Barttin Regulates the Subcellular Localization and Posttranslational Modification of Human Cl-/H+ Antiporter ClC-5

Abstract

Dent disease 1 (DD1) is a renal salt-wasting tubulopathy associated with mutations in the Cl-/H+ antiporter ClC-5. The disease typically manifests with proteinuria, hypercalciuria, nephrocalcinosis, and nephrolithiasis but is characterized by large phenotypic variability of no clear origin. Several DD1 cases have been reported lately with additional atypical hypokalemic metabolic alkalosis and hyperaldosteronism, symptoms usually associated with another renal disease termed Bartter syndrome (BS). Expression of the Bartter-like DD1 mutant ClC-5 G261E in HEK293T cells showed that it is retained in the ER and lacks the complex glycosylation typical for ClC-5 WT. Accordingly, the mutant abolished CLC ionic transport. Such phenotype is not unusual and is often observed also in DD1 ClC-5 mutants not associated with Bartter like phenotype. We noticed, therefore, that one type of BS is associated with mutations in the protein barttin that serves as an accessory subunit regulating the function and subcellular localization of ClC-K channels. The overlapping symptomatology of DD1 and BS, together with the homology between the proteins of the CLC family, led us to investigate whether barttin might also regulate ClC-5 transport. In HEK293T cells, we found that barttin cotransfection impairs the complex glycosylation and arrests ClC-5 in the endoplasmic reticulum. As barttin and ClC-5 are both expressed in the thin and thick ascending limbs of the Henle’s loop and the collecting duct, interactions between the two proteins could potentially contribute to the phenotypic variability of DD1. Pathologic barttin mutants differentially regulated trafficking and processing of ClC-5, suggesting that the interaction between the two proteins might be relevant also for the pathophysiology of BS. Our findings show that barttin regulates the subcellular localization not only of kidney ClC-K channels but also of the ClC-5 transporter, and suggest that ClC-5 might potentially play a role not only in kidney proximal tubules but also in tubular kidney segments expressing barttin. In addition, they demonstrate that the spectrum of clinical, genetic and molecular pathophysiology investigation of DD1 should be extended.

Genetics and phenotypic heterogeneity of Dent disease: the dark side of the moon

“…DD1 and BS may be diagnosed clinically in the same patient (Besbas et al 2005 ; Bogdanović et al 2010 ; Okamoto et al 2012 ), but complete genetic screening was not undertaken, so we cannot know whether this blended phenotype is due to mutations in both the CLCN5 and the BS- or GS-related genes or to an expansion of the DD1 phenotype. It has also been reported that Barttin, the protein altered in Bartter syndrome type 4, appears to regulate the subcellular localization and post-translational modification of ClC-5 (Wojciechowski et al 2018 ). An abnormal interaction between these two proteins in the thick ascending limbs of Henle’s loop and the collecting duct might, therefore, lie behind the Bartter phenotype seen in some DD1 patients.…”

Are the Clinical Presentations (Phenotypes) of Gitelman’s and Bartter’s Syndromes Gene Mutations Driven by Their Effects on Intracellular pH, Their “pH” Enotype?

“…These measurements were possible due to the rescue from accelerated degradation of expressed mutant protein by co-expression of barttin. Wojciechowski et al noted that several cases of Dent disease 1 (DD1), another renal salt-wasting tubulopathy arising from mutations in the Cl − /H + antiporter ClC-5, had atypical hypokalemic metabolic alkalosis and hyperaldosteronism, findings usually associated with BS [7]. This overlap of DD1 and BS phenotypes suggested that barttin might regulate ClC-5 transport and they found, using barttin cotransfection, that barttin impaired ClC-5's complex glycosylation.…”

Functional Study of Novel Bartter’s Syndrome Mutations in ClC-Kb and Rescue by the Accessory Subunit Barttin Toward Personalized Medicine

“…According to our electrophysiological, biochemical, and imaging findings, the overexpression of the accessory subunit barttin succeeded in restoring at least in part the function of the expression-defective mutants G167V and A242E towards wild type levels. It is widely demonstrated that barttin is able to improve the stability of ClC-K channels by promoting complex glycosylation of the nascent proteins, thus facilitating effective exit from the ER and Golgi and sorting to the plasma membrane, besides modulating channel gating (Estévez et al, 2001;Waldegger et al, 2002;Hayama et al, 2003;Scholl et al, 2006;Janssen et al, 2009;Fischer et al, 2010;Wojciechowski et al, 2018a;Wojciechowski et al, 2018b). We could therefore likely infer that the same mechanism underlies BS mutant channels rescue.…”