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Objective: Neonatal diabetes mellitus (NDM) can be caused by gain-of-function ATP-sensitive K(+) (K(ATP)) channel mutations. This realization has led to sulfonylurea therapy replacing insulin injections in many patients. In a murine model of K(ATP)-dependent NDM, hyperglycemia and consequent loss of β-cells are both avoided by chronic sulfonylurea treatment. Interestingly, K(ATP) mutations may underlie remitting-relapsing, transient, or permanent forms of the disease in different patients, but the reason for the different outcomes is unknown.Research Design and Methods: To gain further insight into disease progression and outcome, we examined the effects of very early intervention by injecting NDM mice with high-dose glibenclamide for only 6 days, at the beginning of disease onset, then after the subsequent progression with measurements of blood glucose, islet function, and insulin sensitivity.Results: Although ∼70% of mice developed severe diabetes after treatment cessation, ∼30% were essentially cured, maintaining near-normal blood glucose until killed. Another group of NDM mice was initiated on oral glibenclamide (in the drinking water), and the dose was titrated daily, to maintain blood glucose <200 mg/dL. In this case, ∼30% were also essentially cured; they were weaned from the drug after ∼4 weeks and again subsequently maintained near-normal blood glucose. These cured mice maintain normal insulin content and were more sensitive to insulin than control mice, a compensatory mechanism that together with basal insulin secretion may be sufficient to maintain near-normal glucose levels.Conclusions: At least in a subset of animals, early sulfonylurea treatment leads to permanent remission of NDM. These cured animals exhibit insulin-hypersensitivity. Although untreated NDM mice rapidly lose insulin content and progress to permanently extremely elevated blood glucose levels, early tight control of blood glucose may permit this insulin-hypersensitivity, in combination with maintained basal insulin secretion, to provide long-term remission. [ABSTRACT FROM AUTHOR]

Objective: The ATP-sensitive K(+) channel (K(ATP)) controls insulin secretion from the islet. Gain- or loss-of-function mutations in channel subunits underlie human neonatal diabetes and congenital hyperinsulinism (HI), respectively. In this study, we sought to identify the mechanistic basis of K(ATP)-induced HI in two probands and to characterize the clinical course.Research Design and Methods: We analyzed HI in two probands and characterized the course of clinical treatment in each, as well as properties of mutant K(ATP) channels expressed in COSm6 cells using Rb efflux and patch-clamp methods.Results: We identified mutation V290M in the pore-forming Kir6.2 subunit in each proband. In vitro expression in COSm6 cells supports the mutation resulting in an inactivating phenotype, which leads to significantly reduced activity in intact cells when expressed homomerically, and to a lesser extent when expressed heteromerically with wild-type subunits. In one heterozygous proband, a fluoro-DOPA scan revealed a causal focal lesion, indicating uniparental disomy with loss of heterozygosity. In a second family, the proband, homozygous for the mutation, was diagnosed with severe diazoxide-unresponsive hypersinsulinism at 2 weeks of age. The patient continues to be treated successfully with octreotide and amlodipine. The parents and a male sibling are heterozygous carriers without overt clinical HI. Interestingly, both the mother and the sibling exhibit evidence of abnormally enhanced glucose tolerance.Conclusions: V290M results in inactivating K(ATP) channels that underlie HI. Homozygous individuals may be managed medically, without pancreatectomy. Heterozygous carriers also show evidence of enhanced glucose sensitivity, consistent with incomplete loss of K(ATP) channel activity. [ABSTRACT FROM AUTHOR]

Objective: The E23K variant in the Kir6.2 subunit of the ATP-sensitive K(+) channel (K(ATP) channel) is associated with increased risk of type 2 diabetes. The present study was undertaken to increase our understanding of the mechanisms responsible. To avoid confounding effects of hyperglycemia, insulin secretion and action were studied in subjects with the variant who had normal glucose tolerance.Research Design and Methods: Nine subjects with the E23K genotype K/K and nine matched subjects with the E/E genotype underwent 5-h oral glucose tolerance tests (OGTTs), graded glucose infusion, and hyperinsulinemic-euglycemic clamp with stable-isotope-labeled tracer infusions to assess insulin secretion, action, and clearance. A total of 461 volunteers consecutively genotyped for the E23K variant also underwent OGTTs. Functional studies of the wild-type and E23K variant potassium channels were conducted.Results: Insulin secretory responses to oral and intravenous glucose were reduced by approximately 40% in glucose-tolerant subjects homozygous for E23K. Normal glucose tolerance with reduced insulin secretion suggests a change in insulin sensitivity. The hyperinsulinemic-euglycemic clamp revealed that hepatic insulin sensitivity is approximately 40% greater in subjects with the E23K variant, and these subjects demonstrate increased insulin sensitivity after oral glucose. The reconstituted E23K channels confirm reduced sensitivity to inhibitory ATP and increase in open probability, a direct molecular explanation for reduced insulin secretion.Conclusions: The E23K variant leads to overactivity of the K(ATP) channel, resulting in reduced insulin secretion. Initially, insulin sensitivity is enhanced, thereby maintaining normal glucose tolerance. Presumably, over time, as insulin secretion falls further or insulin resistance develops, glucose levels rise resulting in type 2 diabetes. [ABSTRACT FROM AUTHOR]

The prediction that overactivity of the pancreatic ATP-sensitive K(+) channel (K(ATP) channel) underlies reduced insulin secretion and causes a diabetic phenotype in humans has recently been borne out by genetic studies implicating "activating" mutations in the Kir6.2 subunit of K(ATP) as causal in both permanent and transient neonatal diabetes. Here we characterize the channel properties of Kir6.2 mutations that underlie transient neonatal diabetes (I182V) or more severe forms of permanent neonatal diabetes (V59M, Q52R, and I296L). In all cases, the mutations result in a significant decrease in sensitivity to inhibitory ATP, which correlates with channel "overactivity" in intact cells. Mutations can be separated into those that directly affect ATP affinity (I182V) and those that stabilize the open conformation of the channel and indirectly reduce ATP sensitivity (V59M, Q52R, and I296L). With respect to the latter group, alterations in channel gating are also reflected in a functional "uncoupling" of sulfonylurea (SU) block: SU sensitivity of I182V is similar to that of wild-type mutants, but the SU sensitivity of all gating mutants is reduced, with the I296L mutant being resistant to block by tolbutamide (

Wambach JA, Marshall BA, Koster JC, White NH, Nichols CG. Successful sulfonylurea treatment of an insulin-naïve neonate with diabetes mellitus due to a KCNJ11 mutation. Activating mutations in the KATP-channel cause neonatal diabetes mellitus (NDM), and patients have been safely transitioned from insulin to sulfonylureas. We report a male infant with permanent NDM (PNDM), born to a PNDM mother. Blood glucose began to rise on day of life (DOL) 2, and sulfonylurea (glyburide) therapy was initiated on DOL 5. Glucose was subsequently well controlled and normal at 3 months. A KATP mutation (R201H; KCNJ11) was detected in the infant, the mother, and 6-yr-old sister with PNDM; both were also subsequently transitioned off insulin onto glyburide. To our knowledge, this is the youngest NDM patient to receive oral glyburide and, importantly, the only one deliberately initiated on sulfonylureas. Strikingly, the current dose (0.017 mg/kg/d) is below the reported therapeutic range and approximately 75-fold lower than doses required by the affected sister and mother. Pancreatic insulin disappears in an animal model of KATP-induced NDM, unless glycemia is well controlled, thus, a dramatically lower glyburide requirement in the infant may reflect preserved insulin content because of early sulfonylurea intervention. Safe and effective initiation of glyburide in an insulin-naïve neonatal patient with KATP-dependent PNDM argues for early detection and sulfonylurea intervention. [ABSTRACT FROM AUTHOR]

Cantú syndrome (CS), a multisystem disease with a complex cardiovascular phenotype, is caused by gain-of-function (GoF) variants in the Kir6.1/SUR2 subunits of ATP-sensitive potassium (KATP) channels and is characterized by low systemic vascular resistance, as well as tortuous, dilated, vessels, and decreased pulse-wave velocity. Thus, CS vascular dysfunction is multifactorial, with both hypomyotonic and hyperelastic components. To dissect whether such complexities arise cell autonomously within vascular smooth muscle cells (VSMCs) or as secondary responses to the pathophysiological milieu, we assessed electrical properties and gene expression in human induced pluripotent stem cell-derived VSMCs (hiPSC-VSMCs), differentiated from control and CS patient-derived hiPSCs, and in native mouse control and CS VSMCs. Whole-cell voltage clamp of isolated aortic and mesenteric arterial VSMCs isolated from wild-type (WT) and Kir6.1[V65M] (CS) mice revealed no clear differences in voltage-gated K+ (Kv) or Ca2+ currents. Kv and Ca2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. While pinacidil-sensitive KATP currents in control hiPSC-VSMCs were similar to those in WT mouse VSMCs, they were considerably larger in CS hiPSC-VSMCs. Under current-clamp conditions, CS hiPSC-VSMCs were also hyperpolarized, consistent with increased basal K conductance and providing an explanation for decreased tone and decreased vascular resistance in CS. Increased compliance was observed in isolated CS mouse aortae and was associated with increased elastin mRNA expression. This was consistent with higher levels of elastin mRNA in CS hiPSC-VSMCs and suggesting that the hyperelastic component of CS vasculopathy is a cell-autonomous consequence of vascular KATP GoF. The results show that hiPSC-VSMCs reiterate expression of the same major ion currents as primary VSMCs, validating the use of these cells to study vascular disease. Results in hiPSC-VSMCs derived from CS patient cells suggest that both the hypomyotonic and hyperelastic components of CS vasculopathy are cell-autonomous phenomena driven by KATP overactivity within VSMCs ., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Physiological Society.)

Cantu syndrome (CS) (OMIM #239850) is an autosomal dominant multiorgan system condition, associated with a characteristic facial phenotype, hypertrichosis, and multiple cardiovascular complications. CS is caused by gain-of-function (GOF) variants in KCNJ8 or ABCC9 that encode pore-forming Kir6.1 and regulatory SUR2 subunits of ATP-sensitive potassium (K ATP ) channels. A novel heterozygous ABCC9 variant, c.2440G>T; p.Gly814Trp, was identified in three individuals from a four generation Greek family. The membrane potential in cells stably expressing hKir6.1 and hSUR2B with p.Gly814Trp was hyperpolarized compared to cells expressing WT channels, and inside-out patch-clamp assays of K ATP channels formed with hSUR2B p.Gly814Trp demonstrated a decreased sensitivity to ATP inhibition, confirming a relatively mild GOF effect of this variant. The specific location of the variant reveals an unrecognized functional role of the first glycine in the signature motif of the nucleotide binding domains in ATP-binding cassette (ABC) protein ion channels., (© 2024 The Author(s). American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Cantú syndrome is a multisystem disorder caused by gain-of-function (GOF) mutations in KCNJ8 and ABCC9, the genes encoding the pore-forming inward rectifier Kir6.1 and regulatory sulfonylurea receptor SUR2B subunits, respectively, of vascular ATP-sensitive K+ (KATP) channels. In this study, we investigated changes in the vascular endothelium in mice in which Cantú syndrome-associated Kcnj8 or Abcc9 mutations were knocked in to the endogenous loci. We found that endothelium-dependent dilation was impaired in small mesenteric arteries from Cantú mice. Loss of endothelium-dependent vasodilation led to increased vasoconstriction in response to intraluminal pressure or treatment with the adrenergic receptor agonist phenylephrine. We also found that either KATP GOF or acute activation of KATP channels with pinacidil increased the amplitude and frequency of wave-like Ca2+ events generated in the endothelium in response to the vasodilator agonist carbachol. Increased cytosolic Ca2+ signaling activity in arterial endothelial cells from Cantú mice was associated with elevated mitochondrial [Ca2+] and enhanced reactive oxygen species (ROS) and peroxynitrite levels. Scavenging intracellular or mitochondrial ROS restored endothelium-dependent vasodilation in the arteries of mice with KATP GOF mutations. We conclude that mitochondrial Ca2+ overload and ROS generation, which subsequently leads to nitric oxide consumption and peroxynitrite formation, cause endothelial dysfunction in mice with Cantú syndrome.

The Ca 2+ -activated Cl - channel regulator CLCA1 potentiates the activity of the Ca 2+ -activated Cl - channel (CaCC) TMEM16A by directly engaging the channel at the cell surface, inhibiting its reinternalization and increasing Ca 2+ -dependent Cl - current (I CaCC ) density. We now present evidence of functional pairing between two other CLCA and TMEM16 protein family members, namely CLCA4 and the CaCC TMEM16B. Similar to CLCA1, (i) CLCA4 is a self-cleaving metalloprotease, and the N-terminal portion (N-CLCA4) is secreted; (ii) the von Willebrand factor type A (VWA) domain in N-CLCA4 is sufficient to potentiate I CaCC in HEK293T cells; and (iii) this is mediated by the metal ion-dependent adhesion site motif within VWA. The results indicate that, despite the conserved regulatory mechanism and homology between CLCA1 and CLCA4, CLCA4-dependent I CaCC are carried by TMEM16B, rather than TMEM16A. Our findings show specificity in CLCA/TMEM16 interactions and suggest broad physiological and pathophysiological links between these two protein families., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Loss-of-function mutation of ABCC9, the gene encoding the SUR2 subunit of ATP sensitive-potassium (KATP) channels, was recently associated with autosomal recessive ABCC9-related intellectual disability and myopathy syndrome (AIMS). Here we identify nine additional subjects, from seven unrelated families, harbouring different homozygous loss-of-function variants in ABCC9 and presenting with a conserved range of clinical features. All variants are predicted to result in severe truncations or in-frame deletions within SUR2, leading to the generation of non-functional SUR2-dependent KATP channels. Affected individuals show psychomotor delay and intellectual disability of variable severity, microcephaly, corpus callosum and white matter abnormalities, seizures, spasticity, short stature, muscle fatigability and weakness. Heterozygous parents do not show any conserved clinical pathology but report multiple incidences of intra-uterine fetal death, which were also observed in an eighth family included in this study. In vivo studies of abcc9 loss-of-function in zebrafish revealed an exacerbated motor response to pentylenetetrazole, a pro-convulsive drug, consistent with impaired neurodevelopment associated with an increased seizure susceptibility. Our findings define an ABCC9 loss-of-function-related phenotype, expanding the genotypic and phenotypic spectrum of AIMS and reveal novel human pathologies arising from KATP channel dysfunction., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Gain-of-function of K ATP channels, resulting from mutations in either KCNJ8 (encoding inward rectifier sub-family 6 [Kir6.1]) or ABCC9 (encoding sulphonylurea receptor [SUR2]), cause Cantú syndrome (CS), a channelopathy characterized by excess hair growth, coarse facial appearance, cardiomegaly, and lymphedema. Here, we established a pipeline for rapid analysis of CS mutation consequences in Landing pad HEK 293 cell lines stably expressing wild type (WT) and mutant human Kir6.1 and SUR2B. Thallium-influx and cell membrane potential, reported by fluorescent Tl-sensitive Fluozin-2 and voltage-sensitive bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) dyes, respectively, were used to assess channel activity. In the Tl-influx assay, CS-associated Kir6.1 mutations increased sensitivity to the ATP-sensitive potassium (K ATP ) channel activator, pinacidil, but there was strikingly little effect of pinacidil for any SUR2B mutations, reflecting unexpected differences in the molecular mechanisms of Kir6.1 versus SUR2B mutations. Compared with the Tl-influx assay, the DiBAC4(3) assay presents more significant signal changes in response to subtle K ATP channel activity changes, and all CS mutants (both Kir6.1 and SUR2B), but not WT channels, caused marked hyperpolarization, demonstrating that all mutants were activated under ambient conditions in intact cells. Most SUR2 CS mutations were markedly inhibited by <100 nM glibenclamide, but sensitivity to inhibition by glibenclamide, repaglinide, and PNU37883A was markedly reduced for Kir6.1 CS mutations. Understanding functional consequences of mutations can help with disease diagnosis and treatment. The analysis pipeline we have developed has the potential to rapidly identify mutational consequences, aiding future CS diagnosis, drug discovery, and individualization of treatment. SIGNIFICANCE STATEMENT: We have developed new fluorescence-based assays of channel activities and drug sensitivities of Cantú syndrome (CS) mutations in human Kir6.1/SUR2B-dependent K ATP channels, showing that Kir6.1 mutations increase sensitivity to potassium channel openers, while SUR2B mutations markedly reduce K channel opener (KCO) sensitivity. However, both Kir6.1 and SUR2B CS mutations are both more hyperpolarized than WT cells under basal conditions, confirming pathophysiologically relevant gain-of-function, validating DiBAC4(3) fluorescence to characterize hyperpolarization induced by K ATP channel activity under basal, non KCO-activated conditions., (Copyright © 2023 by The Author(s).)

Inwardly rectifying potassium (Kir) channels open at the 'helix bundle crossing' (HBC), formed by the M2 helices at the cytoplasmic end of the transmembrane pore. Introduced negative charges at the HBC (G178D) in Kir2.2 channels forces opening, allowing pore wetting and free movement of permeant ions between the cytoplasm and the inner cavity. Single-channel recordings reveal striking, pH-dependent, subconductance behaviors in G178D (or G178E and equivalent Kir2.1[G177E]) mutant channels, with well-resolved non-cooperative subconductance levels. Decreasing cytoplasmic pH shifts the probability towards lower conductance levels. Molecular dynamics simulations show how protonation of Kir2.2[G178D], or the D173 pore-lining residues, changes solvation, K + ion occupancy, and K + conductance. Ion channel gating and conductance are classically understood as separate processes. The present data reveal how individual protonation events change the electrostatic microenvironment of the pore, resulting in step-wise alterations of ion pooling, and hence conductance, that appear as 'gated' substates., (© 2023. The Author(s).)

ABCC9-related intellectual disability and myopathy syndrome (AIMS) arises from loss-of-function (LoF) mutations in the ABCC9 gene, which encodes the SUR2 subunit of ATP-sensitive potassium (K ATP ) channels. K ATP channels are found throughout the cardiovascular system and skeletal muscle and couple cellular metabolism to excitability. AIMS individuals show fatigability, muscle spasms, and cardiac dysfunction. We found reduced exercise performance in mouse models of AIMS harboring premature stop codons in ABCC9. Given the roles of K ATP channels in all muscles, we sought to determine how myopathy arises using tissue-selective suppression of K ATP and found that LoF in skeletal muscle, specifically, underlies myopathy. In isolated muscle, SUR2 LoF results in abnormal generation of unstimulated forces, potentially explaining painful spasms in AIMS. We sought to determine whether excessive Ca 2+ influx through Ca V 1.1 channels was responsible for myopathology but found that the Ca 2+ channel blocker verapamil unexpectedly resulted in premature death of AIMS mice and that rendering Ca V 1.1 channels nonpermeable by mutation failed to reverse pathology; results which caution against the use of calcium channel blockers in AIMS., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Elevated blood glucose levels, or hyperglycemia, can increase brain excitability and amyloid-β (Aβ) release, offering a mechanistic link between type 2 diabetes and Alzheimer's disease (AD). Since the cellular mechanisms governing this relationship are poorly understood, we explored whether ATP-sensitive potassium (KATP) channels, which couple changes in energy availability with cellular excitability, play a role in AD pathogenesis. First, we demonstrate that KATP channel subunits Kir6.2/KCNJ11 and SUR1/ABCC8 were expressed on excitatory and inhibitory neurons in the human brain, and cortical expression of KCNJ11 and ABCC8 changed with AD pathology in humans and mice. Next, we explored whether eliminating neuronal KATP channel activity uncoupled the relationship between metabolism, excitability, and Aβ pathology in a potentially novel mouse model of cerebral amyloidosis and neuronal KATP channel ablation (i.e., amyloid precursor protein [APP]/PS1 Kir6.2-/- mouse). Using both acute and chronic paradigms, we demonstrate that Kir6.2-KATP channels are metabolic sensors that regulate hyperglycemia-dependent increases in interstitial fluid levels of Aβ, amyloidogenic processing of APP, and amyloid plaque formation, which may be dependent on lactate release. These studies identify a potentially new role for Kir6.2-KATP channels in AD and suggest that pharmacological manipulation of Kir6.2-KATP channels holds therapeutic promise in reducing Aβ pathology in patients with diabetes or prediabetes.

The Vanilloid thermoTRP (TRPV1-4) subfamily of TRP channels are involved in thermoregulation, osmoregulation, itch and pain perception, (neuro)inflammation and immune response, and tight control of channel activity is required for perception of noxious stimuli and pain. Here we report voltage-dependent modulation of each of human TRPV1, 3, and 4 by the endogenous intracellular polyamine spermine. As in inward rectifier K channels, currents are blocked in a strongly voltage-dependent manner, but, as in cyclic nucleotide-gated channels, the blockade is substantially reduced at more positive voltages, with maximal blockade in the vicinity of zero voltage. A kinetic model of inhibition suggests two independent spermine binding sites with different affinities as well as different degrees of polyamine permeability in TRPV1, 3, and 4. Given that block and relief occur over the physiological voltage range of action potentials, voltage-dependent polyamine block may be a potent modulator of TRPV-dependent excitability in multiple cell types., (© 2023 Maksaev et al.)

Cantú Syndrome (CS) is an autosomal dominant disorder caused by gain-of-function (GoF) mutations in the Kir6.1 and SUR2 subunits of K ATP channels. K ATP overactivity results in a chronic reduction in arterial tone and hypotension, leading to other systemic cardiovascular complications. However, the underlying mechanism of lymphedema, developed by >50% of CS patients, is unknown. We investigated whether lymphatic contractile dysfunction occurs in mice expressing CS mutations in Kir6.1 (Kir6.1[V65M]) or SUR2 (SUR2[A478V], SUR2[R1154Q]). Pressure myograph tests of contractile function of popliteal lymphatic vessels over the physiological pressure range revealed significantly impaired contractile strength and reduced frequency of spontaneous contractions at all pressures in heterozygous Kir6.1[V65M] vessels, compared to control littermates. Contractile dysfunction of intact popliteal lymphatics in vivo was confirmed using near-infrared fluorescence microscopy. Homozygous SUR2[A478V] vessels exhibited profound contractile dysfunction ex vivo, but heterozygous SUR2[A478V] vessels showed essentially normal contractile function. However, further investigation of vessels from all three GoF mouse strains revealed significant disruption in contraction wave entrainment, decreased conduction speed and distance, multiple pacemaker sites, and reversing wave direction. Tests of 2-valve lymphatic vessels forced to pump against an adverse pressure gradient revealed that all CS-associated genotypes were essentially incapable of pumping under an imposed outflow load. Our results show that varying degrees of lymphatic contractile dysfunction occur in proportion to the degree of molecular GoF in Kir6.1 or SUR2. This is the first example of lymphatic contractile dysfunction caused by a smooth muscle ion channel mutation and potentially explains the susceptibility of CS patients to lymphedema., Competing Interests: The authors have no conflicts of interest to report, financial or otherwise.; CGN holds the position of Executive Editor for Function and was blinded from reviewing or making decisions for the manuscript., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Physiological Society.)

Physiological activity of G protein gated inward rectifier K + (GIRK, Kir3) channel, dynamically regulated by three key ligands, phosphoinositol-4,5-bisphosphate (PIP 2 ), Gβγ, and Na + , underlies cellular electrical response to multiple hormones and neurotransmitters in myocytes and neurons. In a reducing environment, matching that inside cells, purified GIRK2 (Kir3.2) channels demonstrate low basal activity, and expected sensitivity to the above ligands. However, under oxidizing conditions, anomalous behavior emerges, including rapid loss of PIP 2 and Na + -dependent activation and a high basal activity in the absence of any agonists, that is now paradoxically inhibited by PIP 2 . Mutagenesis identifies two cysteine residues (C65 and C190) as being responsible for the loss of PIP 2 and Na + -dependent activity and the elevated basal activity, respectively. The results explain anomalous findings from earlier studies and illustrate the potential pathophysiologic consequences of oxidation on GIRK channel function, as well as providing insight to reversed ligand-dependence of Kir and KirBac channels., Competing Interests: The authors declare no conflict of interest. Colin G. Nichols holds the position of Executive Editor for Function and is blinded from reviewing or making decisions for the manuscript., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Physiological Society.)

Cantú syndrome (CS) is caused by the gain of function mutations in the ABCC9 and KCNJ8 genes encoding, respectively, for the sulfonylureas receptor type 2 (SUR2) and the inwardly rectifier potassium channel 6.1 (Kir6.1) of the ATP-sensitive potassium (KATP) channels. CS is a multi-organ condition with a cardiovascular phenotype, neuromuscular symptoms, and skeletal malformations. Glibenclamide has been proposed for use in CS, but even in animals, the drug is incompletely effective against severe mutations, including the Kir6.1 wt/V65M . Patch-clamp experiments showed that zoledronic acid (ZOL) fully reduced the whole-cell KATP currents in bone calvaria cells from wild type (WT/WT) and heterozygous Kir6.1 wt/V65M CS mice, with IC 50 for ZOL block < 1 nM in each case. ZOL fully reduced KATP current in excised patches in skeletal muscle fibers in WT/WT and CS mice, with IC 50 of 100 nM in each case. Interestingly, KATP currents in the bone of heterozygous SUR2 wt/A478V mice were less sensitive to ZOL inhibition, showing an IC 50 of ~500 nM and a slope of ~0.3. In homozygous SUR2 A478V/A478V cells, ZOL failed to fully inhibit the KATP currents, causing only ~35% inhibition at 100 μM, but was responsive to glibenclamide. ZOL reduced the KATP currents in Kir6.1 wt/VM CS mice in both skeletal muscle and bone cells but was not effective in the SUR2 [A478V] mice fibers. These data indicate a subunit specificity of ZOL action that is important for appropriate CS therapies.

Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human-induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype (WT). There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations up-regulated several anterior HOX genes and down-regulated antioxidant genes CAT and GSTA1 throughout chondrogenesis. BMP4 treatment up-regulated several essential hypertrophic genes in WT chondrocytes; however, this hypertrophic maturation response was inhibited in mutant chondrocytes. These results indicate that the TRPV4 mutations alter BMP signaling in chondrocytes and prevent proper chondrocyte hypertrophy, as a potential mechanism for dysfunctional skeletal development. Our findings provide potential therapeutic targets for developing treatments for TRPV4-mediated skeletal dysplasias., Competing Interests: AD, GM, ZH, AS, RT, NS, CN, CW No competing interests declared, WL Patents on TRPV4 inhibitors have been licensed to TRPblue (US Patents 9,701,675; 10,329,265; and 11,014,896). Dr. Liedtke is an executive employee of Regeneron Pharmaceuticals (Tarrytown NY). FG Patents on TRPV4 inhibitors licensed to TRPblue Inc (US Patents 9,701,675; 10,329,265; and 11,014,896), (© 2023, Dicks et al.)

Inwardly rectifier potassium (Kir) channels are a major potassium channel sub-class whose function is regulated by ligand-dependent gating and highly voltage-dependent block by polyamines. With molecular dynamics simulations over previously unattainable timescales, Jogini et al. (J. Gen. Physiol. https://doi.org/10.1085/jgp.202213085) provide unprecedented visualization of K+ conduction through open Kir2.2 channels and of the molecular details of channel block by spermine., (© 2022 Lee and Nichols.)

Cantu Syndrome (CS), [OMIM #239850] is characterized by hypertrichosis, osteochondrodysplasia, and cardiomegaly. CS is caused by gain-of-function (GOF) variants in the KCNJ8 or ABCC9 genes that encode pore-forming Kir6.1 and regulatory SUR2 subunits of ATP-sensitive potassium (K ATP ) channels. Many subjects with CS also present with the complication of lymphedema. A previously uncharacterized, heterozygous ABCC9 variant, p.(Leu1055&#95;Glu1058delinsPro), termed indel1055, was identified in an individual diagnosed with idiopathic lymphedema. The variant was introduced into the equivalent position of rat SUR2A, and inside-out patches were used to characterize the K ATP channels formed by Kir6.2 and WT or mutant SUR2A subunits coexpressed in Cosm6 cells. The indel1055 variant causes gain-of-function of the channel, with an increase of the IC 50 for ATP inhibition compared to WT. Retrospective consideration of this individual reveals clear features of Cantu Syndrome. An additional heterozygous ABCC9 variant, p.(Ile419Thr), was identified in a second individual diagnosed with lymphedema. In this case, there were no additional features consistent with CS, and the properties of p.(Ile416Thr) (the corresponding mutation in rat SUR2A)--containing channels were not different from WT. This proof-of-principle study shows that idiopathic lymphedema may actually be a first presentation of otherwise unrecognized Cantu Syndrome, but molecular phenotyping of identified variants is necessary to confirm relevance., (© 2022. The Author(s), under exclusive licence to European Society of Human Genetics.)

Chronic hyperglycemia increases pancreatic β-cell metabolic activity, contributing to glucotoxicity-induced β-cell failure and loss of functional β-cell mass, potentially in multiple forms of diabetes. In this perspective we discuss the novel paradoxical and counterintuitive concept of inhibiting glycolysis, particularly by targeted inhibition of glucokinase, the first enzyme in glycolysis, as an approach to maintaining glucose sensing and preserving functional β-cell mass, thereby improving insulin secretion, in the treatment of diabetes., (© 2023 by the American Diabetes Association.)

Ubiquitously expressed throughout the body, ATP-sensitive potassium (K ATP ) channels couple cellular metabolism to electrical activity in multiple tissues; their unique assembly as four Kir6 pore-forming subunits and four sulfonylurea receptor (SUR) subunits has resulted in a large armory of selective channel opener and inhibitor drugs. The spectrum of monogenic pathologies that result from gain- or loss-of-function mutations in these channels, and the potential for therapeutic correction of these pathologies, is now clear. However, while available drugs can be effective treatments for specific pathologies, cross-reactivity with the other Kir6 or SUR subfamily members can result in drug-induced versions of each pathology and may limit therapeutic usefulness. This review discusses the background to K ATP channel physiology, pathology, and pharmacology and considers the potential for more specific or effective therapeutic agents.

The K + channel selectivity filter (SF) is defined by TxGYG amino acid sequences that generate four identical K + binding sites (S1-S4). Only two sites (S3, S4) are present in the non-selective bacterial NaK channel, but a four-site K + -selective SF is obtained by mutating the wild-type TVGDGN SF sequence to a canonical K + channel TVGYGD sequence (NaK2K mutant). Using single molecule FRET (smFRET), we show that the SF of NaK2K, but not of non-selective NaK, is ion-dependent, with the constricted SF configuration stabilized in high K + conditions. Patch-clamp electrophysiology and non-canonical fluorescent amino acid incorporation show that NaK2K selectivity is reduced by crosslinking to limit SF conformational movement. Finally, the eukaryotic K + channel TREK2 SF exhibits essentially identical smFRET-reported ion-dependent conformations as in prokaryotic K + channels. Our results establish the generality of K + -induced SF conformational stability across the K + channel superfamily, and introduce an approach to study manipulation of channel selectivity., (© 2023. The Author(s).)

ATP-sensitive potassium channel (K ATP )gain- (GOF) and loss-of-function (LOF) mutations underlie human neonatal diabetes mellitus (NDM) and hyperinsulinism (HI), respectively. While transgenic mice expressing incomplete K ATP LOF do reiterate mild hyperinsulinism, K ATP knockout animals do not exhibit persistent hyperinsulinism. We have shown that islet excitability and glucose homeostasis are regulated by identical K ATP channels in zebrafish. SUR1 truncation mutation (K499X) was introduced into the abcc8 gene to explore the possibility of using zebrafish for modeling human HI. Patch-clamp analysis confirmed the complete absence of channel activity in β-cells from K499X (SUR1 -/- ) fish. No difference in random blood glucose was detected in heterozygous SUR1+/- fish nor in homozygous SUR1 -/- fish, mimicking findings in SUR1 knockout mice. Mutant fish did, however, demonstrate impaired glucose tolerance, similar to partial LOF mouse models. In paralleling features of mammalian diabetes and hyperinsulinism resulting from equivalent LOF mutations, these gene-edited animals provide valid zebrafish models of K ATP -dependent pancreatic diseases.

Background Cardiomegaly caused by left ventricular hypertrophy is a risk factor for development of congestive heart failure, classically associated with decreased systolic and/or diastolic ventricular function. Less attention has been given to the phenotype of left ventricular hypertrophy with enhanced ventricular function and increased cardiac output, which is potentially associated with high-output heart failure. Lack of recognition may pose diagnostic ambiguity and management complexities. Methods and Results We sought to systematically characterize high-output cardiac hypertrophy in subjects with Cantu syndrome (CS), caused by gain-of-function variants in ABCC9 , which encodes cardiovascular K ATP (ATP-sensitive potassium) channel subunits. We studied the cardiovascular phenotype longitudinally in 31 subjects with CS with confirmed ABCC9 variants (median [interquartile range] age 8 years [3-32 years], body mass index 19.9 [16.5-22.9], 16 male subjects). Subjects with CS presented with significant left ventricular hypertrophy (left ventricular mass index 86.7 [57.7-103.0] g/m 2 in CS, n=30; 26.6 [24.1-32.8] g/m 2 in controls, n=17; P <0.0001) and low blood pressure (systolic 94.5 [90-103] mm Hg in CS, n=17; 109 [98-115] mm Hg in controls, n=17; P =0.0301; diastolic 60 [56-66] mm Hg in CS, n=17; 69 [65-72] mm Hg in control, n=17; P =0.0063). Most (21/31) subjects with CS exhibited eccentric hypertrophy with normal left ventricular wall thickness. Congestive heart failure symptoms were evident in 4 of the 5 subjects with CS aged >40 years on long-term follow-up. Conclusions The data define the natural history of high-output cardiac hypertrophy resulting from decreased systemic vascular resistance in subjects with CS, a defining population for long-term consequences of high-output hypertrophy caused by low systemic vascular resistance, and the potential for progression to high-output heart failure.

The interest in astrocytes, the silent brain cells that accumulate polyamines (PAs), is growing. PAs exert anti-inflammatory, antioxidant, antidepressant, neuroprotective, and other beneficial effects, including increasing longevity in vivo. Unlike neurons, astrocytes are extensively coupled to others via connexin (Cx) gap junctions (GJs). Although there are striking modulatory effects of PAs on neuronal receptors and channels, PA regulation of the astrocytic GJs is not well understood. We studied GJ-propagation using molecules of different (i) electrical charge, (ii) structure, and (iii) molecular weight. Loading single astrocytes with patch pipettes containing membrane-impermeable dyes, we observed that (i) even small molecules do not easily permeate astrocytic GJs, (ii) the ratio of the charge to weight of these molecules is the key determinant of GJ permeation, (iii) the PA spermine (SPM) induced the propagation of negatively charged molecules via GJs, (iv) while no effects were observed on propagation of macromolecules with net-zero charge. The GJ uncoupler carbenoxolone (CBX) blocked such propagation. Taken together, these findings indicate that SPM is essential for astrocytic GJ communication and selectively facilitates intracellular propagation via GJs for negatively charged molecules through glial syncytium.

K ATP channels function as negative regulators of active lymphatic pumping and lymph transport. This review summarizes and critiques the evidence for the expression of specific K ATP channel subunits in lymphatic smooth muscle and endothelium, the roles that they play in normal lymphatic function, and their possible involvement in multiple diseases, including metabolic syndrome, lymphedema, and Cantú syndrome. For each of these topics, suggestions are made for directions for future research.

Three decades of research, both in vitro and in vivo, have demonstrated the conformational heterogeneity that is displayed by the amyloid β peptide (Aβ) in Alzheimer's disease (AD). Understanding the distinct properties between Aβ conformations and how conformation may impact cellular activity remain open questions, yet still continue to provide new insights into protein misfolding and aggregation. In particular, there is interest in the group of soluble oligomeric prefibrillar Aβ species comprising lower molecular weight oligomers up to larger protofibrils. In the current study, a number of strategies were utilized to separate Aβ protofibrils and oligomers and show that the smaller Aβ oligomers have a much different conformation than Aβ protofibrils. The differences were consistent for both Aβ40 and Aβ42. Protofibrils bound thioflavin T to a greater extent than oligomers, and were highly enriched in β-sheet secondary structure. Aβ oligomers possessed a more open structure with significant solvent exposure of hydrophobic domains as determined by tryptophan fluorescence and bis-ANS binding, respectively. The protofibril-selective antibody AbSL readily discerned conformational differences between protofibrils and oligomers. The more developed structure for Aβ protofibrils ultimately proved critical for provoking the release of tumor necrosis factor α from microglial cells. The findings demonstrated a dependency on β-sheet structure for soluble Aβ aggregates to cause a microglial inflammatory response. The Aβ aggregation process yields many conformationally-varied species with different levels of β-structure and exposed hydrophobicity. The conformation elements likely determine biological activity and pathogenicity., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Kir6.1 and SUR2 are subunits of ATP-sensitive potassium (K ATP ) channels expressed in a wide range of tissues. Extensive study has implicated roles of these channel subunits in diverse physiological functions. Together they generate the predominant K ATP conductance in vascular smooth muscle and are the target of vasodilatory drugs. Roles for Kir6.1/SUR2 dysfunction in disease have been suggested based on studies of animal models and human genetic discoveries. In recent years, it has become clear that gain-of-function (GoF) mutations in both genes result in Cantú syndrome (CS)-a complex, multisystem disorder. There is currently no targeted therapy for CS, but studies of mouse models of the disease reveal that pharmacological reversibility of cardiovascular and gastrointestinal pathologies can be achieved by administration of the K ATP channel inhibitor, glibenclamide. Here we review the function, structure, and physiological and pathological roles of Kir6.1/SUR2B channels, with a focus on CS. Recent studies have led to much improved understanding of the underlying pathologies and the potential for treatment, but important questions remain: Can the study of genetically defined CS reveal new insights into Kir6.1/SUR2 function? Do these reveal new pathophysiological mechanisms that may be important in more common diseases? And is our pharmacological armory adequately stocked?

β-Cell failure and loss of β-cell mass are key events in diabetes progression. Although insulin hypersecretion in early stages has been implicated in β-cell exhaustion/failure, loss of β-cell mass still occurs in KATP gain-of-function (GOF) mouse models of human neonatal diabetes in the absence of insulin secretion. Thus, we hypothesize that hyperglycemia-induced increased β-cell metabolism is responsible for β-cell failure and that reducing glucose metabolism will prevent loss of β-cell mass. To test this, KATP-GOF mice were crossed with mice carrying β-cell-specific glucokinase haploinsufficiency (GCK+/-), to genetically reduce glucose metabolism. As expected, both KATP-GOF and KATP-GOF/GCK+/- mice showed lack of glucose-stimulated insulin secretion. However, KATP-GOF/GCK+/- mice demonstrated markedly reduced blood glucose, delayed diabetes progression, and improved glucose tolerance compared with KATP-GOF mice. In addition, decreased plasma insulin and content, increased proinsulin, and augmented plasma glucagon observed in KATP-GOF mice were normalized to control levels in KATP-GOF/GCK+/- mice. Strikingly, KATP-GOF/GCK+/- mice demonstrated preserved β-cell mass and identity compared with the marked decrease in β-cell identity and increased dedifferentiation observed in KATP-GOF mice. Moreover KATP-GOF/GCK+/- mice demonstrated restoration of body weight and liver and brown/white adipose tissue mass and function and normalization of physical activity and metabolic efficiency compared with KATP-GOF mice. These results demonstrate that decreasing β-cell glucose signaling can prevent glucotoxicity-induced loss of insulin content and β-cell failure independently of compensatory insulin hypersecretion and β-cell exhaustion., (© 2022 by the American Diabetes Association.)

Cytoplasmic domains frequently promote functional assembly of multimeric ion channels. To investigate structural determinants of this process, we generated the 'T1-chimera' construct of the NaChBac sodium channel by truncating its C-terminal domain and splicing the T1-tetramerisation domain of the Kv1.2 channel to the N terminus. Purified T1-chimera channels were tetrameric, conducted Na + when reconstituted into proteoliposomes, and were functionally blocked by the drug mibefradil. Both the T1-chimera and full-length NaChBac had comparable expression levels in the membrane, whereas a NaChBac mutant lacking a cytoplasmic domain had greatly reduced membrane expression. Our findings support a model whereby bringing the transmembrane regions into close proximity enables their tetramerisation. This phenomenon is found with other channels, and thus, our findings substantiate this as a common assembly mechanism., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Secretion of insulin from pancreatic β-cells is complex, but physiological glucose-dependent secretion is dominated by electrical activity, in turn controlled by ATP-sensitive potassium (KATP) channel activity. Accordingly, loss-of-function mutations of the KATP channel Kir6.2 (KCNJ11) or SUR1 (ABCC8) subunit increase electrical excitability and secretion, resulting in congenital hyperinsulinism (CHI), whereas gain-of-function mutations cause underexcitability and undersecretion, resulting in neonatal diabetes mellitus (NDM). Thus, diazoxide, which activates KATP channels, and sulfonylureas, which inhibit KATP channels, have dramatically improved therapies for CHI and NDM, respectively. However, key findings do not fit within this simple paradigm: mice with complete absence of β-cell KATP activity are not hyperinsulinemic; instead, they are paradoxically glucose intolerant and prone to diabetes, as are older human CHI patients. Critically, despite these advances, there has been little insight into any role of KATP channel activity changes in the development of type 2 diabetes (T2D). Intriguingly, the CHI progression from hypersecretion to undersecretion actually mirrors the classical response to insulin resistance in the progression of T2D. In seeking to explain the progression of CHI, multiple lines of evidence lead us to propose that underlying mechanisms are also similar and that development of T2D may involve loss of KATP activity., (© 2022 by the American Diabetes Association.)

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

Background/aims: Mutations in KCNJ11, the gene encoding the Kir6.2 subunit of pancreatic and neuronal KATP channels, are associated with a spectrum of neonatal diabetes diseases., Methods: Variant screening was used to identify the cause of neonatal diabetes, and continuous glucose monitoring was used to assess effectiveness of sulfonylurea treatment. Electrophysiological analysis of variant KATP channel function was used to determine molecular basis., Results: We identified a previously uncharacterized KCNJ11 mutation, c.988T>C [p.Tyr330His], in an Italian child diagnosed with sulfonylurea-resistant permanent neonatal diabetes and developmental delay (intermediate DEND). Functional analysis of recombinant KATP channels reveals that this mutation causes a drastic gain-of-function, due to a reduction in ATP inhibition. Further, we demonstrate that the Tyr330His substitution causes a significant decrease in sensitivity to the sulfonylurea, glibenclamide., Conclusions: In this subject, the KCNJ11 (c.988T>C) mutation provoked neonatal diabetes, with mild developmental delay, which was insensitive to correction by sulfonylurea therapy. This is explained by the molecular loss of sulfonylurea sensitivity conferred by the Tyr330His substitution and highlights the need for molecular analysis of such mutations., (© 2022 S. Karger AG, Basel.)

ATP-sensitive potassium channels (K ATP channels) are hetero-octameric nucleotide-gated ion channels that couple cellular metabolism to excitability in various tissues. In the heart, K ATP channels are activated during ischaemia and potentially during adrenergic stimulation. In the vasculature, they are normally active at a low level, reducing vascular tone, but the ubiquitous nature of these channels leads to complex and poorly understood channelopathies as a result of gain- or loss-of-function mutations. Zebrafish (ZF) models of these channelopathies may provide insights to the link between molecular dysfunction and complex pathophysiology, but this requires understanding the tissue dependence of channel activity and subunit specificity. Thus far, direct analysis of ZF K ATP expression and functional properties has only been performed in pancreatic β-cells. Using a comprehensive combination of genetically modified fish, electrophysiology and gene expression analysis, we demonstrate that ZF cardiac myocytes (CM) and vascular smooth muscle (VSM) express functional K ATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. However, in contrast to mammalian cardiovascular K ATP channels, ZF channels are insensitive to potassium channel opener drugs (pinacidil, minoxidil) in both chambers of the heart and in VSM. The results provide a first characterization of the molecular properties of fish K ATP channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9-related Intellectual Disability and Myopathy syndrome. KEY POINTS: Zebrafish cardiac myocytes (CM) and vascular smooth muscle (VSM) express functional K ATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. In contrast to mammalian cardiovascular K ATP channels, zebrafish channels are insensitive to potassium channel opener drugs (pinacidil, minoxidil) in both chambers of the heart and in VSM. We provide a first characterization of the molecular properties of fish K ATP channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9-related Intellectual Disability and Myopathy syndrome., (© 2021 The Authors. The Journal of Physiology © 2021 The Physiological Society.)

Background and Purpose: K ATP channels are negative regulators of lymphatic vessel excitability and contractility and are proposed to be targets for immune cell products that inhibit lymph transport. Previous studies in rat and guinea pig mesenteric lymphatics found that NO-mediated inhibition of lymphatic contraction was prevented or reversed by the K ATP channel inhibitor, glibenclamide. We revisited this hypothesis using mouse lymphatic vessels and K ATP channel knockout mice., Experimental Approach: Mouse popliteal lymphatics were isolated, and contractility was assessed using pressure myography. K + channel expression was determined by PCR analysis of FACS-purified lymphatic smooth muscle cells., Key Results: The NO-producing agonist, ACh, and the NO donor, NONOate, both produced dose-dependent inhibition of spontaneous lymphatic contractions that were blocked by the soluble GC inhibitor, ODQ, or the PKG inhibitor, Rp-8-Br-PET-cGMPS. Surprisingly, the inhibitory effects of both were preserved in K ir 6.1 -/- vessels, suggesting that K ATP channels did not mediate NO-induced responses. We hypothesized a role for BK channels, given their prominence in arterial smooth muscle. Indeed, BK channels were expressed in mouse lymphatic smooth muscle and NS11021 (a BK channel activator) caused dilation and reduced contraction frequency, whereas iberiotoxin and penitrem A (BK channel inhibitors) produced right-ward shifts in NONOate concentration-response curves., Conclusion and Implications: Inhibition of mouse lymphatic contractions by NO primarily involves activation of BK channels, rather than K ATP channels. Thus, BK channels are a potential target for therapeutic reversal of lymph pump inhibition by NO generated by immune cell activation of iNOS in chronic lymphoedema., (© 2021 The British Pharmacological Society.)

(1) Background: Cantu syndrome (CS) arises from gain-of-function (GOF) mutations in the ABCC9 and KCNJ8 genes, which encode ATP-sensitive K + (KATP) channel subunits SUR2 and Kir6.1, respectively. Most CS patients have mutations in SUR2, the major component of skeletal muscle KATP, but the consequences of SUR2 GOF in skeletal muscle are unknown. (2) Methods: We performed in vivo and ex vivo characterization of skeletal muscle in heterozygous SUR2[A478V] (SUR2 wt/AV ) and homozygous SUR2[A478V] (SUR2 AV/AV ) CS mice. (3) Results: In SUR2 wt/AV and SUR2 AV/AV mice, forelimb strength and diaphragm amplitude movement were reduced; muscle echodensity was enhanced. KATP channel currents recorded in Flexor digitorum brevis fibers showed reduced MgATP-sensitivity in SUR2 wt/AV , dramatically so in SUR2 AV/AV mice; IC 50 for MgATP inhibition of KATP currents were 1.9 ± 0.5 × 10 -5 M in SUR2 wt/AV and 8.6 ± 0.4 × 10 -6 M in WT mice and was not measurable in SUR2 AV/AV . A slight rightward shift of sensitivity to inhibition by glibenclamide was detected in SUR2 AV/AV mice. Histopathological and qPCR analysis revealed atrophy of soleus and tibialis anterior muscles and up-regulation of atrogin-1 and MuRF1 mRNA in CS mice. (4) Conclusions: SUR2[A478V] "knock-in" mutation in mice impairs KATP channel modulation by MgATP, markedly so in SUR2 AV/AV , with atrophy and non-inflammatory edema in different skeletal muscle phenotypes.

Potassium (K+) channels are membrane proteins with the remarkable ability to very selectively conduct K+ ions across the membrane. High-resolution structures have revealed that dehydrated K+ ions permeate through the narrowest region of the pore, formed by the backbone carbonyls of the signature selectivity filter (SF) sequence TxGYG. However, the existence of nonselective channels with similar SF sequences, as well as effects of mutations in other regions on selectivity, suggest that the SF is not the sole determinant of selectivity. We changed the selectivity of the KirBac1.1 channel by introducing mutations at residue I131 in transmembrane helix 2 (TM2). These mutations increase Na+ flux in the absence of K+ and introduce significant proton conductance. Consistent with K+ channel crystal structures, single-molecule FRET experiments show that the SF is conformationally constrained and stable in high-K+ conditions but undergoes transitions to dilated low-FRET states in high-Na+/low-K+ conditions. Relative to wild-type channels, I131M mutants exhibit marked shifts in the K+ and Na+ dependence of SF dynamics to higher K+ and lower Na+ concentrations. These results illuminate the role of I131, and potentially other structural elements outside the SF, in controlling ion selectivity, by suggesting that the physical interaction of these elements with the SF contributes to the relative stability of the constrained K+-induced SF configuration versus nonselective dilated conformations., (© 2021 Matamoros and Nichols.)

Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits, the most common mutations being SUR2[R1154Q] and SUR2[R1154W], carried by approximately 30% of patients. We used CRISPR/Cas9 genome engineering to introduce the equivalent of the human SUR2[R1154Q] mutation into the mouse ABCC9 gene. Along with minimal CS disease features, R1154Q cardiomyocytes and vascular smooth muscle showed much lower KATP current density and pinacidil activation than WT cells. Almost complete loss of SUR2-dependent protein and KATP in homozygous R1154Q ventricles revealed underlying diazoxide-sensitive SUR1-dependent KATP channel activity. Surprisingly, sequencing of SUR2 cDNA revealed 2 distinct transcripts, one encoding full-length SUR2 protein; and the other with an in-frame deletion of 93 bases (corresponding to 31 amino acids encoded by exon 28) that was present in approximately 40% and approximately 90% of transcripts from hetero- and homozygous R1154Q tissues, respectively. Recombinant expression of SUR2A protein lacking exon 28 resulted in nonfunctional channels. CS tissue from SUR2[R1154Q] mice and human induced pluripotent stem cell-derived (hiPSC-derived) cardiomyocytes showed only full-length SUR2 transcripts, although further studies will be required in order to fully test whether SUR2[R1154Q] or other CS mutations might result in aberrant splicing and variable expressivity of disease features in human CS.