Dataset Information

Cl^- and H⁺ coupling properties and subcellular localizations of wildtype and disease-associated variants of the voltage-gated Cl^-/H⁺ exchanger ClC-5.

ABSTRACT: Dent disease 1 (DD1) is caused by mutations in the CLCN5 gene encoding a voltage-gated electrogenic nCl^-/H⁺ exchanger ClC-5. Using ion-selective microelectrodes and Xenopus oocytes, here we studied Cl^-/H⁺ coupling properties of WT ClC-5 and four DD1-associated variants (S244L, R345W, Q629*, and T657S), along with trafficking and localization of ClC-5. WT ClC-5 had a 2Cl^-/H⁺ exchange ratio at a V_h of +40 mV with a [Cl^-]_out of 104 mm, but the transport direction did not reverse with a [Cl^-]_out of 5 mm, indicating that ClC-5-mediated exchange of two Cl^- out for one H⁺ in is not permissible. We hypothesized that ClC-5 and H⁺-ATPase are functionally coupled during H⁺-ATPase-mediated endosomal acidification, crucial for ClC-5 activation by depolarizing endosomes. ClC-5 transport that provides three net negative charges appeared self-inhibitory because of ClC-5's voltage-gated properties, but shunt conductance facilitated further H⁺-ATPase-mediated endosomal acidification. Thus, an on-and-off "burst" of ClC-5 activity was crucial for preventing Cl^- exit from endosomes. The subcellular distribution of the ClC-5:S244L variant was comparable with that of WT ClC-5, but the variant had a much slower Cl^- and H⁺ transport and displayed an altered stoichiometry of 1.6:1. The ClC-5:R345W variant exhibited slightly higher Cl^-/H⁺ transport than ClC-5:S244L, but co-localized with early endosomes, suggesting decreased ClC-5:R345W membrane trafficking is perhaps in a fully functional form. The truncated ClC-5:Q629* variant displayed the lowest Cl^-/H⁺ exchange and was retained in the endoplasmic reticulum and cis-Golgi, but not in early endosomes, suggesting the nonsense mutation affects ClC-5 maturation and trafficking.

SUBMITTER: Chang MH

PROVIDER: S-EPMC7008381 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Cl- and H+ coupling properties and subcellular localizations of wildtype and disease-associated variants of the voltage-gated Cl-/H+ exchanger ClC-5.

Chang Min-Hwang MH Brown Matthew R MR Liu Yiran Y Gainullin Vladimir G VG Harris Peter C PC Romero Michael F MF Lieske John C JC

The Journal of biological chemistry 20191218 6

Dent disease 1 (DD1) is caused by mutations in the CLCN5 gene encoding a voltage-gated electrogenic nCl-/H+ exchanger ClC-5. Using ion-selective microelectrodes and Xenopus oocytes, here we studied Cl-/H+ coupling properties of WT ClC-5 and four DD1-associated variants (S244L, R345W, Q629*, and T657S), along with trafficking and localization of ClC-5. WT ClC-5 had a 2Cl-/H+ exchange ratio at a Vh of +40 mV ...[more]

PMID: 31852738

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Project description: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.

Dataset Information

Cl^- and H⁺ coupling properties and subcellular localizations of wildtype and disease-associated variants of the voltage-gated Cl^-/H⁺ exchanger ClC-5.

Publications

Cl<sup>-</sup> and H<sup>+</sup> coupling properties and subcellular localizations of wildtype and disease-associated variants of the voltage-gated Cl<sup>-</sup>/H<sup>+</sup> exchanger ClC-5.

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