Yi-Ying Kuo

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.

While specific transcription factors are known to regulate cell fate decisions, the degree to which they can stimulate formation of specific cell organelles is less clear. We used a multi-omics comparison of the transcriptomes of ciliated and non-ciliated embryonic cells to identify transcription factors upregulated in ciliated cells, and conditional genetics in mouse embryos and stem cells to demonstrate that SP5/8 regulate cilia formation and gene expression. In Sp5/8 mutant embryos primary and motile cilia are shorter than normal and reduced in number across cell types, contributing to situs inversus and hydrocephalus. Moreover, expression of SP8 is sufficient to induce primary cilia in unciliated cells. This work opens new avenues for studying cilia assembly using stem cell models and offers new insights into human ciliopathies.

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.

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.