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.

Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells.

The Ca2+-activated Cl- channel regulator CLCA1 potentiates the activity of the Ca2+-activated Cl- channel (CaCC) TMEM16A by directly engaging the channel at the cell surface, inhibiting its reinternalization and increasing Ca2+-dependent Cl- current (ICaCC) 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 ICaCC 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 ICaCC 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.

Novel loss-of-function variants expand ABCC9-related intellectual disability and myopathy syndrome.

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.

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.

Rapid Characterization of the Functional and Pharmacological Consequences of Cantú Syndrome KATP Channel Mutations in Intact Cells.

Gain-of-function of KATP 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 (KATP) 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 KATP 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 KATP 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 KATP channel activity under basal, non KCO-activated conditions.

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.

Potassium channel selectivity filter dynamics revealed by single-molecule FRET.

Potassium (K) channels exhibit exquisite selectivity for conduction of K+ ions over other cations, particularly Na+. High-resolution structures reveal an archetypal selectivity filter (SF) conformation in which dehydrated K+ ions, but not Na+ ions, are perfectly coordinated. Using single-molecule FRET (smFRET), we show that the SF-forming loop (SF-loop) in KirBac1.1 transitions between constrained and dilated conformations as a function of ion concentration. The constrained conformation, essential for selective K+ permeability, is stabilized by K+ but not Na+ ions. Mutations that render channels nonselective result in dilated and dynamically unstable conformations, independent of the permeant ion. Further, while wild-type KirBac1.1 channels are K+ selective in physiological conditions, Na+ permeates in the absence of K+. Moreover, whereas K+ gradients preferentially support 86Rb+ fluxes, Na+ gradients preferentially support 22Na+ fluxes. This suggests differential ion selectivity in constrained versus dilated states, potentially providing a structural basis for this anomalous mole fraction effect.

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.

Polyamines and potassium channels: A 25-year romance.

Potassium channels that exhibit the property of inward rectification (Kir channels) are present in most cells. Cloning of the first Kir channel genes 25 years ago led to recognition that inward rectification is a consequence of voltage-dependent block by cytoplasmic polyamines, which are also ubiquitously present in animal cells. Upon cellular depolarization, these polycationic metabolites enter the Kir channel pore from the intracellular side, blocking the movement of K+ ions through the channel. As a consequence, high K+ conductance at rest can provide very stable negative resting potentials, but polyamine-mediated blockade at depolarized potentials ensures, for instance, the long plateau phase of the cardiac action potential, an essential feature for a stable cardiac rhythm. Despite much investigation of the polyamine block, where exactly polyamines get to within the Kir channel pore and how the steep voltage dependence arises remain unclear. This Minireview will summarize current understanding of the relevance and molecular mechanisms of polyamine block and offer some ideas to try to help resolve the fundamental issue of the voltage dependence of polyamine block.

Cantu syndrome-associated SUR2 (ABCC9) mutations in distinct structural domains result in KATP channel gain-of-function by differential mechanisms.

The complex disorder Cantu syndrome (CS) arises from gain-of-function mutations in either KCNJ8 or ABCC9, the genes encoding the Kir6.1 and SUR2 subunits of ATP-sensitive potassium (KATP) channels, respectively. Recent reports indicate that such mutations can increase channel activity by multiple molecular mechanisms. In this study, we determined the mechanism by which KATP function is altered by several substitutions in distinct structural domains of SUR2: D207E in the intracellular L0-linker and Y985S, G989E, M1060I, and R1154Q/R1154W in TMD2. We engineered substitutions at their equivalent positions in rat SUR2A (D207E, Y981S, G985E, M1056I, and R1150Q/R1150W) and investigated functional consequences using macroscopic rubidium (86Rb+) efflux assays and patch-clamp electrophysiology. Our results indicate that D207E increases KATP channel activity by increasing intrinsic stability of the open state, whereas the cluster of Y981S/G985E/M1056I substitutions, as well as R1150Q/R1150W, augmented Mg-nucleotide activation. We also tested the responses of these channel variants to inhibition by the sulfonylurea drug glibenclamide, a potential pharmacotherapy for CS. None of the D207E, Y981S, G985E, or M1056I substitutions had a significant effect on glibenclamide sensitivity. However, Gln and Trp substitution at Arg-1150 significantly decreased glibenclamide potency. In summary, these results provide additional confirmation that mutations in CS-associated SUR2 mutations result in KATP gain-of-function. They help link CS genotypes to phenotypes and shed light on the underlying molecular mechanisms, including consequences for inhibitory drug sensitivity, insights that may inform the development of therapeutic approaches to manage CS.

Cantú syndrome: Findings from 74 patients in the International Cantú Syndrome Registry.

Cantú syndrome (CS), first described in 1982, is caused by pathogenic variants in ABCC9 and KCNJ8, which encode the regulatory and pore forming subunits of ATP-sensitive potassium (KATP ) channels, respectively. Multiple case reports of affected individuals have described the various clinical features of CS, but systematic studies are lacking. To define the effects of genetic variants on CS phenotypes and clinical outcomes, we have developed a standardized REDCap-based registry for CS. We report phenotypic features and associated genotypes on 74 CS subjects, with confirmed ABCC9 variants in 72 of the individuals. Hypertrichosis and a characteristic facial appearance are present in all individuals. Polyhydramnios during fetal life, hyperflexibility, edema, patent ductus arteriosus (PDA), cardiomegaly, dilated aortic root, vascular tortuosity of cerebral arteries, and migraine headaches are common features, although even with this large group of subjects, there is incomplete penetrance of CS-associated features, without clear correlation to genotype.

Glibenclamide treatment in a Cantú syndrome patient with a pathogenic ABCC9 gain-of-function variant: Initial experience.

Cantú syndrome (CS), characterized by hypertrichosis, distinctive facial features, and complex cardiovascular abnormalities, is caused by pathogenic variants in ABCC9 and KCNJ8 genes. These genes encode gain-of-function mutations in the regulatory (SUR2) and pore-forming (Kir6.1) subunits of KATP channels, respectively, suggesting that channel-blocking sulfonylureas could be a viable therapy. Here we report a neonate with CS, carrying a heterozygous ABCC9 variant (c.3347G>A, p.Arg1116His), born prematurely at 32 weeks gestation. Initial echocardiogram revealed a large patent ductus arteriosus (PDA), and high pulmonary pressures with enlarged right ventricle. He initially received surfactant and continuous positive airway pressure ventilation and was invasively ventilated for 4 weeks, until PDA ligation. After surgery, he still had ongoing bilevel positive airway pressure (BiPAP) requirement, but was subsequently weaned to nocturnal BiPAP. He was treated for pulmonary hypertension with Sildenafil, but failed to make further clinical improvement. A therapeutic glibenclamide trial was commenced in week 11 (initial dose of 0.05 mg-1 kg-1 day-1 in two divided doses). After 1 week of treatment, he began to tolerate time off BiPAP when awake, and edema improved. Glibenclamide was well tolerated, and the dose was slowly increased to 0.15 mg-1 kg-1 day-1 over the next 12 weeks. Mild transient hypoglycemia was observed, but there was no cardiovascular dysfunction. Confirmation of therapeutic benefit will require studies of more CS patients but, based on this limited experience, consideration should be given to glibenclamide as CS therapy, although problems associated with prematurity, and complications of hypoglycemia, might limit outcome in critically ill neonates with CS.

Transient Notch Activation Induces Long-Term Gene Expression Changes Leading to Sick Sinus Syndrome in Mice.

RATIONALE: Notch signaling programs cardiac conduction during development, and in the adult ventricle, injury-induced Notch reactivation initiates global transcriptional and epigenetic changes. OBJECTIVE: To determine whether Notch reactivation may stably alter atrial ion channel gene expression and arrhythmia inducibility. METHODS AND RESULTS: To model an injury response and determine the effects of Notch signaling on atrial electrophysiology, we transiently activate Notch signaling within adult myocardium using a doxycycline-inducible genetic system (inducible Notch intracellular domain [iNICD]). Significant heart rate slowing and frequent sinus pauses are observed in iNICD mice when compared with controls. iNICD mice have structurally normal atria and preserved sinus node architecture, but expression of key transcriptional regulators of sinus node and atrial conduction, including Nkx2-5 (NK2 homeobox 5), Tbx3, and Tbx5 are dysregulated. To determine whether the induced electrical changes are stable, we transiently activated Notch followed by a prolonged washout period and observed that, in addition to decreased heart rate, atrial conduction velocity is persistently slower than control. Consistent with conduction slowing, genes encoding molecular determinants of atrial conduction velocity, including Scn5a (Nav1.5) and Gja5 (connexin 40), are persistently downregulated long after a transient Notch pulse. Consistent with the reduction in Scn5a transcript, Notch induces global changes in the atrial action potential, including a reduced dVm/dtmax. In addition, programmed electrical stimulation near the murine pulmonary vein demonstrates increased susceptibility to atrial arrhythmias in mice where Notch has been transiently activated. Taken together, these results suggest that transient Notch activation persistently alters ion channel gene expression and atrial electrophysiology and predisposes to an arrhythmogenic substrate. CONCLUSIONS: Our data provide evidence that Notch signaling regulates transcription factor and ion channel gene expression within adult atrial myocardium. Notch reactivation induces electrical changes, resulting in sinus bradycardia, sinus pauses, and a susceptibility to atrial arrhythmias, which contribute to a phenotype resembling sick sinus syndrome.

K(ATP) channel gain-of-function leads to increased myocardial L-type Ca(2+) current and contractility in Cantu syndrome.

Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) KATP channel subunits. We show that patients with CS, as well as mice with constitutive (cGOF) or tamoxifen-induced (icGOF) cardiac-specific Kir6.1 GOF subunit expression, have enlarged hearts, with increased ejection fraction and increased contractility. Whole-cell voltage-clamp recordings from cGOF or icGOF ventricular myocytes (VM) show increased basal L-type Ca(2+) current (LTCC), comparable to that seen in WT VM treated with isoproterenol. Mice with vascular-specific expression (vGOF) show left ventricular dilation as well as less-markedly increased LTCC. Increased LTCC in KATP GOF models is paralleled by changes in phosphorylation of the pore-forming α1 subunit of the cardiac voltage-gated calcium channel Cav1.2 at Ser1928, suggesting enhanced protein kinase activity as a potential link between increased KATP current and CS cardiac pathophysiology.

Conserved functional consequences of disease-associated mutations in the slide helix of Kir6.1 and Kir6.2 subunits of the ATP-sensitive potassium channel.

Cantu syndrome (CS) is a condition characterized by a range of anatomical defects, including cardiomegaly, hyperflexibility of the joints, hypertrichosis, and craniofacial dysmorphology. CS is associated with multiple missense mutations in the genes encoding the regulatory sulfonylurea receptor 2 (SUR2) subunits of the ATP-sensitive K+ (KATP) channel as well as two mutations (V65M and C176S) in the Kir6.1 (KCNJ8) subunit. Previous analysis of leucine and alanine substitutions at the Val-65-equivalent site (Val-64) in Kir6.2 indicated no major effects on channel function. In this study, we characterized the effects of both valine-to-methionine and valine-to-leucine substitutions at this position in both Kir6.1 and Kir6.2 using ion flux and patch clamp techniques. We report that methionine substitution, but not leucine substitution, results in increased open state stability and hence significantly reduced ATP sensitivity and a marked increase of channel activity in the intact cell irrespective of the identity of the coassembled SUR subunit. Sulfonylurea inhibitors, such as glibenclamide, are potential therapies for CS. However, as a consequence of the increased open state stability, both Kir6.1(V65M) and Kir6.2(V64M) mutations essentially abolish high-affinity sensitivity to the KATP blocker glibenclamide in both intact cells and excised patches. This raises the possibility that, at least for some CS mutations, sulfonylurea therapy may not prove to be successful and highlights the need for detailed pharmacogenomic analyses of CS mutations.

Modulation of TMEM16A channel activity by the von Willebrand factor type A (VWA) domain of the calcium-activated chloride channel regulator 1 (CLCA1).

Calcium-activated chloride channels (CaCCs) are key players in transepithelial ion transport and fluid secretion, smooth muscle constriction, neuronal excitability, and cell proliferation. The CaCC regulator 1 (CLCA1) modulates the activity of the CaCC TMEM16A/Anoctamin 1 (ANO1) by directly engaging the channel at the cell surface, but the exact mechanism is unknown. Here we demonstrate that the von Willebrand factor type A (VWA) domain within the cleaved CLCA1 N-terminal fragment is necessary and sufficient for this interaction. TMEM16A protein levels on the cell surface were increased in HEK293T cells transfected with CLCA1 constructs containing the VWA domain, and TMEM16A-like currents were activated. Similar currents were evoked in cells exposed to secreted VWA domain alone, and these currents were significantly knocked down by TMEM16A siRNA. VWA-dependent TMEM16A modulation was not modified by the S357N mutation, a VWA domain polymorphism associated with more severe meconium ileus in cystic fibrosis patients. VWA-activated currents were significantly reduced in the absence of extracellular Mg2+, and mutation of residues within the conserved metal ion-dependent adhesion site motif impaired the ability of VWA to potentiate TMEM16A activity, suggesting that CLCA1-TMEM16A interactions are Mg2+- and metal ion-dependent adhesion site-dependent. Increase in TMEM16A activity occurred within minutes of exposure to CLCA1 or after a short treatment with nocodazole, consistent with the hypothesis that CLCA1 stabilizes TMEM16A at the cell surface by preventing its internalization. Our study hints at the therapeutic potential of the selective activation of TMEM16A by the CLCA1 VWA domain in loss-of-function chloride channelopathies such as cystic fibrosis.

Conformational changes at cytoplasmic intersubunit interactions control Kir channel gating.

The defining structural feature of inward-rectifier potassium (Kir) channels is the unique Kir cytoplasmic domain. Recently we showed that salt bridges located at the cytoplasmic domain subunit interfaces (CD-Is) of eukaryotic Kir channels control channel gating via stability of a novel inactivated closed state. The cytoplasmic domains of prokaryotic and eukaryotic Kir channels show similar conformational rearrangements to the common gating ligand, phosphatidylinositol bisphosphate (PIP2), although these exhibit opposite coupling to opening and closing transitions. In Kir2.1, mutation of one of these CD-I salt bridge residues (R204A) reduces apparent PIP2 sensitivity of channel activity, and here we show that Ala or Cys substitutions of the functionally equivalent residue (Arg-165) in the prokaryotic Kir channel KirBac1.1 also significantly decrease sensitivity of the channel to PIP2 (by 5-30-fold). To further understand the structural basis of CD-I control of Kir channel gating, we examined the effect of the R165A mutation on PIP2-induced changes in channel function and conformation. Single-channel analyses indicated that the R165A mutation disrupts the characteristic long interburst closed state of reconstituted KirBac1.1 in giant liposomes, resulting in a higher open probability due to more frequent opening bursts. Intramolecular FRET measurements indicate that, relative to wild-type channels, the R165A mutation results in splaying of the cytoplasmic domains away from the central axis and that PIP2 essentially induces opposite motions of the major ß-sheet in this channel mutant. We conclude that the removal of stabilizing CD-I salt bridges results in a collapsed state of the Kir domain.