A new regulator of the vacuolar H+-ATPase in the kidney

Xu et al. identify Slc26a11, a novel member of the Slc26 anion exchanger family, as an electrogenic (Cl−)n/HCO3− exchanger. Functional characterization of this transporter suggests that Slc26a11 mediates classical electroneutral Cl−/HCO3− exchange but also exhibits an electrogenic Cl− conductance. In the kidney, Slc26a11 colocalizes with the vacuolar H+-ATPase in intercalated cells, emphasizing the cooperation of the proton pump with chloride transporters

What is the Performance of Incubators? The Point of View of Coached Entrepreneurs

This article examines the performance of incubators because their economic model implies constantly finding external sources of financing. In order to evaluate the performance of incubators in France, we questioned 404 entrepreneurs in 80 incubators. The results show the social utility of incubators in France. Indeed, they encourage entrepreneurs to pass to the act of creation, but also contribute to the success of incubated firms. Moreover, these companies create more jobs than other start-ups. However, the services provided by incubators could be more developed and focus more on the assistance in order to find potential investors. Lastly, the work quality of an incubator as perceived by entrepreneurs is largely dependant on its director. This fact can explain important variations of performance between incubators

A Chloride Channel at the Basolateral Membrane of the Distal-convoluted Tubule: a Candidate ClC-K Channel

The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na+-Cl− cotransporter located at the apical membrane, and Na+, K+ ATPase at the basolateral side. Cl− transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl− channels on microdissected mouse DCTs using the patch-clamp technique. A channel of ∼9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NPo in cell-attached patches was not modified when tubules were preincubated in the presence of 10−5 M forskolin, but the channel was inhibited by phorbol ester (10−6 M). In addition, NPo was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM (NPo increased threefold), or pH increased from 6.4 to 8.0 (NPo increased 15-fold). Selectivity experiments conducted on inside-out patches showed that the Na+ to Cl− relative permeability was 0.09, and the anion selectivity sequence Cl− ∼ I−> Br− ∼ NO3− > F−. Intracellular NPPB (10−4 M) and DPC (10−3 M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl− channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family

Heterogeneity in the processing of ClC-5 mutants related to Dent disease

International audienceMutations in the electrogenic Cl-/H+ exchanger ClC-5 gene CLCN5 are frequently associated with Dent disease, an X-linked recessive disorder affecting the proximal tubules. Here, we investigate the consequences in X. laevis oocytes and in HEK293 cells of 9 previously reported, pathogenic, missense mutations of ClC-5, most of them which are located in regions forming the subunit interface. Two mutants trafficked normally to the cell surface and to early endosomes, and displayed complex glycosylation at the cell surface like wild-type ClC 5, but exhibited reduced currents. Three mutants displayed improper N-glycosylation, and were non-functional due to being retained and degraded at the endoplasmic reticulum. Functional characterization of four mutants allowed us to identify a novel mechanism leading to ClC-5 dysfunction in Dent disease. We report that these mutant proteins were delayed in their processing and that the stability of their complex glycosylated form was reduced, causing lower cell surface expression. The early endosome distribution of these mutants was normal. Half of these mutants displayed reduced currents, whereas the other half showed abolished currents. Our study revealed distinct cellular mechanisms accounting for ClC-5 loss-of-function in Dent disease

Etude des canaux potassium et chlorure de la membrane basolatérale du tubule distal de souris

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Analyse fonctionnelle de nouvelles mutations pathogènes du canal chlorure CLC-KB impliquées dans le syndrome de Bartter

Le syndrome de Bartter de type III résulte de mutations du gène CLCNKB codant le canal chlorure CLC-KB. Un grand nombre de mutations a été répertorié, mais peu d entre elles ont été caractérisées fonctionnellement. Devant ce manque de données, nous nous sommes fixés comme objectif de procéder à une analyse fonctionnelle des mutations de CLCNKB pour tenter d approfondir davantage les mécanismes de régulation de CLC-KB. Ce travail a nécessité l emploi de trois systèmes d expression hétérologue, les ovocytes de Xenopus laevis et les lignées cellulaires rénales HEK293T et MDCK, afin de procéder à des analyses électrophysiologiques et des expériences de biologie cellulaire. Nous démontrons que toutes les mutations étudiées altèrent l expression membranaire de la protéine et que la conductance résiduelle est proportionnée à cette expression. Ainsi, la réduction des courants provient d une réduction du nombre de canaux et non d une altération majeure de la conduction ou de la régulation. Nous rapportons également que certaines mutations modifient la sensibilité au pH et au calcium extracellulaires du CLC-KB. Cette altération peut avoir un impact pathologique majeur. En conclusion, cette étude fonctionnelle montre le rôle essentiel du CLC-KB dans le maintien de la balance sodée et laisse entrevoir un espoir thérapeutique à long terme de restauration des anomalies précisément identifiées. Enfin, l analyse de mutations est un outil extrêmement puissant pour les études structure-fonction. Associée à la recherche de nouveaux partenaires, elle devrait permettre de découvrir les régulations physiologiques de ce canal, pour l heure presqu inconnues.Bartter s syndrome type III results from loss-of-function mutations in the CLCNKB gene that encodes the CLC-KB chloride channel. To date, few CLCNKB mutations have been fully functionally investigated. In this study, we have investigated the functional consequences of novel CLCNKB mutations (collected by the Department of Genetics of the European Georges Pompidou Hospital) in Xenopus laevis oocytes, HEK293T and MDCK cells, in terms of electrical activity and surface expression. We have demonstrated that all mutations decreased plasma membrane expression and conductance of CLC-KB in similar proportions, indicating that altered cell surface expression is the main functional defect caused by CLCNKB mutations. However, some mutations also alter external pH and calcium sensitivity. The physiological relevance of these regulations is open to question, but clearly, their alteration could have a pathological impact. In conclusion, this functional study highlights the essential role of CLC-KB in the preservation of sodium balance and provides new clues for finding specific therapeutic drugs that would be able to restore sufficient function of CLC-KB in patients with Bartter s syndrome type III. Moreover, functional analysis of mutations is an extremely powerful tool for structure-function studies. In association with the search of new partners, it should allow discovering the physiological regulations of this channel that are almost unknown.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF