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1.
Front Pharmacol ; 11: 327, 2020.
Article in English | MEDLINE | ID: mdl-32256370

ABSTRACT

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.

2.
Hum Mutat ; 39(9): 1273-1283, 2018 09.
Article in English | MEDLINE | ID: mdl-29935101

ABSTRACT

Myotonia congenita (MC) is a skeletal-muscle hyperexcitability disorder caused by loss-of-function mutations in the ClC-1 chloride channel. Mutations are scattered over the entire sequence of the channel protein, with more than 30 mutations located in the poorly characterized cytosolic C-terminal domain. In this study, we characterized, through patch clamp, seven ClC-1 mutations identified in patients affected by MC of various severities and located in the C-terminal region. The p.Val829Met, p.Thr832Ile, p.Val851Met, p.Gly859Val, and p.Leu861Pro mutations reside in the CBS2 domain, while p.Pro883Thr and p.Val947Glu are in the C-terminal peptide. We showed that the functional properties of mutant channels correlated with the clinical phenotypes of affected individuals. In addition, we defined clusters of ClC-1 mutations within CBS2 and C-terminal peptide subdomains that share the same functional defect: mutations between 829 and 835 residues and in residue 883 induced an alteration of voltage dependence, mutations between 851 and 859 residues, and in residue 947 induced a reduction of chloride currents, whereas mutations on 861 residue showed no obvious change in ClC-1 function. This study improves our understanding of the mechanisms underlying MC, sheds light on the role of the C-terminal region in ClC-1 function, and provides information to develop new antimyotonic drugs.


Subject(s)
Chloride Channels/genetics , DNA Mutational Analysis , Mutation/genetics , Myotonia Congenita/genetics , Adolescent , Adult , Amino Acids/genetics , Female , Humans , Ion Channel Gating/genetics , Male , Middle Aged , Myotonia Congenita/drug therapy , Myotonia Congenita/physiopathology , Patch-Clamp Techniques , Peptides/genetics , Protein Domains/genetics
3.
G Ital Nefrol ; 35(3)2018 May.
Article in Italian | MEDLINE | ID: mdl-29786180

ABSTRACT

Bartter syndromes (BS) types 1-5 are rare salt-losing tubulopathies presenting with overlapping clinical phenotypes including marked salt wasting and hypokalemia leading to polyuria, polydipsia, volume contraction, muscle weakness and growth retardation. These diseases are due to an impairment of sodium, potassium, chloride reabsorption caused by mutations in genes encoding for ion channel or transporters expressed in specific nephron tubule segments. Particularly, BS type 3 is a clinically heterogeneous form caused by mutations in CLCNKB gene which encodes the ClC-Kb chloride channel involved in NaCl reabsorption in the renal tubule. Specific therapy for BS is lacking and the only pharmacotherapy up today available is purely symptomatic and characterized by limiting side effects. The improvement of our understanding of the phenotype/genotype correlation and of the precise pathogenic mechanisms associated with BS type 3 as well as the pharmacological characterization of ClC-K chloride channels are fundamental to design therapies tailored upon patients' mutation. This mini review focused on recent studies representing relevant forward steps in the field as well as noteworthy examples of how basic and clinical research can cooperate to gain insight into the pathophysiology of this renal channelopathy, paving the way for a personalized therapy.


Subject(s)
Bartter Syndrome/drug therapy , Rare Diseases/drug therapy , Bartter Syndrome/epidemiology , Bartter Syndrome/genetics , Bartter Syndrome/physiopathology , Chloride Channels/deficiency , Chloride Channels/genetics , Chlorides/metabolism , Drug Design , Genes, Recessive , Genetic Association Studies , Humans , Ion Channels/genetics , Ion Channels/metabolism , Ion Transport , Nephrons/metabolism , Pharmacogenetics , Potassium/metabolism , Precision Medicine , Rare Diseases/epidemiology , Rare Diseases/genetics , Sodium/metabolism
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