Pharmacovigilance database search discloses ClC-K channels as a novel target of the AT1 receptor blockers valsartan and olmesartan.
ABSTRACT: BACKGROUND AND PURPOSE:Human ClC-K chloride channels are highly attractive targets for drug discovery as they have a variety of important physiological functions and are associated with genetic disorders. These channels are crucial in the kidney as they control chloride reabsorption and water diuresis. In addition, loss-of-function mutations of CLCNKB and BSND genes cause Bartter's syndrome (BS), whereas CLCNKA and CLCNKB gain-of-function polymorphisms predispose to a rare form of salt sensitive hypertension. Both disorders lack a personalized therapy that is in most cases only symptomatic. The aim of this study was to identify novel ClC-K ligands from drugs already on the market, by exploiting the pharmacological side activity of drug molecules available from the FDA Adverse Effects Reporting System database. EXPERIMENTAL APPROACH:We searched for drugs having a Bartter-like syndrome as a reported side effect, with the assumption that BS could be causatively related to the block of ClC-K channels. The ability of the selected BS-causing drugs to bind and block ClC-K channels was then validated through an integrated experimental and computational approach based on patch clamp electrophysiology in HEK293 cells and molecular docking simulations. KEY RESULTS:Valsartan and olmesartan were able to block ClC-Ka channels and the molecular requirements for effective inhibition of these channels have been identified. CONCLUSION AND IMPLICATIONS:These results suggest additional mechanisms of action for these sartans further to their primary AT1 receptor antagonism and propose these compounds as leads for designing new potent ClC-K ligands.
Project description:Type III and IV Bartter syndromes (BS) are rare kidney tubulopathies caused by loss-of-function mutations in the CLCNKB and BSND genes coding respectively for the ClC-Kb chloride channels and accessory subunit barttin. ClC-K channels are expressed in the Henle's loop, distal convoluted tubule, and cortical collecting ducts of the kidney and contribute to chloride absorption and urine concentration. In our Italian cohort, we identified two new mutations in CLCNKB, G167V and G289R, in children affected by BS and previously reported genetic variants, A242E, a chimeric gene and the deletion of the whole CLCNKB. All the patients had hypokalemia and metabolic alkalosis, increased serum renin and aldosterone levels and were treated with a symptomatic therapy. In order to define the molecular mechanisms responsible for BS, we co-expressed ClC-Kb wild type and channels with point mutations with barttin in HEK 293 cells and characterized chloride currents through the patch-clamp technique. In addition, we attempted to revert the functional defect caused by BS mutations through barttin overexpression. G167V and A242E channels showed a drastic current reduction compared to wild type, likely suggesting compromised expression of mutant channels at the plasma membrane. Conversely, G289R channel was similar to wild type raising the doubt that an additional mutation in another gene or other mechanisms could account for the clinical phenotype. Interestingly, increasing ClC-K/barttin ratio augmented G167V and A242E mutants' chloride current amplitudes towards wild type levels. These results confirm a genotype-phenotype correlation in BS and represent a preliminary proof of concept that molecules functioning as molecular chaperones can restore channel function in expression-defective ClC-Kb mutants.
Project description:BACKGROUND:Bartter's syndrome is a heterogeneous disorder characterized by deficient renal reabsorption of sodium and chloride, and hypokalemic metabolic alkalosis with hyper-reninemia and hyperaldosteronemia. Bartter syndrome type III (BS type III), due to mutations in the CLCNKB gene, is highly variable. The aim of our study was to describe the clinical presentation in a Chinese girl with BS type III and to explore mutations or SNPs of CLCNKB gene in her family. CASE PRESENTATION:The clinic data of the patient was collected. Mutations or SNPs were investigated by sequencing of the exon of CLCNKB gene. The clinic analysis confirmed the diagnosis of BS type III. The coexistence of 13 reported SNPs and 11 novel SNPs of CLCNKB gene were found in the patient and her parent. a novel heterozygous C to G transition at nucleotide 2471 in exon 20 of CLCNKB gene harbored uniquely by the patient were revealed. CONCLUSION:A novel heterozygous C to G mutation at nucleotide 2471 of CLCNKB gene and some new SNPs were identified in a Chinese girl with BS type III having persistent hypokalemia. The novel mutation and SNPs make the genetic background of the patient more complicated.
Project description:CLC-K chloride channels are expressed in the kidney and the inner ear, where they are involved in NaCl reabsorption and endolymph production, respectively. These channels require the beta subunit barttin for proper function. Mutations in ClC-Kb and barttin, lead to Bartter's syndrome. Block of CLC-K channels by acid pH was described in a previous work, and we had identified His-497 as being responsible for the acidic block of CLC-K channels. Here, we show that ClC-K currents are blocked also by alkaline pH with an apparent pK value of ?8.7 for ClC-K1. Using noise analysis, we demonstrate that alkaline block is mediated by an allosteric reduction of the open probability. By an extensive mutagenic screen we identified K165, a highly conserved residue in the extracellular vestibule of the channel, as the major element responsible for the alkaline pH modulation. Deprotonation of K165 underlies the alkaline block. However, MTS modification of the K165C mutant demonstrated that not only the charge but also the chemical and sterical properties of lysine 165 are determinants of CLC-K gating.
Project description:Bartter syndrome (BS) is classified into 5 genotypes according to underlying mutant genes and BS III is caused by loss-of-function mutations in the CLCNKB gene encoding for basolateral ClC-Kb. BS III is the most common genotype in Korean patients with BS and W610X is the most common CLCNKB mutation in Korean BS III. In this study, we tested the hypothesis that the CLCNKB W610X mutation can be rescued in vitro using aminoglycoside antibiotics, which are known to induce translational read-through of a nonsense mutation. The CLCNKB cDNA was cloned into a eukaryotic expression vector and the W610X nonsense mutation was generated by site-directed mutagenesis. Cultured polarized MDCK cells were transfected with the vectors, and the read-through was induced using an aminoglycoside derivative, G418. Cellular expression of the target protein was monitored via immunohistochemistry. While cells transfected with the mutant CLCNKB failed to express ClC-Kb, G418 treatment of the cells induced the full-length protein expression, which was localized to the basolateral plasma membranes. It is demonstrated that the W610X mutation in CLCNKB can be a good candidate for trial of translational read-through induction as a therapeutic modality.
Project description:BACKGROUND:Bartter syndrome (BS) is a rare autosomal recessive disorder of salt reabsorption at the thick ascending limb of the Henle loop, characterized by hypokalemia, salt loss, metabolic alkalosis, hyperreninemic hyperaldosteronism with normal blood pressure. BS type III, often known as classic BS (CBS), is caused by loss-of-function mutations in CLCNKB (chloride voltage-gated channel Kb) encoding basolateral ClC-Kb. CASE PRESENTATION:We reported a 15-year-old CBS patient with a compound heterozygous mutation of CLCNKB gene. She first presented with vomiting, hypokalemic metabolic alkalosis at the age of 4?months, and was clinically diagnosed as CBS. Indomethacin, spironolactone and oral potassium were started from then. During follow-up, the serum electrolyte levels were generally normal, but the patient showed failure to thrive and growth hormone (GH) deficiency was diagnosed. The recombinant human GH therapy was performed, and the growth velocity was improved. When she was 14, severe proteinuria and chronic kidney disease (CKD) were developed. Renal biopsy showed focal segmental glomerulosclerosis (FSGS) with juxtaglomerular apparatus cell hyperplasia, and genetic testing revealed a point deletion of c.1696delG (p. Glu566fs) and a fragment deletion of exon 2-3 deletions in CLCNKB gene. Apart from the CBS, ostium secundum atrial septal defect (ASD) was diagnosed by echocardiography. CONCLUSIONS:This is the first report of this compound heterozygous of CLCNKB gene in BS Children. Our findings contribute to a growing list of CLCNKB mutations associated with CBS. Some recessive mutations can induce CBS in combination with other mutations.
Project description:BACKGROUND: ClC-Kb and ClC-Ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of ClC-Kb leads to Bartter's Syndrome, a kidney disease. A point mutation in ClC-Kb, R538P, linked to Bartter's Syndrome and located in the C-terminal cytoplasmic domain was hypothesized to alter electrophysiological properties due to its proximity to an important membrane-embedded helix. METHODOLOGY/PRINCIPAL FINDINGS: Two-electrode voltage clamp experiments were used to examine the electrophysiological properties of the mutation R538P in both ClC-Kb and ClC-Ka. R538P selectively abolishes extracellular calcium activation of ClC-Kb but not ClC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the ClC-Kb C-terminal domain had either a different oligomeric status or dimerization interface than that of ClC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the ClC-Kb C-terminal domain and used multi-angle light scattering together with a cysteine-crosslinking approach to show that the dimerization interface is conserved between the ClC-Kb and ClC-Ka C-terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface. CONCLUSIONS: The R538P mutation in ClC-Kb, which leads to Bartter's Syndrome, abolishes calcium activation of the channel. This suggests that a significant conformational change--ranging from the cytoplasmic side of the protein to the extracellular side of the protein--is involved in the Ca(2+)-activation process for ClC-Kb, and shows that the cytoplasmic domain is important for the channel's electrophysiological properties. In the highly similar ClC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca(2+). This selective outcome indicates that ClC-Ka and ClC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure.
Project description:Chloride permeability in the thin and thick ascending limbs of the loop of Henle is a crucial component for urine concentration, with ClC-K/barttin chloride channels as a central component in establishing the cortico-medullary osmotic gradient. Barttin is an accessory subunit of human ClC-K channels, promoting trafficking of the ClC-K/barttin complex to the plasma membrane, increasing channel stability, and switching ClC-K/barttin channels into an active state. Barttin undergoes post-translational palmitoylation, which is essential for channel activation. Here, we identified DHHC7 as a major barttin palmitoyl-acyltransferase, the depletion of which reduced barttin palmitoylation and the macroscopic current amplitudes in cells expressing ClC-K/barttin channels. To investigate a functional role of barttin palmitoylation in vivo, we established Zdhhc7-/- mice. Although barttin palmitoylation was significantly decreased in the kidneys of Zdhhc7-/- animals, it did not result in any pathological consequences for kidney structure or function under physiological conditions. However, when Zdhhc7-/- animals were fed a low-salt diet, they developed hyponatremia and mild metabolic alkalosis, symptoms characteristic of human Bartter syndrome (BS) Type IV with mild progression. Notably, decreased barttin palmitoylation was also found for the R8L barttin mutant identified in human BS Type IV. These data suggest that barttin palmitoylation plays an important role in chloride channel dysfunction in certain variants of BS. Thus, our study provides evidence of the downregulation of barttin palmitoylation as a possible mechanism in the etiology of BS, making the restoration of barttin palmitoylation a promising clinical strategy for the treatment of Type IV BS.
Project description:Bartter syndrome (BS) is an inherited renal tubular disorder characterized by low or normal blood pressure, hypokalemic metabolic alkalosis, and hyperreninemic hyperaldosteronism. Type III BS is caused by loss-of-function mutations in CLCNKB encoding basolateral ClC-Kb. The clinical phenotype of patients with CLCNKB mutations has been known to be highly variable, and cases that are difficult to categorize as type III BS or other hereditary tubulopathies, such as Gitelman syndrome, have been rarely reported. We report a case of a 10-year-old Korean boy with atypical clinical findings caused by a novel CLCNKB mutation. The boy showed intermittent muscle cramps with laboratory findings of hypokalemia, severe hypomagnesemia, and nephrocalcinosis. These findings were not fully compatible with those observed in cases of BS or Gitelman syndrome. The CLCNKB mutation analysis revealed a heterozygous c.139G>A transition in exon 13 [p.Gly(GGG)465Glu(GAG)]. This change is not a known mutation; however, the clinical findings and in silico prediction results indicated that it is the underlying cause of his presentation.
Project description:Type III Bartter syndrome (BS) is an autosomal recessive renal tubule disorder caused by loss-of-function mutations in the CLCNKB gene, which encodes the chloride channel protein ClC-Kb. In this study, we carried out a complete clinical and genetic characterization in a cohort of 30 patients, one of the largest series described. By comparing with other published populations, and considering that 80% of our patients presented the p.Ala204Thr Spanish founder mutation presumably associated with a common phenotype, we aimed to test the hypothesis that allelic differences could explain the wide phenotypic variability observed in patients with type III BS.Clinical data were retrieved from the referral centers. The exon regions and flanking intronic sequences of the CLCNKB gene were screened for mutations by polymerase chain reaction (PCR) followed by direct Sanger sequencing. Presence of gross deletions or duplications in the region was checked for by MLPA and QMPSF analyses.Polyuria, polydipsia and dehydration were the main common symptoms. Metabolic alkalosis and hypokalemia of renal origin were detected in all patients at diagnosis. Calciuria levels were variable: hypercalciuria was detected in 31% of patients, while 23% had hypocalciuria. Nephrocalcinosis was diagnosed in 20% of the cohort. Two novel CLCNKB mutations were identified: a small homozygous deletion (c.753delG) in one patient and a small deletion (c.1026delC) in another. The latter was present in compound heterozygosis with the already previously described p.Glu442Gly mutation. No phenotypic association was obtained regarding the genotype.A poor correlation was found between a specific type of mutation in the CLCNKB gene and type III BS phenotype. Importantly, two CLCNKB mutations not previously described were found in our cohort.
Project description:CLC-K channels belong to the CLC gene family, which comprises both Cl(-) channels and Cl(-)/H(+) antiporters. They form homodimers which additionally co-assemble with the small protein barttin. In the kidney, they are involved in NaCl reabsorption; in the inner ear they are important for endolymph production. Mutations in CLC-Kb lead to renal salt loss (Bartter's syndrome); mutations in barttin lead additionally to deafness. CLC-K channels are interesting potential drug targets. CLC-K channel blockers have potential as alternative diuretics, whereas CLC-K activators could be used for the treatment of patients with Bartter's syndrome. Several small organic acids inhibit CLC-K channels from the outside by binding to a site in the external vestibule of the ion conducting pore. Benzofuran derivatives with affinities better than 10 ?M have been discovered. Niflumic acid (NFA) exhibits a complex interaction with CLC-K channels. Below ?1?mM, NFA activates CLC-Ka, whereas at higher concentrations NFA inhibits channel activity. The co-planarity of the rings of the NFA molecule is essential for its activating action. Mutagenesis has led to the identification of potential regions of the channel that interact with NFA. CLC-K channels are also modulated by pH and [Ca(2+)](ext). The inhibition at low pH has been shown to be mediated by a His-residue at the beginning of helix Q, the penultimate transmembrane helix. Two acidic residues from opposite subunits form two symmetrically related intersubunit Ca(2+) binding sites, whose occupation increases channel activity. The relatively high affinity CLC-K blockers may already serve as leads for the development of useful drugs. On the other hand, the CLC-K potentiator NFA has a quite low affinity, and, being a non-steroidal anti-inflammatory drug, can be expected to exert significant side effects. More specific and more potent activators will be needed and it will be important to understand the molecular mechanisms that underlie NFA activation.