Personalized Therapeutics for KATP-Dependent Pathologies.

Ubiquitously expressed throughout the body, ATP-sensitive potassium (KATP) 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 KATP channel physiology, pathology, and pharmacology and considers the potential for more specific or effective therapeutic agents.

A novel ABCC9 variant in a Greek family with Cantu syndrome affecting multiple generations highlights the functional role of the SUR2B NBD1.

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 (KATP) 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 KATP 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.

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.

Multiple vascular anomalies and refractory pericardial effusion in a young patient with Cantu syndrome: a case report and review of the literature.

BACKGROUND: Cantu syndrome is a rare and complex multisystem disorder characterized by hypertrichosis, facial dysmorphism, osteochondroplasia and cardiac abnormalities. With only 150 cases reported worldwide, Cantu syndrome is now gaining wider recognition due to molecular testing and a growing body of literature that further characterizes the syndrome and some of its most important features. Cardiovascular pathology previously described in the literature include cardiomegaly, pericardial effusion, vascular dilation and tortuosity, and other congenital heart defects. However, cardiovascular involvement is highly variable amongst individuals with Cantu syndrome. In some instances, it can be extensive and severe requiring surgical management and long term follow up. CASE PRESENTATION: Herein we report a case of a fourteen-year-old female who presented with worsening pericardial effusion of unknown etiology, and echocardiographic findings of concentric left ventricular hypertrophy, a mildly dilated aortic root and ascending aorta. Her medical history was notable for hemoptysis and an episode of pulmonary hemorrhage secondary to multiple aortopulmonary collaterals that were subsequently embolized in early childhood. She was initially managed with Ibuprofen and Colchicine but continued to worsen, and ultimately required a pericardial window for the management of refractory pericardial effusion. Imaging studies obtained on subsequent visits revealed multiple dilated and tortuous blood vessels in the head, neck, chest, and pelvis. A cardiomyopathy molecular studies panel was sent, and a pathogenic variant was identified in the ABCC9 gene, confirming the molecular diagnosis of autosomal dominant Cantu syndrome. CONCLUSIONS: Vascular anomalies and significant cardiac involvement are often present in Cantu syndrome, however there are currently no established screening recommendations or surveillance protocols in place. The triad of hypertrichosis, facial dysmorphism, and unexplained cardiovascular involvement in any patient should raise suspicion for Cantu syndrome and warrant further investigation. Initial cardiac evaluation and follow up should be indicated in any patient with a clinical and/or molecular diagnosis of Cantu syndrome. Furthermore, whole body imaging should be utilized to evaluate the extent of vascular involvement and dictate long term monitoring and care.

Kir6.1 and SUR2B in Cantú syndrome.

Kir6.1 and SUR2 are subunits of ATP-sensitive potassium (KATP) 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 KATP 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 KATP 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?

KATP channels in lymphatic function.

KATP channels function as negative regulators of active lymphatic pumping and lymph transport. This review summarizes and critiques the evidence for the expression of specific KATP 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.

ATP-sensitive potassium channels in zebrafish cardiac and vascular smooth muscle.

ATP-sensitive potassium channels (KATP channels) are hetero-octameric nucleotide-gated ion channels that couple cellular metabolism to excitability in various tissues. In the heart, KATP 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 KATP expression and functional properties has only been performed in pancreatic ß-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 KATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. However, in contrast to mammalian cardiovascular KATP 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 KATP 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 KATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. In contrast to mammalian cardiovascular KATP 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 KATP channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9-related Intellectual Disability and Myopathy syndrome.

Diverse clinical manifestations of Cantú syndrome: The first case series in Vietnam.

Cantú syndrome (CS) is an extremely rare autosomal dominant hereditary disease characterized by congenital hypertrichosis, distinct coarse facial features, cardiac defects, and other abnormalities in the skeletal and neurological systems. At present, cases with pathognomonic clinical manifestations are increasingly confirmed by genetic analysis. Two causative genes for CS are the well-known ABCC9 and the more rarely reported KCNJ8. Here, we report three Vietnamese children with CS, confirmed through genetic testing, presenting de novo ABCC9 mutations. The patients shared some common clinical manifestations, including congenital hypertrichosis, distinctive facial features, and a history of polyhydramnios during pregnancy. Concerning the various cardiac and neurological problems in the lifetime of patients with CS, an accurate diagnosis and appropriate management, especially genetic counseling, should be clinically applied in CS. Thus, our findings might modestly contribute to the global CS data, providing practical insights into CS manifestations.

Cantù syndrome: Report of a patient with a novel variant in KCNJ8 and revision of literature.

Cantù syndrome (CS) is a rare multisystemic disorder, characterized by congenital hypertrichosis, macrocephaly, facial dysmorphisms, cardiomegaly, vascular, and skeletal anomalies. From the cognitive point of view, most of the patients show a mild speech delay and a few of them present intellectual disability and learning difficulties. To date, most CS-reported cases are caused by heterozygous ABCC9 gene mutations. Only three patients with CS and heterozygous KCNJ8 gene variants have been reported. The authors here present the fourth case of CS with a variant in KCNJ8 in a 6-month-old baby. Diagnosis was reached through Trio-Whole Exome analysis that revealed a de novo missense variant in KCNJ8.

Behavioral and cognitive functioning in individuals with Cantú syndrome.

Cantú syndrome (CS) is caused by pathogenic variants in ABCC9 and KCNJ8 encoding the regulatory and pore-forming subunits of ATP-sensitive potassium (KATP ) channels. CS is characterized by congenital hypertrichosis, distinctive facial features, peripheral edema, and cardiac and neurodevelopmental abnormalities. Behavioral and cognitive issues have been self-reported by some CS individuals, but results of formal standardized investigations have not been published. To assess the cognitive profile, social functioning, and psychiatric symptoms in a large group of CS subjects systematically in a cross-sectional manner, we invited 35 individuals (1-69 years) with confirmed ABCC9 variants and their relatives to complete various commonly applied standardized age-related questionnaires, including the Kaufman brief intelligence test 2, the social responsiveness scale-2, and the Achenbach system of empirically based assessment. The majority of CS individuals demonstrated average verbal and nonverbal intelligence compared to the general population. Fifteen percent of cases showed social functioning strongly associated with a clinical diagnosis of autism spectrum disorder. Both externalizing and internalizing problems were also present in this cohort. In particular, anxiety, anxiety or attention deficit hyperactivity disorder, and autism spectrum behaviors were predominantly observed in the younger subjects in the cohort (≥25%), but this percentage decreased markedly in adults.

ATP-sensitive potassium channels: key players in pathophysiology of many diseases.

ATP-sensitive potassium channels have been an intensively studied type of protein complexes incorporated in the cell membrane for several decades. Their unique function makes them special, as they create a connection between the metabolic state and membrane voltage of the cell. This position of a bridge involved in many cellular cascades allow them to participate in various processes at often surprising positions in nearly all organ systems of the body, from the pancreas, heart muscle or retina, to the central nervous system. This review summarizes the most important roles of ATP-sensitive potassium channels focusing on their possible clinical use within particular organ systems.

Kir6.1-dependent KATP channels in lymphatic smooth muscle and vessel dysfunction in mice with Kir6.1 gain-of-function.

KEY POINTS: Spontaneous contractions are essential for normal lymph transport and these contractions are exquisitely sensitive to the KATP channel activator pinacidil. KATP channel Kir6.1 and SUR2B subunits are expressed in mouse lymphatic smooth muscle (LSM) and form functional KATP channels as verified by electrophysiological techniques. Global deletion of Kir6.1 or SUR2 subunits results in severely impaired lymphatic contractile responses to pinacidil. Smooth muscle-specific expression of Kir6.1 gain-of-function mutant (GoF) subunits results in profound lymphatic contractile dysfunction and LSM hyperpolarization that is partially rescued by the KATP inhibitor glibenclamide. In contrast, lymphatic endothelial-specific expression of Kir6.1 GoF has essentially no effect on lymphatic contractile function. The high sensitivity of LSM to KATP channel GoF offers an explanation for the lymphoedema observed in patients with Cantú syndrome, a disorder caused by gain-of-function mutations in genes encoding Kir6.1 or SUR2, and suggests that glibenclamide may be an appropriate therapeutic agent. ABSTRACT: This study aimed to understand the functional expression of KATP channel subunits in distinct lymphatic cell types, and assess the consequences of altered KATP channel activity on lymphatic pump function. KATP channel subunits Kir6.1 and SUR2B were expressed in mouse lymphatic muscle by PCR, but only Kir6.1 was expressed in lymphatic endothelium. Spontaneous contractions of popliteal lymphatics from wild-type (WT) (C57BL/6J) mice, assessed by pressure myography, were very sensitive to inhibition by the SUR2-specific KATP channel activator pinacidil, which hyperpolarized both mouse and human lymphatic smooth muscle (LSM). In vessels from mice with deletion of Kir6.1 (Kir6.1-/- ) or SUR2 (SUR2[STOP]) subunits, contractile parameters were not significantly different from those of WT vessels, suggesting that basal KATP channel activity in LSM is not an essential component of the lymphatic pacemaker, and does not exert a strong influence over contractile strength. However, these vessels were >100-fold less sensitive than WT vessels to pinacidil. Smooth muscle-specific expression of a Kir6.1 gain-of-function (GoF) subunit resulted in severely impaired lymphatic contractions and hyperpolarized LSM. Membrane potential and contractile activity was partially restored by the KATP channel inhibitor glibenclamide. In contrast, lymphatic endothelium-specific expression of Kir6.1 GoF subunits had negligible effects on lymphatic contraction frequency or amplitude. Our results demonstrate a high sensitivity of lymphatic contractility to KATP channel activators through activation of Kir6.1/SUR2-dependent channels in LSM. In addition, they offer an explanation for the lymphoedema observed in patients with Cantú syndrome, a disorder caused by gain-of-function mutations in genes encoding Kir6.1/SUR2.

Generalized hypertrichosis syndromes in Mexico.

Hypertrichosis is a rare condition characterized by excessive hair in areas of the body that are not predominantly androgen dependent. We can identify three main syndromes with congenital generalized hypertrichosis terminalis described in Mexico. The first is X-linked generalized hypertrichosis, an ultra-rare disease, with few cases reported to date. The second is Cantú syndrome, also known as hypertrichotic osteochondrodysplasia, which has a wide spectrum of clinical manifestations and is caused by pathogenic variants in ABCC9 and KCNJ8. The third is congenital hypertrichosis terminalis with or without gingival hyperplasia, which displays other features and involves several associated genes. The first two syndromes were described by the Mexican geneticist José María Cantú, and the concept of atavistic genes was invoked to explain the emergence of this outstanding trait. By understanding the genetic and pathophysiological basis of hypertrichosis, we can offer effective treatment to patients and help solve esthetic problems related to hair growth.

Three-dimensional facial morphology in Cantú syndrome.

Cantú syndrome (CS) was first described in 1982, and is caused by pathogenic variants in ABCC9 and KCNJ8 encoding regulatory and pore forming subunits of ATP-sensitive potassium (KATP ) channels, respectively. It is characterized by congenital hypertrichosis, osteochondrodysplasia, extensive cardiovascular abnormalities and distinctive facial anomalies including a broad nasal bridge, long philtrum, epicanthal folds, and prominent lips. Many genetic syndromes, such as CS, involve facial anomalies that serve as a significant clue in the initial identification of the respective disorder before clinical or molecular diagnosis are undertaken. However, an overwhelming number of CS patients receive misdiagnoses based on an evaluation of coarse facial features. By analyzing three-dimensional images of CS faces, we quantified facial dysmorphology in a cohort of both male and female CS patients with confirmed ABCC9 variants. Morphometric analysis of different regions of the face revealed gender-specific significant differences in face shape. Moreover, we show that 3D facial photographs can distinguish between CS and other genetic disorders with specific facial dysmorphologies that have been mistaken for CS-associated anomalies in the past, hence assisting in an earlier clinical and molecular diagnosis. This optimizes genetic counseling and reduces stress for patients and parents by avoiding unnecessary misdiagnosis.

Glibenclamide and HMR1098 normalize Cantú syndrome-associated gain-of-function currents.

Cantú syndrome (CS) is caused by dominant gain-of-function mutation in ATP-dependent potassium channels. Cellular ATP concentrations regulate potassium current thereby coupling energy status with membrane excitability. No specific pharmacotherapeutic options are available to treat CS but IKATP channels are pharmaceutical targets in type II diabetes or cardiac arrhythmia treatment. We have been suggested that IKATP inhibitors, glibenclamide and HMR1098, normalize CS channels. IKATP in response to Mg-ATP, glibenclamide and HMR1098 were measured by inside-out patch-clamp electrophysiology. Results were interpreted in view of cryo-EM IKATP channel structures. Mg-ATP IC50 values of outward current were increased for D207E (0.71 ± 0.14 mmol/L), S1020P (1.83 ± 0.10), S1054Y (0.95 ± 0.06) and R1154Q (0.75 ± 0.13) channels compared to H60Y (0.14 ± 0.01) and wild-type (0.15 ± 0.01). HMR1098 dose-dependently inhibited S1020P and S1054Y channels in the presence of 0.15 mmol/L Mg-ATP, reaching, at 30 µmol/L, current levels displayed by wild-type and H60Y channels in the presence of 0.15 mmol/L Mg-ATP. Glibenclamide (10 µmol/L) induced similar normalization. S1054Y sensitivity to glibenclamide increases strongly at 0.5 mmol/L Mg-ATP compared to 0.15 mmol/L, in contrast to D207E and S1020P channels. Experimental findings agree with structural considerations. We conclude that CS channel activity can be normalized by existing drugs; however, complete normalization can be achieved at supraclinical concentrations only.

Increased tolerance to stress in cardiac expressed gain-of-function of adenosine triphosphate-sensitive potassium channel subunit Kir6.1.

BACKGROUND: The adenosine triphosphate-sensitive potassium (KATP) channel opener diazoxide (DZX) prevents myocyte volume derangement and reduced contractility secondary to stress. KATP channels are composed of pore-forming (Kir6.1 or Kir6.2) and regulatory (sulfonylurea receptor, SUR1 or SUR2) subunits. Gain of function (GOF) of Kir6.1 subunits has been implicated in cardiac pathology in Cantu syndrome in humans (cardiomegaly, lymphedema, and pericardial effusions). We hypothesized that GOF of Kir6.1 subunits would result in altered myocyte response to stress. MATERIALS AND METHODS: Isolated cardiac myocytes from wild type (WT) and transgenic Kir6.1GOF mice were exposed to Tyrode's physiologic solution for 20 min, test solution (Tyrode's or stress [hyperkalemic cardioplegia {CPG, known myocyte stress}] +/- KATP channel opener DZX), followed by Tyrode's for 20 min. Myocyte volume and contractility were measured and compared. RESULTS: WT myocytes demonstrated significant swelling in response to stress, but significantly less swelling was seen in Kir6.1GOF myocytes. DZX prevented swelling secondary to CPG in WT but resulted in a nonsignificant reduction in swelling in Kir6.1GOF myocytes. Both WT and Kir6.1GOF myocytes demonstrated a reduction in contractility during stress, although this was only significant in Kir6.1GOF myocytes. DZX was not associated with an improvement in contractility in Kir6.1GOF myocytes following stress. CONCLUSIONS: Similar to previous results in Kir6.1(-/-) myocytes, Kir6.1GOF myocytes demonstrate resistance (less volume derangement) to stress of cardioplegia. Understanding the role of Kir6.1 in myocyte response to stress may aid in the treatment of patients with Cantu syndrome and warrants further investigation.

Cantú syndrome resulting from activating mutation in the KCNJ8 gene.

ATP-sensitive potassium (KATP ) channels, composed of inward-rectifying potassium channel subunits (Kir6.1 and Kir6.2, encoded by KCNJ8 and KCNJ11, respectively) and regulatory sulfonylurea receptor (SUR1 and SUR2, encoded by ABCC8 and ABCC9, respectively), couple metabolism to excitability in multiple tissues. Mutations in ABCC9 cause Cantú syndrome (CS), a distinct multiorgan disease, potentially via enhanced KATP channel activity. We screened KCNJ8 in an ABCC9 mutation-negative patient who also exhibited clinical hallmarks of CS (hypertrichosis, macrosomia, macrocephaly, coarse facial appearance, cardiomegaly, and skeletal abnormalities). We identified a de novo missense mutation encoding Kir6.1[p.Cys176Ser] in the patient. Kir6.1[p.Cys176Ser] channels exhibited markedly higher activity than wild-type channels, as a result of reduced ATP sensitivity, whether coexpressed with SUR1 or SUR2A subunits. Our results identify a novel causal gene in CS, but also demonstrate that the cardinal features of the disease result from gain of KATP channel function, not from a Kir6-independent SUR2 function.

Aortic aneurysm and craniosynostosis in a family with Cantu syndrome.

Cantu syndrome is an autosomal dominant overgrowth syndrome associated with facial dysmorphism, congenital hypertrichosis, and cardiomegaly. Some affected individuals show bone undermodeling of variable severity. Recent investigations revealed that the disorder is caused by a mutation in ABCC9, encoding a regulatory SUR2 subunit of an ATP-sensitive potassium channel mainly expressed in cardiac and skeletal muscle as well as vascular smooth muscle. We report here on a Japanese family with this syndrome. An affected boy and his father had a novel missense mutation in ABCC9. Each patient had a coarse face and hypertrichosis. However, cardiomegaly was seen only in the boy, and macrosomia only in the father. Skeletal changes were not evident in either patient. Craniosynostosis in the boy and the development of aortic aneurysm in the father are previously undescribed associations with Cantu syndrome.