Kir6.1 and SUR2B in Cantú syndrome

Abstract

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?

FROM DRUGS, TO CURRENTS, TO GENES, NOW BACK

Over 60 years ago, in a New Jersey Schering Corporation research laboratory, a novel class of antihypertensive drug was discovered. This agent, a thiazide diuretic derivative, was notable in that it provoked blood pressure lowering effects without evidence of diuresis (1). Around the same time, researchers at The Upjohn Company in Kalamazoo, Michigan were themselves narrowing in on a new molecule capable of powerful vasodilation alongside an unexpected side effect of excessive hair growth (hypertrichosis). These discoveries, of diazoxide and minoxidil, respectively, heralded a new frontier for vasoactive drugs which, though it was not known at the time, exerted their blood pressure lowering effects via activation of ATP-sensitive potassium (K_ATP) channels. In the intervening decades, the discovery, first, of ATP-sensitive potassium channels in vascular smooth muscle (VSM), then of the genes that encode these channels, and, finally, of the human diseases which arise from mutation of these genes, brings us to today.

K_ATP channels are potassium selective ion channels regulated by intracellular nucleotides, including ATP and ADP (6). These channels form octameric complexes in which a tetrameric assembly of 4 pore-forming Kir6 subunits is surrounded by four regulatory SUR proteins (2–4). The Kir6 proteins are members of the inwardly rectifying potassium (Kir) channel subfamily but, unlike other Kirs, they coassemble with sulfonylurea receptor (SUR) subunits—the target of the sulfonylurea K_ATP channel inhibitors. SUR proteins are members of the ATP-binding cassette transporter family with no known transport function, which have been coopted as accessory proteins to regulate K_ATP channel function. Activity of the channel complex is determined by the intracellular [ATP]/[ADP] ratio and thus channels link cellular metabolism to electrical behaviors: ATP binds to Kir6 to inhibit channels (5) and, in the presence of physiological Mg²⁺, ATP and ADP bind to the nucleotide-binding domains of SUR to increase channel activity (6, 7).

There are two major isoforms of both Kir6 and SUR subunits (Fig. 1), encoded by paralogous genes arising from an evolutionary gene duplication event occurring sometime between Urbilateria and vertebrate divergence given that, for example, the invertebrate Drosophila melanogaster and the spider mite (Tetranychus urticae) genomes only contain 1 SUR homolog (8, 9). In humans the KCNJ8 and ABCC9 genes, which encode Kir6.1 and SUR2, respectively, are positioned adjacent to each other on chromosome 12, with the related, KCNJ11 and ABCC8, encoding Kir6.2 and SUR1, respectively, located on chromosome 11. SUR transcripts are subject to alternative splicing with resultant variants exhibiting differing functional properties (10–16). Of particular note, SUR2 undergoes alternative splicing to two major variants, SUR2A and SUR2B, which vary in their carboxy-terminal 42 amino acid exon. K_ATP channel subunits exhibit differential expression patterns resulting in distinct channel properties in different tissues, as extensively reviewed elsewhere (10, 15).

Figure 1.

Kir6.1/SUR2 channel genes and structure. A: K_ATP channel encoding genes, ABCC8 and KCNJ11 encoding Kir6.2 and SUR1 are located on human chromosome 11p15.1; ABCC9 and KCNJ8 are located on chromosome 12p12.q. ABCC9 transcripts are subject to differential splicing to SUR2A and SUR2B by alternative inclusion of terminal 42 amino acid exons. B: cartoon structures of Kir6.1, a membrane of the 2-transmembrane domain family of inwardly rectifying potassium (Kir) channels, and SUR2 a member of the ABC-transporter family.

Studies of knockout mice and recombinant channels indicate that the combination of Kir6.1 and SUR2B forms the predominant composition of K_ATP channels in vascular smooth muscle and that these channel complexes represent the major target for vasodilatory potassium channel openers (KCOs) including diazoxide, minoxidil, and pinacidil (17–20). These channels exhibit a number of key properties, which distinguish them from other K_ATP channels found in the pancreas (formed of Kir6.2/SUR1) or striated muscle (Kir6.2/SUR2A) in that Kir6.1/SUR2B channels are activated by both pinacidil and diazoxide, display low open probability in excised patches in the absence of Mg²⁺-nucleotides, and are only inhibited at high (millimolar) concentrations of ATP (21–23). ATP- and KCO-sensitive potassium currents with varying properties have been recorded in different vascular tissues from multiple species (as reviewed extensively in 22) which likely reflects some additional heterogeneity in the identity of the underlying channels (24, 25).

Kir6.1 AND SUR2B: FROM CLONING TO CHANNELOPATHY

KCNJ8 was first cloned by the group of Susumu Seino in 1995, and mRNA expression was reported in all primary tissues analyzed, leading to the suggestion that the subunit is ubiquitously expressed (11). However, since subsequent studies have shown that KCNJ8 is expressed in VSM and endothelial cells, the identification of the transcripts in any vascularized tissue may arise from the vessels within, and modern expression databases show weak or no Kir6.1 expression in many tissues (Fig. 2) (26, 27). The SUR2A and SUR2B splice variants were subsequently cloned and studies showed that the pharmacological properties of recombinant channels containing these subunits mirrored the native striated and smooth muscle K_ATP responses, respectively (10, 15).

Figure 2.

Tissue mRNA expression of KCNJ8. Adapted from the ARCHS4 web resource (https://maayanlab.cloud/archs4/gene/KCNJ8) (26) with permission.

Two decades after their cloning, the first definitive demonstration of the pathological consequences of mutations in KCNJ8 and ABCC9 in humans was provided by the identification of multiple missense mutations in both genes which converge in the same clinical presentation, Cantú syndrome (CS) (28–33). CS is a rare, heritable disorder characterized by hypertrichosis, distinct facial features, and a range of striking cardiovascular abnormalities, including decreased systemic vascular resistance and drastic cardiomegaly (34, 35). Extensive characterization has shown that CS mutations in both KCNJ8 and ABCC9 result in gain-of-function (GoF) of recombinant channels (28, 30,31, 36–38). Thus, 60 years after the discovery of KCOs that provoke vasodilation and excessive hair growth, activatory mutations in the genes encoding their target channels were found to lead to the same clinical outcomes. In fact, this link between excessive potassium channel activity and CS was, in a remarkable spot of clinical detective work, predicted years before the genetic basis was discovered, due to the striking overlap in features with the known adverse effects of KCOs (39).

With knowledge of the molecular effects of CS-associated mutations, recent efforts have been directed at determining how Kir6.1/SUR2 channel GoF causes the diverse, multiorgan pathophysiology observed in CS. These studies have clear relevance for attempts to counter CS pathophysiology, which is currently with no specific therapy but also have the potential to broaden our understanding of Kir6.1/SUR2 channel functions in diverse processes.

Kir6.1/SUR2B PARTNERS IN PHYSIOLOGY

Kir6.1/SUR2B channels are expressed in a range of tissues (Figs. 2 and 3). Much previous study has focused on channel function in VSM but, given the multiorgan abnormalities observed in CS, future studies could establish additional roles in diverse tissues. K_ATP channels, in general, serve to couple cellular metabolism to excitability (6). For instance, postprandial elevations in ATP generation in pancreatic β-cells lead to ATP-induced inhibition of Kir6.2/SUR1 channels, depolarization of the cell membrane, activation of voltage-gated calcium channels, and consequent insulin release (40). In striated muscle, Kir6.2/SUR2A channel activation in conditions of metabolic stress is critical for myo-protection (41). Activation of VSM K_ATP by hypoxia provides a clear potential link between metabolic stress and channel activity (22). However, unlike the Kir6.2-containing channels in β-cells and striated muscle, Kir6.1-containing K_ATP channels exhibit reduced sensitivity to intracellular ATP (21, 22). As smooth muscle Kir6.1/SUR2B channels are subject to regulation by a wide range of vaso-active signaling molecules and pathways [reviewed elsewhere (23, 42), and see Fig. 4], it is tempting to speculate that vascular smooth muscles channels are less strict metabolic sensors and more integrators of diverse vaso-regulatory molecules.

Figure 3.

Tissue mRNA expression of ABCC9. Adapted from the ARCHS4 web resource (https://maayanlab.cloud/archs4/gene/ABCC9) (26) with permission.

Figure 4.

Regulatory pathways for Kir6.1/SUR2B VSM K_ATP channels. K_ATP channels in VSM are activated by a range of mechanisms, including endothelial cell (EC)-derived nitric oxide (NO), and eicosanoids (EETs); Gαs-coupled receptors for adenosine, vasoactive intestinal peptide (VIP), GLP-1, and calcitonin gene-related peptide (CGRP), which increase channel activity via protein kinase A (PKA)-mediated phosphorylation of SUR2 and Kir6.1; lipid modulation including PIP2 and palmitoylation of Cys176; and metabolic regulation including increased intracellular ADP/ATP ratios, hypoxia, and acidification. Channels are inhibited by Gaq-coupled receptors for norepinephrine (NE), endothelin-1 (ET-1), angiotensin II (Ang II), vasopressin (AVP), serotonin (5-HT), histamine, and neuropeptide Y (NPY) via PKC phosphorylation of Kir6.1; increased intracellular ATP concentrations; and reactive oxygen species and superoxide involving glutathionylation (GSH) of Cys176 (23, 43–53). VSM, vascular smooth muscle.

Ultimately, activation of K_ATP in VSM results in membrane hyperpolarization, reduced voltage-gated calcium channel activity, and vasodilation (54). Global Kir6.1 and SUR2 knockout mice are hypertensive and exhibit coronary artery vasospasm (18, 19). Specific deletion of Kir6.1 in smooth muscle recapitulates a hypertensive phenotype, but with no indication of the coronary artery dysfunction observed in the global knockout (20), and specific expression of transgenic SUR2B in smooth muscle fails to rescue global SUR2 knockout mice from vasospasm (55). These findings may suggest a role for Kir6.1/SUR2 channels in the regulation of the coronary vasculature outside of smooth muscle, perhaps involving channels in the endothelium (56, 57), which is electrically coupled to smooth muscle and is the source of vaso-active molecules such as endothelin and nitric oxide. Endothelial K_ATP channels likely consist of Kir6.1 and Kir6.2 in complex with SUR2B and specific deletion of Kir6.1 in the endothelium results in increased vasoconstrictive ET-1 release and coronary perfusion pressure (56–58). Alternatively, a recent study suggested that coronary vasodilation results from activation of cardiomyocyte K_ATP channels, which is communicated to the microvasculature via gap junctions and elevations in extracellular K⁺ concentrations (59). Elsewhere, K_ATP channel activity is also critical for the vascular reactivity involved in functional hyperemia in skeletal muscle beds (22, 60–62), and recent studies demonstrate a role of endothelial and pericyte K_ATP channels, activated by extracellular adenosine and a purinergic receptor/PKA pathway, in cerebral blood flow and neurovascular coupling (63). Although details remain incomplete, this vast literature most clearly points to roles for K_ATP channels in general, and Kir6.1 and SUR2B proteins specifically, in the regulation of vascular tone.

Upregulation and activation of Kir6.1-containing K_ATP channels in vascular smooth muscle may partly underlie the drastic peripheral vasodilation and vascular hyporeactivity observed in septic shock (64–66), and the K_ATP channel inhibitor glibenclamide can reverse endotoxin-induced hypotension in canine and swine models (67, 68). These findings prompted trials of glibenclamide to treat septic shock in humans, but they failed to demonstrate clinical benefits, potentially attributable to differences between endotoxemia in animal models and septic shock in humans, to the relatively low doses used in clinical studies, or to differing routes of administration used in animal studies (69, 70). Interestingly, both mutant Kir6.1-null mice and flies in which Kir6.1 expression was knocked down by siRNA exhibit reduced survival in experimental models of endotoxemia (71, 72). These data suggest that Kir6.1-containing K_ATP channels may be protective in infection, perhaps due to critical VSM roles in regulating coronary perfusion (66). A recent study also reported increased NPRL3-inflammosome activation in Kir6.1-KO mice and increased IL-1β release from bone-derived macrophages, though this latter result was directly contradicted in other studies and, ultimately, there are, as yet, no clearly defined roles for Kir6.1 or SUR2 containing K_ATP channels in immune responses (73, 74).

In cardiac ventricular myocytes, Kir6.2 and the SUR2A splice variant comprise the major sarcolemmal K_ATP channels, and knockout of either gene abolishes K_ATP channel activity (75, 76). However, Kir6.1 is also expressed in ventricular myocytes according to immune-localization studies (77) and may play some currently underrecognized roles. Notably, unlike Kir6.2/SUR2A channels, Kir6.1/SUR2B channels do not typically show spontaneous activation on patch excision (21, 54), so inside-out patch clamp experiments may fail to identify these channels without careful experimental design. Beyond the scope of this review, there has been much confusion about the molecular identity—and even the fundamental existence—of a mitochondrial K_ATP channel, and Kir6.1 and SUR2 proteins have been suggested to underlie such channels at various times, though this now appears unlikely to be the case (18, 78, 79).

There are compelling data supporting a distinct K_ATP channel make-up in the specialized cardiac conduction system (CCS). CCS K_ATP channels display quite distinct properties from ventricular cardiomyocyte channels, including reduced inhibition by cytosolic ATP, lower single channel conductances, and increased sensitivity to diazoxide activation (80). These properties are consistent with CCS channels containing Kir6.1 and SUR2B, and Kir6.1 knockout is associated atrioventricular (AV) block (18, 81). Sinoatrial node (SAN) cells also express K_ATP channels with lower single channel conductances than ventricular myocyte channels (82). Conditional knockout of Kir6.1 in SAN cells resulted in the loss of tolbutamide-sensitive K_ATP currents in a subset of cells (81), but K_ATP currents in the SAN are also reduced in global Kir6.2 knockout, so it is possible that channels are mixtures of Kir6.1, Kir6.2, SUR2, and SUR1 subunits, at least in mice (83, 84). K_ATP activation by hypoxia and metabolic poisoning can cause bradycardia and AV block (83, 85), yet intriguingly deletion of Kir6.1 in SAN also results in bradycardia, alongside action potential prolongation (81). Thus both activation and loss of K_ATP can induce bradycardia and conduction abnormalities, where acute activation can provoke maximal diastolic hyperpolarization, and loss of Kir6.1-currents prolongs the action potential, both with bradycardic outcomes.

Kir6.1- and SUR2-containing K_ATP channels are also found elsewhere, including in the dermal papilla and sheath of human hair follicles where they may act to control cell proliferation and hair growth (86–88) and in microglia, astrocytes, and neurons in the brain (89). Potassium channels sensitive to ATP, KCOs, and sulfonylurea inhibitors, which regulate salt and fluid reabsorption, are found in renal epithelial cells, but these channels are formed by Kir1.1 and CFTR complexes (90–94). Kir6.1 and SUR2B are reportedly expressed in both renal and alveolar epithelial cells but unequivocal evidence for a physiological role of Kir6.1/SUR2B channels, as would be provided from gene knockout mice, is, to our knowledge, still lacking (95–98).

Kir6.1/SUR2B GoF MUTATIONS IN CS

Although the pathological effects of mutations in KCNJ11 (Kir6.2) and ABCC8 (SUR1) were first recognized >25 years ago (99–103), we are just beginning to dissect the complex consequences of Kir6.1/SUR2 dysfunction in human disease. A major breakthrough was provided by the identification of multiple missense variants in ABCC9 in a number of patients diagnosed with Cantú syndrome (28, 29). This was followed soon after by identification of additional patients with CS with heterozygous variants in KCNJ8 (31, 33). Analysis of recombinant channels showed that CS-associated amino acid substitutions result in K_ATP channel gain-of-function (GoF) in each case (28, 30, 31, 36–38). Suddenly, it became clear that overactivity of Kir6.1 and SUR2-containing K_ATP channels converged in a genetically defined clinical constellation.

The growing evidence supporting a causal effect of GoF mutations in both Kir6.1 and SUR2 in CS casts doubt on previous associations of KCNJ8 variants with cardiac electrophysiological defects such as atrial and ventricular fibrillation, and Brugada-, J-wave, and early repolarization syndromes (as reviewed recently in 105). For example, Kir6.1 [S422L] was reported to cause GoF of recombinant channels and was associated with Brugada syndrome (106–108), but subsequent studies have failed to confirm GoF in recombinant channels (31), or in mouse cardiomyocytes overexpressing mutant transgenes (109), and this variant is found in 4% of Ashkenazi Jews, showing it is not likely to have a highly penetrant pathological effect. Perhaps a more compelling issue is that if Kir6.1 [S422L] does cause GoF of K_ATP channels, why does it not cause CS? Cell- or tissue-specific effects of the mutation are possible but have not been demonstrated. Similarly curiously, a number of reportedly GoF mutations in ABCC9, not found in CS individuals, were recently identified in sudden unexpected death (110), potentially caused by K_ATP-dependent cardiac electrophysiological derangements. Loss-of-function mutations in KCNJ8 and ABCC9 are associated with sudden infant death syndrome and ABCC9-related intellectual disability and myopathy syndrome (AIMS), respectively (111, 112).

CS (OMIM 239850) was first described in 1982 by the eponymous Dr. J. M. Cantú and colleagues who identified four individuals in Mexico with generalized hypertrichosis, macrosomia at birth, cardiomegaly, and skeletal abnormalities (35). This initial report suggested a recessive inheritance pattern, as the features were observed in two siblings but not their parents. We now understand that the disorder is inherited in an autosomal dominant manner, but the severity of features can vary significantly, even within families, which can obscure diagnosis without genetic evidence (34, 113). Distinct classifications of acromegaloid facial appearance syndrome (AFA; OMIM 102150) and hypertrichosis with acromegaloid facial features (HAFF; OMIM 135400) have been made for some ABCC9 mutations, but these conditions are likely part of the CS spectrum (113, 114). An international registry of patients with CS (34), hosted at Washington University in St. Louis, now includes >90 genetically confirmed individuals. Although CS is certainly a rare disease, the lack of knowledge of the disorder and missed (or mis-) diagnosis likely mean that this tally is a severe underestimate. Anecdotally, we know of six individuals with CS in the greater St. Louis area (population: 3 million), a frequency that would extrapolate to ∼ 15,000 people worldwide. Commonly reported misdiagnoses include lysosomal storage disorders, Beckwith–Weidemann syndrome (due to coarse facial features), or Pompe disease (based on neonatal cardiomegaly) (34). Fascinatingly, there is also some clinical overlap with a growing number of other GoF potassium channelopathies, including developmental delay, hypotonia, craniofacial dysmorphology, skeletal abnormalities, and hypertrichosis in FHEIG (KCNK4 mutations) and Zimmerman–Laband syndrome (KCNN3, KCNH1 mutations), which may further confound diagnosis but could point to common electrophysiological mechanisms (115–119).

In the initial studies, a small number of individuals with “CS” were reported without mutations in ABCC9 or KCNJ8 (28, 29, 31). Whether this means additional genes can also cause the identical syndrome or whether the unaccounted-for individuals actually have a different disorder is not known. How to classify a “disease” is a slippery problem, but we suggest that there is now sufficient evidence to state that GoF mutations in either ABCC9 or KCNJ8 are definitive criteria for CS diagnosis. Currently, the features observed in ABCC9- and KCNJ8-associated CS appear to be conserved which points to abnormalities tracing back to tissues where expression of these two subunits overlaps.

THE MOLECULAR BASIS OF CS

In 2017, K_ATP channels were catapulted into the “Resolution Revolution,” as three groups revealed cryo-EM structures of the pancreatic (Kir6.2/SUR1) channel (120–123). These structures provided long-sought molecular snapshots of the K_ATP channel complex and, in the process, confirmed many postulations from decades of detailed structure-function studies, including the structure of the cytoplasmic inhibitory ATP binding site on Kir6.2 and its close proximity with residues involved in PIP2 binding; the nucleotide-binding domains of SUR1 in both dimerized (120) and undimerized states; and the binding site of the antidiabetic K_ATP channel inhibitor, glibenclamide (glyburide; GLIB) (123). Most recently, the Shyng group has published the first cryo-EM structures of Kir6.1/SUR2B channels (Fig. 5) (124).

Figure 5.

Kir6.1/SUR2B structural features. A: top, cartoon of Kir6.1/SUR2B proteins color coded according to cryo-EM model (bottom). The “propeller” structure (pdb: 7MIT) (124) shows the Kir6.1 ATP binding sire (ATP shown as spheres), the Kir6.1 N-terminus (yellow), Kir6.1 C-terminus region implicated in PNU-37883 inhibition (shown as blue sticks), glibenclamide binding site (solid teal oval), and residues in TMD2 implicated in KCO binding (dashed blue circle) (125, 126). CS mutation sites in Kir6.1 (labeled) and throughout SUR2 shown as spheres. The Kir6.1 tetramer is shown alongside a SUR2B monomer (3 SUR2B subunits omitted for simplicity). B: top, primary amino-acid sequence of the extracellular “turret” region of Kir6.1 and Kir6.2. Bottom, unique turret region of Kir6.1 highlighted in orange. C: close-up view of glibenclamide binding site showing close apposition of Tyr1209 (pdb: 7MJO; human numbering; equivalent of Y1205 in rat clone) (124).

Several new concepts for the structural basis of K_ATP channel gating emerge from these structures. First, the N-terminus of Kir6 subunits is seen to bind within the vestibule of SUR subunits when the NBDs are not dimerized, constraining rotational movement of the Kir6.2 cytoplasmic domain, which may inhibit channel opening and stabilize the binding of inhibitors (123, 127, 128), a concept supported by previous functional studies that showed that N-terminal deletions in Kir6.2 reduced the effects of inhibitors (129–131). Second, major rigid-body rotational movements of the TMD1 and TMD2 regions of SUR can occur that 1) disrupt the interface between Kir6 and the L0 linker in SUR (120, 124) and 2) promote an interaction between the C-terminus of Kir6 and the NBD domain of the SUR subunit (in a dynamic tripartite interaction also involving the NBD1-TMD2 linker), consistent with functional studies of these regions (120, 124, 132, 133). This movement might represent a transduction pathway for Mg²⁺-nucleotide activation of the channel gate, which, it is suggested, could ultimately involve upward and rotational movements of the Kir6 cytoplasmic domain, and a widening of the inner helix gate—as shown in a recent “open” structure of Kir6.2/SUR1 complexes that include two gain-of-function mutations (124, 134), the Kir6.2 ATP binding site G334D mutation, and the open-state stability C166S mutation (analogous to the C176S Cantú syndrome mutation in Kir6.1). This structure reveals an increase in the ion pathway radius at the “inner helix gate,” from wild type (WT) (∼1 Å) to mutant (∼3.3 Å), potentially sufficient to allow partially hydrated K⁺ ions to pass. There was also a distortion of the inhibitory ATP binding site on Kir6.2 (134), potentially explaining why ATP preferentially binds to closed channels (135). As with all good structural studies, as many questions are provided as answers and these new ideas require additional functional studies to further enhance our understanding of the complex gating of K_ATP.

Functional analysis of recombinant K_ATP channels shows that CS-associated mutations have GoF effects via multiple mechanisms: some mutations act to increase the unliganded open probability of channels and hence reduce ATP inhibition allosterically, whereas other mutations augment the activatory effects of Mg²⁺-nucleotides binding to SUR2 (30, 36, 37, 136). Examples of the former mechanism included the Kir6.1 [V65M] and Kir6.1 [C176S] substitutions (which are both located close to the inner helix gate) and the SUR2 [D207E] substitution (30, 31, 37). A third Kir6.1 variant E189K is located in the intracellular C-terminal domain. The equivalent residue in Kir6.2 (E179) appears to interact with Kir6.2 [R176] in a short helical structure and may contribute to stabilizing the PIP2 and ATP binding site, though this interaction is missing in the Kir6.1/SUR2B structure (123, 124). Substitutions of Kir6.2 [E179] can reduce ATP inhibition both with and without effects on open probability (137). Most recently, a Kir6.1 [E332K] mutation has been identified in a CS infant and requires functional characterization (32). Variants in SUR2 are spread throughout the protein, in transmembrane and nucleotide-binding domains, and the L0-linker (Fig. 5). As noted by Sung and colleagues, multiple mutations cluster in TMD2 including a number on TM12 which follows the NBD1-TMD2 linker implicated in Mg²⁺-nucleotide activation, and the neighboring TM13-15 (124) (Fig. 5). To date, all mutations in SUR2 are found in the region common to both SUR2A and SUR2B. Given that much of the pathophysiology in CS appears to be traced back to SUR2B-containing tissues (see below), it will be interesting to see whether any CS-associated mutations appear in either of the terminal exons that define SUR2A and SUR2B.

THE PATHOPHYSIOLOGICAL CONSEQUENCES OF CS

Presentation of the diverse features of CS varies among affected individuals, but consistent findings are distinctive facies, hypertrichosis, and decreased systemic vascular resistance (SVR) alongside high-output cardiomegaly (34) (see also Fig. 6). Additional common cardiovascular abnormalities include aortic root dilation, tortuous vessels with persistence of fetal vessels, including bronchoaortal collaterals and patent ductus arteriosus (PDA), pulmonary hypertension (PH), edema, and pericardial effusion. Some of these, such as chronically decreased SVR, edema, and PH, are known effects of K_ATP overactivity induced by KCOs, but other features—including the drastic cardiomegaly observed—are not trivially predicted. How Kir6.1/SUR2 channel overactivity results in many features of CS, such as craniofacial dysmorphology, skeletal abnormalities, joint hyperflexibility, and immunological dysfunction, are also unexplained.

Figure 6.

CS-associated pathologies. A: multisystem pathologies observed in CS (images adapted from https://bioicons.com). Working models for cardiovascular (B) and GI (C) abnormalities in CS arising from GoF of vascular (B) or GI (C) smooth muscle K_ATP channels, and reversal by sulfonylureas (138, 139). CS, Cantú syndrome; GI, gastrointestinal tract; GoF, gain-of-function.

To provide answers, we have begun to dissect CS pathophysiology through investigations of novel murine and zebrafish models of CS in which CRISPR/Cas9 genome engineering was used to introduce human disease-associated mutations into ABCC9 and KCNJ8 (140–142). SUR2 [A478V], SUR2 [R1154Q], and Kir6.1 [V65M] mutant mice (using human residue numbering) and SUR2 [C1043Y], SUR2 [G989E], and Kir6.1 [V65M] fish recapitulate key cardiovascular features of CS, including cardiomegaly. The severity of the cardiac remodeling correlates with magnitude of the molecular effects of mutations in recombinant channels and vascular smooth muscle cells and was most severe in both Kir6.1 [V65M] mice and fish (30, 31, 36, 138, 140, 141, 143, 144). Notably, although SUR2 mutations provoke GoF of the predominant cardiac K_ATP channels in excised patch clamp experiments, Kir6.1 mutations do not—consistent with the established prominence of Kir6.2 as the pore-forming subunit in cardiomyocytes. Importantly, however, both SUR2- and Kir6.1-mutant mouse lines, as well as humans with both KCNJ8 and ABCC9 mutations, exhibit cardiomegaly (105, 140). Significantly, GoF mutations in Kir6.2 (expressed in cardiomyocytes and pancreatic β cells) cause neonatal diabetes, which is not typically associated with cardiac hypertrophy.

The earlier findings suggest that remodeling to the high-output hypertrophic cardiac state arises secondary to K_ATP GoF outside of the heart, likely acting as a compensation for chronically low SVR. Given that transgenic overexpression of GoF Kir6 subunits in smooth muscle results in chronic vasodilation (54), we hypothesized that K_ATP GoF induced by CS-associated mutations in both Kir6.1 and SUR2 would converge in chronic VSM hypoexcitability and vasodilation, and in turn provoke cardiac remodeling. To test this, we crossed SUR2 [A478V] mice with transgenic mice expressing dominant-negative pore mutant (Kir6.1-AAA; SM-DN) subunits specifically in smooth muscle and showed functional knockdown was sufficient to markedly reverse cardiac hypertrophy (138). Further study demonstrated that CS mice exhibit upregulation of renin-angiotensin signaling (RAS), pharmacological blockade of which reverses cardiac hypertrophy (143). This link between genetic K_ATP GoF and cardiac remodeling via RAS upregulation echoes the previously reported finding that chronic administration of the KCO minoxidil in rats leads to cardiac remodeling via RAS upregulation (145, 146). These studies indicate that cardiac remodeling in CS arises secondary to VSM K_ATP GoF and chronic vasodilation, which triggers sustained RAS elevation, driving blood volume expansion and hypertrophic remodeling (Fig. 6B). Significantly, even though basal cardiac output is elevated in CS mice and human subjects, exercise intolerance is also observed (34, 143). Although primary skeletal muscle fatigability or pathology cannot be ruled out (147, 148), this may reflect reduced cardiac reserve and requires further investigation.

VSM K_ATP GoF appears necessary for the full cardiac remodeling observed in Cantú mice, but transgenic expression of GoF Kir6.1 mutant subunits specifically in smooth muscle provokes only relatively mild cardiac changes (149). This may point to additional roles for K_ATP outside of contractile VSM in cardiac CS pathology. A possible candidate tissue is the kidney, where 1) K_ATP activation in renal epithelial cells could promote sodium reabsorption and 2) K_ATP activation has been shown to promote renin-secretion from specialized juxtaglomerular cells (150, 151). These actions may compound systemic RAS feedback triggered by low SVR and further expand blood volume, leading to cardiac dilation and hypertrophy. Pulmonary hypertension in CS appears paradoxical, as K_ATP channel activation in pulmonary vascular smooth muscle provokes vasodilation (22, 152) but may be explained by volume overload of the pulmonary vasculature, left-heart structural abnormalities, bronchopulmonary dysplasia due to prematurity, or PDA in certain CS individuals (153).

Functional Kir6.1 and SUR2 expression also overlap in visceral smooth muscle, including multiple regions of the gastrointestinal (GI) tract, and patients with CS commonly report GI dysmotility, which can seriously impact quality of life (34, 139). In Cantú mice, intercrossing heterozygous Kir6.1 [V65M] mice yields homozygous pups, which die shortly after weaning to solid food, but death can be prevented by weaning onto a gel-diet (139). Postmortem study of solid-diet-fed homozygous mice reveals intestinal blockage and distension suggestive of GI dysmotility. K_ATP channels in the GI system likely display low basal activity, as glibenclamide administration has no major effect on tension in isolated intestinal rings from WT mice, but it does increase basal contractility in rings from Cantú mice, and can restore GI motility in the Kir6.1 [V65M] GI tract (139). Moreover, sharp electrode recordings from intestinal segments from SUR2 [478V] and Kir6.1 [V65M] mice revealed marked hyperpolarization of smooth muscle cells, and in the more severe Kir6.1 [V65M] mice, essentially abolished slow-wave amplitude without significant effect on slow-wave frequency (139). These findings are consistent with CS mutations impacting GI smooth muscle (but not the GI pacemaker system comprised of Interstitial Cells of Cajal) to impair motility (Fig. 6C).

The study of genetically defined rare diseases may establish new biomedical paradigms. These mouse studies show that smooth muscle K_ATP GoF underlies both CV and GI abnormalities in CS. It is tempting to speculate that CS may represent a cardinal example of the pathological effect of smooth muscle hypoexcitability and that other molecular dysfunctions leading to similar electrical consequences (i.e., chronic membrane hyperpolarization) might lead to similar pathologies, such as high-output heart failure and patent ductus arteriosus (143, 154). VSM contractility is regulated by myriad ion channels and perhaps genetic or acquired dysfunction in any could lead to high-output hypertrophic cardiac remodeling. Perhaps SM K_ATP activity in the GI system is increased, contributing to dysmotility, in other pathological states. A new role for K_ATP channels in lymphatic smooth muscle is also emerging, as described elsewhere in this edition by Davis and Nichols. Abnormalities in other smooth muscle-lined tissues, such as the respiratory tract, point to the unexplored importance of K_ATP function in development.

As mentioned earlier, the hair-growth-promoting properties of minoxidil were clear from initial development. Today minoxidil is used as a vasodilator for resistant hypertension, but only as a last line, due in part to the coincident hirsutism (155). This “side effect” was itself soon promoted as a desired effect, however, and the drug was marketed in over-the-counter hair growth formulas. Of concern, if excessive doses are used, even topically, there is the potential for minoxidil to have systemic effects—precipitating a possible “drug-induced Cantú syndrome.” Indeed PH, edema, coarsening of facial features, and even reopening of the ductus arteriosus are associated with the Kir6.1/SUR2B active KCOs minoxidil and diazoxide (156–158). Multiple KCOs induce and prolong anagen, the rapidly dividing stage of hair, in cultured follicles to promote growth, which can be reversed by the K_ATP inhibitor tolbutamide (86–88, 159). It is possible that CS-associated mutations have the same hair growth cycle effects, though this requires clarification. Thinking more broadly about the electrical consequences, K_ATP activation versus voltage-gated calcium channel (VGCC) activation would be expected to have opposing electrophysiological effects, and interestingly Timothy syndrome, caused by gain-of-function mutations in the VGCC, Ca_V1.2 is associated with baldness at birth (160), potentially the reverse mechanistic phenomenon.

Other features commonly observed in CS individuals include migraines, skeletal abnormalities, and arrhythmias. In the case of neurological features, it is not known whether there are primary central nervous system defects. It is possible that Kir6.1/SUR2 mutations evoke altered neurovascular coupling, or even altered glymphatic functions, which may have secondary neurological impacts. A key role or K_ATP channels in the cerebral vasculature is emerging, where channels in the brain capillary endothelium and in pericytes have been shown to be activated by extracellular adenosine, a vasodilatory mediator released from firing neurons in a mechanism which serves to couple increased blood flow to active brain regions (63). Patients with CS exhibit increased basal cerebral blood flow and marked dilation of the large cerebral vessels (161), and we hypothesize that this may be associated with reduced dynamic control in NVC. Intriguingly, a high prevalence of white matter hyperintensities is reported in cerebral MRI imaging of both patients with CS and AIMS (161), which might reflect inadequate blood flow management and increased incidence of local ischemia. In a series of studies on human volunteers, the powerful migraine-inducing effects of KCOs have been demonstrated (162–164). These studies suggest that K_ATP overactivity is a substrate of migraine formation, which may be caused by dilation of cerebral arteries and acutely increased cerebral blood flow. Cantú mice provide a powerful tool for better understanding of the roles of K_ATP in the cerebral vasculature, and future studies might help to dissect the pathological role of K_ATP dysfunction both in CS and broader diseases.

Early reports on individuals with CS described a range of skeletal and joint abnormalities, including osteopenia, thickening of the calvaria, pectus carinatum, scoliosis, Erlenmeyer-flask-like bones, and hyperflexible joints (34, 35). Knowledge about the roles of K_ATP channels in bone and joint tissues is scarce, though pharmacological effects of K_ATP channel modulators have been shown in cell lines and human chrondrocytes (165–167). Skeletal abnormalities are also observed in AIMS and dissecting roles for K_ATP in diverse musculoskeletal tissues promises to be a fruitful direction for future research (112).

Subsets of individuals with CS have been subject to electrocardiogram monitoring revealing a high incidence of atrioventricular (AV) block, whereas arrhythmia diagnoses were self-reported in 18% of subjects in the International Cantú Syndrome Registry (ICSR) (34, 149). The nature, extent, and mechanistic basis of CS-associated cardiac arrhythmia require elucidation. It is possible that arrhythmias could arise from direct electrical effects of K_ATP channel activation in the heart due to proarrhythmic action potential shortening, but no QT shortening is observed in humans or Cantú mice (140, 149). Mice with transgenic GoF K_ATP subunits display augmented l-type calcium channel activity, which may compensate for K_ATP activation but predispose to arrhythmia (149, 168).

Due to low patient numbers, a full picture of the CS’s natural history is still lacking. Importantly, however, in the ICSR, four female subjects (all over 45 yr of age) have been diagnosed with high-output heart failure, suggesting that long-term cardiac health is impaired (G. Singh, personal correspondence). There are few registered older male individuals with CS, which may indicate worsened survival but might also be explained by decreased identification of male subjects. Most registered individuals are children or young adults, which might also reflect impaired survival or increased diagnosis, widespread genetic testing, and improved modern perinatal care.

THERAPEUTIC POSSIBILITIES IN CS

There is currently no specific therapy for CS. There have been multiple incidences of individuals with CS being treated with conventional therapies for cardiomyopathy or heart failure, including diuretics and RAS inhibitors. Based on our current understanding of CS pathophysiology, we predict that RAS inhibitors may well reverse cardiac hypertrophy, but in doing so, will essentially decompensate the CV system and exacerbate the primary pathological decreases in SVR (Fig. 6B). Strikingly, angiotensin-converting enzyme inhibitors have been associated with orthopnea and worsened CS symptoms in multiple individuals and should likely be avoided (G. Singh, personal correspondence). Clearly, targeted therapies for CS are desirable.

A range of K_ATP channel inhibiting drugs has been developed as antidiabetic therapies, due to their action on pancreatic K_ATP channels. Importantly, some, such as the sulfonylurea glibenclamide, also inhibit cardiovascular K_ATP channels, suggesting they may be repurposed for treating CS (169). Recently, we demonstrated that a 4-wk treatment of GLIB, administered through subcutaneous, slow-release pellets, resulted in reversal of the lowered systemic vascular resistance and cardiac hypertrophy observed in heterozygous SUR2 [A478V] mice (138). Importantly, very high doses of glibenclamide (∼19 mg/kg/day) were required. Moderately high doses of 1 mg/kg/day, close to the highest doses administered to patients with neonatal diabetes (170), were without effect on CV pathophysiology in the Cantú mice. The high-dose requirement likely reflects the low sensitivity of VSM Kir6.1/SUR2B K_ATP channels for sulfonylureas and will inevitably lead to inhibition of pancreatic Kir6.2/SUR1 K_ATP channels. The new K_ATP channel structures reveal the basis for this different sensitivity: GLIB binds within the core transmembrane region of SURs, between transmembrane helices (TM) 7, 8, 11, 15, and 17, and in close proximity with S1238 in SUR1 (on TM16). The equivalent residue in SUR2 is the more bulky Y1205 (rat numbering) which causes a steric slash with GLIB (Fig. 5C) (124). Previous studies have shown that substituting these residues can switch sensitivity between the high-affinity SUR1 and low-affinity SUR2 (171, 172).

In mice, we showed that even these high doses of glibenclamide provoke only transient, and spontaneously resolving, decreases in blood glucose (138), consistent with previous studies (173). The transient nature of the blood glucose decrease likely arises from downregulation of insulin secretion despite chronic β-cell depolarization (138). Whether a similar process occurs in humans is not known, but some pancreatic effects are likely unavoidable if GLIB is repurposed for CS, and blood glucose will require careful monitoring. It is possible that blood glucose lowering could be mitigated by coadministration of GLIB with octreotide, but this requires preclinical testing. Although GLIB normalized cardiac size and blood pressure (BP) in SUR2 [A478V] mice, GLIB had only a partial effect on blood pressure, and failed to reverse cardiac phenotypes, in Kir6.1 [V65M] mice (138). The V65M substitution, near to the inner helix gate, markedly increases channel open-state stability, decoupling SUR-dependent regulation of the gate and reducing channel sensitivity to GLIB (30). In addition, the severity of the phenotype in the Kir6.1[V65M] mice, due to the strong GoF nature of the mutation, may make abnormalities resistant or slow to reverse.

In the future, drugs that selectively target Kir6.1/SUR2B channels (i.e., the smooth muscle subunits), over Kir6.2- or SUR1-containing K_ATP channels would be desirable for the treatment of CS and avoid pancreatic effects. Certain known drugs have been shown to demonstrate favorable selectivity for Kir6.1/SUR2B channels, including the antidiabetic biguanide, phenformin, which has minimal effects on Kir6.2/SUR2A channels, and the morpholinoguanidine, PNU-37883 (174, 175). Both these inhibitors appear to act via the Kir6 subunits, with the C-terminus of Kir6.1, in particular, critical for PNU-37883 (174–176). Phenformin inhibition is low potency and canine experiments reveal a poor therapeutic window for PNU-37883 with cardiotoxicity limiting its use (177), but these molecules may provide starting points for optimization. An ideal drug would also be unaffected by CS gating mutations, which might require a pore-blocking mechanism of action. Interestingly, the unique Kir6.1 extracellular “turret” region was resolved in the recent cryo-EM structure (124) (Fig. 5B). This turret motif might represent a targetable domain for Kir6.1 selective modulators, potentially including peptide- or toxin-based approaches, analogous to a recently discovered Kir6.2 toxin (178). With new knowledge of the Kir6.1/SUR2B structure, a new era of precision K_ATP pharmacology beckons in which structural biology and in silico approaches can be leveraged to identify selective modulators that can in turn be tested for efficacy and selectivity in mouse and zebrafish Cantú models.

However, translation of preclinical pharmacology to patients will require resolving a number of significant complications. It is not known whether efficacious doses of sulfonylureas can be reached in humans without adverse effects on pancreatic (Kir6.2/SUR1) channels or cardiac/skeletal muscle (Kir6.2/SUR2A) K_ATP channels, or other mechanisms. Acute hypoglycemic glibenclamide poisoning has been reported at doses as low as 2.5 mg (179). Whether gradual increments in dose can lead to higher tolerance remains to be established and any such approach will require care. An additional conceptual challenge is posed for the cardiologist: in CS, cardiac hypertrophy is generally associated with enhanced ventricular contractility and fractional shortening. Reversal of the disorder would thus be expected to involve reduction in cardiac contractility and output—a highly undesirable effect in classic cardiomegaly but one which in CS might be an indication of clinical efficacy. Large-scale clinical studies should include careful assessment of plasma drug concentrations, blood glucose, and vascular (including measurements of SVR) and cardiac responses. Glibenclamide (0.15 mg/kg/day) has been trialed in a single CS infant exhibiting PDA, pulmonary hypertension, and right ventricle hypertrophy, who required bilevel positive airway pressure (BiPAP) therapy (136). Treatment coincided with improved pulmonary function and reduced edema (136), and only mild, transient, clinically insignificant hypoglycemia. Hypertrichosis was unaffected at the time of reporting even after ∼7 mo of the maximal dose, though this may not be too surprising as K_ATP inhibition could only be expected to inhibit new hair growth and not enhance hair loss. To quote Aristotle, “one swallow does not a summer make,” but this child’s case is an encouraging sign that a meaningful treatment for this complex condition may not be too far away.

CONCLUSIONS

The history of research on Kir6.1/SUR2 channels has led from studies of the effects of drugs on systemic physiology, to the identification of native currents, the cloning of the Kir6.1/SUR2 genes and detailed characterization of recombinant proteins, to investigations of physiological roles in animal models, atomic insights into channel structures, and to human genetic disease requiring targeted therapy. Ongoing investigations of Cantú syndrome promise to reveal new mechanisms in diverse pathologies, including the cardiovascular, musculoskeletal, and neurovascular systems, driven by electrical abnormalities. Demonstration of the clinical safety and efficacy of repurposed sulfonylureas would represent a major milestone for this orphan disease, but future identification of more targeted therapies may provide significant benefits for subjects with CS specifically, and potentially find broader use in more common diseases arising from electrical dysfunction.

GRANTS

This work was supported by NIH grant (R35 HL140024, to CGN). CM was supported by American Heart Association Postdoctoral Fellowship 19POST34380407 and NIH Award K99 HL150277.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

C.M. and C.G.N. drafted manuscript; C.M. and C.G.N. edited and revised manuscript; C.M. and C.G.N. approved final version of manuscript.