, and carbamazepine. We found all three drugs bind within a common pocket in SUR1. Further, we found the N-terminus of Kir6.2 inserted within the central cavity of the SUR1 ABC core, adjacent the drug binding pocket. The findings reveal a common mechanism by which diverse compounds stabilize the Kir6.2 N-terminus within the SUR1 ABC core, allowing it to act as a firm \"handle\" for the assembly of metastable mutant SUR1-Kir6.2 complexes.

biophysics↗

bioRxiv2017

Anti-diabetic drug binding site in KATP channels revealed by Cryo-EM

Sulfonylureas are anti-diabetic medications that act by inhibiting pancreatic KATP channels composed of SUR1 and Kir6.2. The mechanism by which these drugs interact with and inhibit the channel has been extensively investigated, yet it remains unclear where the drug binding pocket resides. Here, we present a cryo-EM structure of the channel bound to a high-affinity sulfonylurea drug glibenclamide and ATP at 3.8[A] resolution, which reveals in unprecedented details of the ATP and glibenclamide binding sites. Importantly, the structure shows for the first time that glibenclamide is lodged in the transmembrane bundle of the SUR1-ABC core connected to the first nucleotide binding domain near the inner leaflet of the lipid bilayer. Mutation of residues predicted to interact with glibenclamide in our model led to reduced sensitivity to glibenclamide. Our structure provides novel mechanistic insights of how sulfonylureas and ATP interact with the KATP channel complex to inhibit channel activity.

biochemistry↗

bioRxiv2016

Cryo-EM structure of the ATP-sensitive potassium channel illuminates mechanisms of assembly and gating

ATP-sensitive potassium (KATP) channels are metabolic sensors that couple cell energetics to membrane excitability. In pancreatic {beta}-cells, channels formed by SUR1 and Kir6.2 regulate insulin secretion and are the targets of antidiabetic sulfonylureas. Here, we used cryo-EM to elucidate structural basis of channel assembly and gating. The structure, determined in the presence of ATP and the sulfonylurea glibenclamide, at ~6[A] resolution reveals a closed Kir6.2 tetrameric core with four peripheral SUR1s each anchored to a Kir6.2 by its N-terminal transmembrane domain (TMD0). Intricate interactions between TMD0, the loop following TMD0, and Kir6.2 near the proposed PIP2 binding site, and where ATP density is observed, suggest SUR1 may contribute to ATP and PIP2 binding to enhance Kir6.2 sensitivity to both. The SUR1-ABC core is found in an unusual inward-facing conformation whereby the two nucleotide binding domains are misaligned along a two-fold symmetry axis, revealing a possible mechanism by which glibenclamide inhibits channel activity.

biochemistry↗