Mutations in the chloride-proton antiporter, ClC-5, cause Dent’s disease, a kidney disease defined by excessive loss of proteins in the urine. ClC-5 resides on early endosomal membranes in proximal tubule epithelial cells, where it facilitates protein receptor-mediated endocytosis. Loss-of-function mutations in ClC-5 produce proximal tubule defects in protein reabsorption. This study characterized an epithelial cell phenotype for nonsense ClC-5 mutations, R648X and R704X. Both ClC-5 mutants displayed defective biosynthesis, mistrafficking and ER localization. This study showed that ClC-5 mutations, R718X and C221R, which are also misprocessed and ER retained, are targeted for proteasomal degradation as a means to be efficiently eliminated from the ER. In addition, we have shown that a missense mutation in ClC- 5, C221R, causes a global conformational change in the antiporter, which likely reflects protein misfolding, as evident by enhanced susceptibility to trypsin proteolysis. We have characterized ClC-5 disease-causing mutations in an epithelial cell model of the proximal tubule.

Mutations in the chloride-proton antiporter, ClC-5, cause Dent’s disease, a kidney disease defined by excessive loss of proteins in the urine. ClC-5 resides on early endosomal membranes in proximal tubule epithelial cells, where it facilitates protein receptor-mediated endocytosis. Loss-of-function mutations in ClC-5 produce proximal tubule defects in protein reabsorption. This study characterized an epithelial cell phenotype for nonsense ClC-5 mutations, R648X and R704X. Both ClC-5 mutants displayed defective biosynthesis, mistrafficking and ER localization. This study showed that ClC-5 mutations, R718X and C221R, which are also misprocessed and ER retained, are targeted for proteasomal degradation as a means to be efficiently eliminated from the ER. In addition, we have shown that a missense mutation in ClC- 5, C221R, causes a global conformational change in the antiporter, which likely reflects protein misfolding, as evident by enhanced susceptibility to trypsin proteolysis. We have characterized ClC-5 disease-causing mutations in an epithelial cell model of the proximal tubule.

The chloride/proton antiporter ClC-5 is primarily expressed in the kidney where it aids in re-absorption of proteins from the glomerular filtrate. Functional disruption of ClC-5 causes Dent’s Disease – a renal condition characterized by proteinuria and kidney failure in a third of all cases. The majority of disease-causing mutations translate into premature truncations of the carboxy-terminal (Ct) region of ClC-5 and are predicted to disrupt the protein-protein interactions mediated by this domain. In this thesis, direct ATP binding to the two cystathionine β-synthase (CBS) domains of ClC-5 was demonstrated. ATP binding enhanced the global compactness of the ClC-5 Ct region likely through a clamping motion of the CBS domains around the nucleotide. Along with ATP, the sodium proton exchange regulatory factor 2 (NHERF2) also binds ClC-5; however, the molecular mechanism behind this interaction was unknown as ClC-5 lacked the PDZ binding motif traditionally localized at the Ct end of bait proteins. Here, we also identified a class I PDZ binding motif (657-660; TSII) within the internal sequence of ClC-5. Despite the buried position of this motif in the Ct peptide’s X-ray crystal structure (PDB: 2J9L), the high propensity of this region for dynamic flexibility prompted us to test whether it could mediate NHERF2 interactions. In support of this hypothesis, we demonstrated that the motif is transiently available to interact directly with NHERF2 in vivo and to enable an enhancement in receptor-mediated endocytosis in mammalian cells. Collectively, these results gave further evidence that the intracellular Ct region of ClC-5 serves as a hub to mediate interactions essential for its maturation, stability, and trafficking in renal epithelium, as well as providing further insights into the molecular basis of Dent’s Disease.

Les canaux chlorure sont impliqués dans divers processus indispensables à la vie cellulaire, tel que la stabilisation du potentiel de membrane, la régulation du volume ou du pH, ainsi que dans les transports transépithéliaux de sels. Depuis une douzaine d'années, l'application de la biologie moléculaire à l'étude des canaux chlorure a permis d'identifier plusieurs familles de gènes. La variété fonctionnelle des canaux chlorure peut donc s'expliquer par leur diversité moléculaire. Actuellement, la détermination des rôles physiologiques des canaux chlorure clonés constitue un enjeu majeur de leur étude.<br /><br />Récemment, une stratégie de clonage par homologie nous a permis d'isoler deux nouveaux membres de la famille des ClC, canaux chlorure dépendants du voltage: le xClC-5 et le xClC-3. La caractérisation fonctionnelle et la détermination de la localisation tissulaire et cellulaire de ces protéines a constitué l'objectif de notre étude. Pour cela, l'ovocyte de xénope et la lignée cellulaire HEK 293 ont été utilisés comme systèmes d'expression. L'analyse électrophysiologique, menée par une technique de voltage-clamp en double micro-électrodes, nous a permis de caractériser le xClC-5 du point de vue électrophysiologique et de déterminer sa sensibilité au pH, aux ions métalliques et aux inhibiteurs de tyrosine kinases. L'utilisation d'anticorps nous a permis d'étudier la glycosylation des protéines et d'examiner leur distribution tissulaire et/ou leur localisation cellulaire. Nos résultats sont discutés par rapport à ceux d'autres équipes, en prêtant une attention particulière aux modèles fonctionnels possibles, qui émergent actuellement pour les protéines ClC-5 et ClC-3.

The chloride/proton antiporter ClC-5 is primarily expressed in the kidney where it aids in re-absorption of proteins from the glomerular filtrate. Functional disruption of ClC-5 causes Dent’s Disease – a renal condition characterized by proteinuria and kidney failure in a third of all cases. The majority of disease-causing mutations translate into premature truncations of the carboxy-terminal (Ct) region of ClC-5 and are predicted to disrupt the protein-protein interactions mediated by this domain. In this thesis, direct ATP binding to the two cystathionine β-synthase (CBS) domains of ClC-5 was demonstrated. ATP binding enhanced the global compactness of the ClC-5 Ct region likely through a clamping motion of the CBS domains around the nucleotide. Along with ATP, the sodium proton exchange regulatory factor 2 (NHERF2) also binds ClC-5; however, the molecular mechanism behind this interaction was unknown as ClC-5 lacked the PDZ binding motif traditionally localized at the Ct end of bait proteins. Here, we also identified a class I PDZ binding motif (657-660; TSII) within the internal sequence of ClC-5. Despite the buried position of this motif in the Ct peptide’s X-ray crystal structure (PDB: 2J9L), the high propensity of this region for dynamic flexibility prompted us to test whether it could mediate NHERF2 interactions. In support of this hypothesis, we demonstrated that the motif is transiently available to interact directly with NHERF2 in vivo and to enable an enhancement in receptor-mediated endocytosis in mammalian cells. Collectively, these results gave further evidence that the intracellular Ct region of ClC-5 serves as a hub to mediate interactions essential for its maturation, stability, and trafficking in renal epithelium, as well as providing further insights into the molecular basis of Dent’s Disease.

Le tubule proximal participe à la diurèse en modifiant la composition de l'ultrafiltrat glomérulaire. Grâce à de nombreux transports transépithéliaux, il le glucose, les acides aminés et les protéines de bas poids moléculaires, ainsi que 80 % des ions HPO42- ou HCO3-, 60 % des ions Na+, Cl-, K+, Ca2+, 75 % de l’eau et 30 % des ions Mg2+ ultrafiltrés.Durant ma thèse, j'ai étudié les rôles physiologiques et physiopathologiques de deux protéines de transport exprimées dans le tubule proximal.Dans le cadre de ma première étude, j'ai évalué in vivo la fonction rénale de souris n'exprimant pas une protéine appelée Kir4.2, dont le rôle est inconnu. Nos résultats montrent que Kir4.2, associée à Kir5.1, forme un canal potassique basolatéral Kir4.2/Kir5.1 dans le tubule proximal. L'absence de Kir4.2 provoque chez la souris une acidose tubulaire proximale isolée, consécutive à une ammoniogénèse altérée. De fait, la perte de fonctionnalité de Kir4.2 pourrait être à l'origine d'acidoses tubulaires proximales isolées familiales idiopathiques.Dans le cadre de ma seconde étude, j'ai analysé in vitro la fonctionnalité d'un mutant pathogène de l'échangeur 2Cl-/H+ ClC-5 impliqué dans la maladie de Dent. Cette maladie, caractérisée par une protéinurie de bas poids moléculaire associées à divers troubles du tubule proximal, serait liée à un défaut d'acidification des endosomes précoces par ClC-5. Toutefois, le mutant de ClC-5 que nous avons étudié, converti en canal chlorure, acidifie autant les endosomes précoces que le ClC-5 sauvage. Surprenants, ces résultats suggèrent que la maladie de Dent puisse être causée par un défaut d'accumulation d'ions chlorure dans l'endosome. / The proximal tubule is involved in diuresis by modifying the content of the glomerular ultrafiltrate. Using a variety of transepithelial transports systems, it reabsorbs all ultrafiltrated glucose, amino-acids and low molecular weight proteins, as well as 80% of HPO42- and HCO3- ions, about 60% of Na+, Cl-, K+, and Ca2+ ions, 75% of water and 30% of Mg2+.During this thesis, I determined the physiological and physiopathological roles of two transport proteins present in proximal tubule. Firstly, I evaluated the renal function of mice invalidated for the Kir4.2 protein, whose role was undetermined. Our results show that Kir4.2, in association with Kir5.1, form a Kir4.2/Kir5.1 potassium channel at the basolateral membrane of proximal tubular cells. Furthermore, Kir4.2-null mice exhibit a reduced ammoniagenesis leading to an isolated proximal renal tubular acidosis. This study provides the gene encoding Kir4.2 as a candidate gene for the yet unexplained autosomal dominant isolated proximal renal tubular acidosis.Secondly, I evaluated in vitro the functional consequences of a pathogenic mutation of the 2Cl-/H+ exchanger ClC-5, involved in Dent’s disease. This disease, characterized by a low-molecular-weigth-proteinuria in the context of a general proximal tubule dysfunction, is currently thought to be due to an acidification defect of early endosomes linked to a loss of function of ClC-5. Surprisingly, our results show that ClC-5, converted into a chloride channel by this mutation, indeed acidifies the early endosomes as well as the ClC-5 wild-type. Thus, Dent’s disease may originate from a defect in the accumulation of chloride ions into the early endosomes.