Non-radioactive Rb+ Efflux Assay for Screening KATP Channel Modulators.

ATP-sensitive potassium (KATP) 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, KATP channels influence a broad spectrum of physiological processes, including insulin secretion and cardiovascular functions. KATP 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 KATP channel subtypes with distinct tissue distributions and physiological functions. Mutations in the genes encoding KATP channel subunits underlie various human diseases. Targeted treatment for these diseases requires subtype-specific KATP channel modulators. Rubidium ions (Rb+) also pass through KATP channels, and Rb+ efflux assays can be used to assess KATP 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 KATP channel activity in Rb+ efflux assays. In this chapter, we describe a detailed protocol for Rb+ efflux assays designed to identify new KATP channel modulators with potential therapeutic utilities.

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 ß-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.

Dynamic duo: Kir6 and SUR in KATP channel structure and function.

KATP 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 KATP gating. SUR is an ATP binding cassette (ABC) protein family homolog that lacks membrane transport activity but is essential for KATP 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 KATP cryoEM structures.

Structure of an open K ATP channel reveals tandem PIP 2 binding sites mediating the Kir6.2 and SUR1 regulatory interface.

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 ß-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 indicated 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 the cryoEM structure of a K ATP channel harboring the neonatal diabetes mutation Kir6.2-Q52R, bound to natural C18:0/C20:4 long-chain PIP 2 in an 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.

KATP channel mutations in congenital hyperinsulinism: Progress and challenges towards mechanism-based therapies.

Congenital hyperinsulinism (CHI) is the most common cause of persistent hypoglycemia in infancy/childhood and is a serious condition associated with severe recurrent attacks of hypoglycemia due to dysregulated insulin secretion. Timely diagnosis and effective treatment are crucial to prevent severe hypoglycemia that may lead to life-long neurological complications. In pancreatic ß-cells, adenosine triphosphate (ATP)-sensitive K+ (KATP) channels are a central regulator of insulin secretion vital for glucose homeostasis. Genetic defects that lead to loss of expression or function of KATP channels are the most common cause of HI (KATP-HI). Much progress has been made in our understanding of the molecular genetics and pathophysiology of KATP-HI in the past decades; however, treatment remains challenging, in particular for patients with diffuse disease who do not respond to the KATP channel activator diazoxide. In this review, we discuss current approaches and limitations on the diagnosis and treatment of KATP-HI, and offer perspectives on alternative therapeutic strategies.

KATP channels in focus: Progress toward a structural understanding of ligand regulation.

KATP channels are hetero-octameric complexes of four inward rectifying potassium channels, Kir6.1 or Kir6.2, and four sulfonylurea receptors, SUR1, SUR2A, or SUR2B from the ABC transporter family. This unique combination enables KATP channels to couple intracellular ATP/ADP ratios, through gating, with membrane excitability, thus regulating a broad range of cellular activities. The prominence of KATP channels in human physiology, disease, and pharmacology has long attracted research interest. Since 2017, a steady flow of high-resolution KATP cryoEM structures has revealed complex and dynamic interactions between channel subunits and their ligands. Here, we highlight insights from recent structures that begin to provide mechanistic explanations for decades of experimental data and discuss the remaining knowledge gaps in our understanding of KATP channel regulation.

Mechanistic insights on KATP channel regulation from cryo-EM structures.

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.

Ligand-mediated Structural Dynamics of a Mammalian Pancreatic KATP Channel.

Regulation of pancreatic KATP channels involves orchestrated interactions of their subunits, Kir6.2 and SUR1, and ligands. Previously we reported KATP 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 KATP 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 PIP2 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 PIP2 binding. 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.

Localized islet nuclear enlargement hyperinsulinism (LINE-HI) due to ABCC8 and GCK mosaic mutations.

Objective: Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycemia in children. In addition to typical focal or diffuse HI, some cases with diazoxide-unresponsive congenital HI have atypical pancreatic histology termed Localized Islet Nuclear Enlargement (LINE) or mosaic HI, characterized by histologic features similar to diffuse HI, but confined to only a region of pancreas. Our objective was to characterize the phenotype and genotype of children with LINE-HI. Design: The phenotype and genotype features of 12 children with pancreatic histology consistent with LINE-HI were examined. Methods: We compiled clinical features of 12 children with LINE-HI and performed next-generation sequencing on specimens of pancreas from eight of these children to look for mosaic mutations in genes known to be associated with diazoxide-unresponsive HI (ABCC8, KCNJ11, and GCK). Results: Children with LINE-HI had lower birth weights and later ages of presentation compared to children with typical focal or diffuse HI. Partial pancreatectomy in LINE-HI cases resulted in euglycemia in 75% of cases; no cases have developed diabetes. Low-level mosaic mutations were identified in the pancreas of six cases with LINE-HI (three in ABCC8, three in GCK). Expression studies confirmed that all novel mutations were pathogenic. Conclusion: These results indicate that post-zygotic low-level mosaic mutations of known HI genes are responsible for some cases of LINE-HI that lack an identifiable germ-line mutation and that partial pancreatectomy may be curative for these cases.

Subcellular trafficking and endocytic recycling of KATP channels.

Sarcolemmal/plasmalemmal ATP-sensitive K+ (KATP) channels have key roles in many cell types and tissues. Hundreds of studies have described how the KATP channel activity and ATP sensitivity can be regulated by changes in the cellular metabolic state, by receptor signaling pathways and by pharmacological interventions. These alterations in channel activity directly translate to alterations in cell or tissue function, that can range from modulating secretory responses, such as insulin release from pancreatic ß-cells or neurotransmitters from neurons, to modulating contractile behavior of smooth muscle or cardiac cells to elicit alterations in blood flow or cardiac contractility. It is increasingly becoming apparent, however, that KATP channels are regulated beyond changes in their activity. Recent studies have highlighted that KATP channel surface expression is a tightly regulated process with similar implications in health and disease. The surface expression of KATP channels is finely balanced by several trafficking steps including synthesis, assembly, anterograde trafficking, membrane anchoring, endocytosis, endocytic recycling, and degradation. This review aims to summarize the physiological and pathophysiological implications of KATP channel trafficking and mechanisms that regulate KATP channel trafficking. A better understanding of this topic has potential to identify new approaches to develop therapeutically useful drugs to treat KATP channel-related diseases.

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 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.

Production and purification of ATP-sensitive potassium channel particles for cryo-electron microscopy.

ATP-sensitive potassium (KATP) 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 ß-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 KATP 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 KATP channel particles. In this chapter, we describe a method for expressing KATP channels in mammalian cell culture, solubilizing the channel in detergent micelles and purifying KATP channels using an affinity tag to the SURx subunit for cryoEM structural studies.

AKAP79/150 coordinates leptin-induced PKA signaling to regulate KATP channel trafficking in pancreatic ß-cells.

The adipocyte hormone leptin regulates glucose homeostasis both centrally and peripherally. A key peripheral target is the pancreatic ß-cell, which secretes insulin upon glucose stimulation. Leptin is known to suppress glucose-stimulated insulin secretion by promoting trafficking of KATP channels to the ß-cell surface, which increases K+ conductance and causes ß-cell hyperpolarization. We have previously shown that leptin-induced KATP channel trafficking requires protein kinase A (PKA)-dependent actin remodeling. However, whether PKA is a downstream effector of leptin signaling or PKA plays a permissive role is unknown. Using FRET-based reporters of PKA activity, we show that leptin increases PKA activity at the cell membrane and that this effect is dependent on N-methyl-D-aspartate receptors, CaMKKß, and AMPK, which are known to be involved in the leptin signaling pathway. Genetic knockdown and rescue experiments reveal that the increased PKA activity upon leptin stimulation requires the membrane-targeted PKA-anchoring protein AKAP79/150, indicating that PKA activated by leptin is anchored to AKAP79/150. Interestingly, disrupting protein phosphatase 2B (PP2B) anchoring to AKAP79/150, known to elevate basal PKA signaling, leads to increased surface KATP channels even in the absence of leptin stimulation. Our findings uncover a novel role of AKAP79/150 in coordinating leptin and PKA signaling to regulate KATP channel trafficking in ß-cells, hence insulin secretion. The study further advances our knowledge of the downstream signaling events that may be targeted to restore insulin secretion regulation in ß-cells defective in leptin signaling, such as those from obese individuals with type 2 diabetes.

Functional characterization of ABCC8 variants of unknown significance based on bioinformatics predictions, splicing assays, and protein analyses: Benefits for the accurate diagnosis of congenital hyperinsulinism.

ABCC8 encodes the SUR1 subunit of the ß-cell ATP-sensitive potassium channel whose loss of function causes congenital hyperinsulinism (CHI). Molecular diagnosis is critical for optimal management of CHI patients. Unfortunately, assessing the impact of ABCC8 variants on RNA splicing remains very challenging as this gene is poorly expressed in leukocytes. Here, we performed bioinformatics analysis and cell-based minigene assays to assess the impact on splicing of 13 ABCC8 variants identified in 20 CHI patients. Next, channel properties of SUR1 proteins expected to originate from minigene-detected in-frame splicing defects were analyzed after ectopic expression in COSm6 cells. Out of the analyzed variants, seven induced out-of-frame splicing defects and were therefore classified as recessive pathogenic, whereas two led to skipping of in-frame exons. Channel functional analysis of the latter demonstrated their pathogenicity. Interestingly, the common rs757110 SNP increased exon skipping in our system suggesting that it may act as a disease modifier factor. Our strategy allowed determining the pathogenicity of all selected ABCC8 variants, and CHI-inheritance pattern for 16 out of the 20 patients. This study highlights the value of combining RNA and protein functional approaches in variant interpretation and reveals the minigene splicing assay as a new tool for CHI molecular diagnostics.

Leptin modulates pancreatic ß-cell membrane potential through Src kinase-mediated phosphorylation of NMDA receptors.

The adipocyte-derived hormone leptin increases trafficking of KATP and Kv2.1 channels to the pancreatic ß-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca2+ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in ß-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase-mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, ß-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate ß-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.

Ion Channels of the Islets in Type 2 Diabetes.

Ca2+ is an essential signal for pancreatic ß-cell function. Ca2+ plays critical roles in numerous ß-cell pathways such as insulin secretion, transcription, metabolism, endoplasmic reticulum function, and the stress response. Therefore, ß-cell Ca2+ handling is tightly controlled. At the plasma membrane, Ca2+ entry primarily occurs through voltage-dependent Ca2+ channels. Voltage-dependent Ca2+ channel activity is dependent on orchestrated fluctuations in the plasma membrane potential or voltage, which are mediated via the activity of many ion channels. During the pathogenesis of type 2 diabetes the ß-cell is exposed to stressful conditions, which result in alterations of Ca2+ handling. Some of the changes in ß-cell Ca2+ handling that occur under stress result from perturbations in ion channel activity, expression or localization. Defective Ca2+ signaling in the diabetic ß-cell alters function, limits insulin secretion and exacerbates hyperglycemia. In this review, we focus on the ß-cell ion channels that control Ca2+ handling and how they impact ß-cell dysfunction in type 2 diabetes.

Pharmacological chaperones of ATP-sensitive potassium channels: Mechanistic insight from cryoEM structures.

ATP-sensitive potassium (KATP) channels are uniquely evolved protein complexes that couple cell energy levels to cell excitability. They govern a wide range of physiological processes including hormone secretion, neuronal transmission, vascular dilation, and cardiac and neuronal preconditioning against ischemic injuries. In pancreatic ß-cells, KATP channels composed of Kir6.2 and SUR1, encoded by KCNJ11 and ABCC8, respectively, play a key role in coupling blood glucose concentration to insulin secretion. Mutations in ABCC8 or KCNJ11 that diminish channel function result in congenital hyperinsulinism. Many of these mutations principally hamper channel biogenesis and hence trafficking to the cell surface. Several small molecules have been shown to correct channel biogenesis and trafficking defects. Here, we review studies aimed at understanding how mutations impair channel biogenesis and trafficking and how pharmacological ligands overcome channel trafficking defects, particularly highlighting recent cryo-EM structural studies which have shed light on the mechanisms of channel assembly and pharmacological chaperones.

Novel dominant KATP channel mutations in infants with congenital hyperinsulinism: Validation by in vitro expression studies and in vivo carrier phenotyping.

Inactivating mutations in the genes encoding the two subunits of the pancreatic beta-cell KATP channel, ABCC8 and KCNJ11, are the most common finding in children with congenital hyperinsulinism (HI). Interpreting novel missense variants in these genes is problematic, because they can be either dominant or recessive mutations, benign polymorphisms, or diabetes mutations. This report describes six novel missense variants in ABCC8 and KCNJ11 that were identified in 11 probands with congenital HI. One of the three ABCC8 mutations (p.Ala1458Thr) and all three KCNJ11 mutations were associated with responsiveness to diazoxide. Sixteen family members carried the ABCC8 or KCNJ11 mutations; only two had hypoglycemia detected at birth and four others reported symptoms of hypoglycemia. Phenotype testing of seven adult mutation carriers revealed abnormal protein-induced hypoglycemia in all; fasting hypoketotic hypoglycemia was demonstrated in four of the seven. All of six mutations were confirmed to cause dominant pathogenic defects based on in vitro expression studies in COSm6 cells demonstrating normal trafficking, but reduced responses to MgADP and diazoxide. These results indicate a combination of in vitro and in vivo phenotype tests can be used to differentiate dominant from recessive KATP channel HI mutations and personalize management of children with congenital HI.