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13 results on '"Mathilde Keck"'

1. A screening pipeline identifies a broad-spectrum inhibitor of bacterial AB toxins with cross protection against influenza A virus H1N1 and SARS-CoV-2

Author

Yu Wu, Nassim Mahtal, Léa Swistak, Sara Sagadiev, Mridu Acharya, Caroline Demeret, Sylvie van der Werf, Florence Guivel-Benhassine, Olivier Schwartz, Serena Petracchini, Amel Mettouchi, Eléa Paillares, Lucie Caramelle, Pierre Couvineau, Robert Thai, Peggy Barbe, Mathilde Keck, Priscille Brodin, Arnaud Machelart, Valentin Sencio, François Trottein, Martin Sachse, Gaëtan Chicanne, Bernard Payrastre, Florian Ville, Victor Kreis, Michel-Robert Popoff, Ludger Johannes, Jean-Christophe Cintrat, Julien Barbier, Daniel Gillet, and Emmanuel Lemichez

Subjects
Diphtheria toxin, biology, Toxin, Endosome, Chemistry, Toxin transport, Shiga toxin, medicine.disease_cause, Microbiology, PIKFYVE, AB toxin, biology.protein, Influenza A virus, medicine
Abstract

A challenge for the development of host-targeted anti-infectives against a large spectrum of AB-like toxin-producing bacteria encompasses the identification of chemical compounds corrupting toxin transport through both endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of small chemical compounds blocking active Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, followed by orthogonal screens against two AB toxins hijacking defined endolysosomal (Diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule N-(3,3-diphenylpropyl)-1-propyl-4-piperidinamine, referred to as C910. This compound induces the swelling of EEA1-positive early endosomes, in absence of PIKfyve kinase inhibition, and disturbs the trafficking of CNF1 and the B-subunit of Shiga toxin along the endolysosomal or retrograde pathways, respectively. Together, we show that C910 protects cells against 8 bacterial AB toxins including large clostridial glucosylating toxins from Clostridium difficile. Of interest, C910 also reduced viral infection in vitro including influenza A virus subtype H1N1 and SARS-CoV-2. Moreover, parenteral administration of C910 to the mice resulted in its accumulation in lung tissues and reduced lethal influenza infection.

Published
2021
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2. Analysis of CLCNKB mutations at dimer-interface, calcium binding site and pore reveals a variety of functional alterations in ClC-Kb channel leading to Bartter syndrome

Author

Marc Paulais, Imene Sakhi, Mathilde Keck, Yohan Bignon, Olga Andrini, Gabrielle Planelles, Naziha Bakouh, Sara Bitam, Nadia Frachon, Jacques Teulon, Centre de Recherche des Cordeliers (CRC), Université Pierre et Marie Curie - Paris 6 (UPMC)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Métabolisme et physiologie rénales (ERL 8228), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP), Institut NeuroMyoGène (INMG), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-École pratique des hautes études (EPHE), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Espace et action, Université de Lyon-Université de Lyon-Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM), Neuropeptides centraux et régulations hydrique et cardiovasculaire, Université Pierre et Marie Curie - Paris 6 (UPMC)-Collège de France (CdF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Paulais, Marc, Centre National de la Recherche Scientifique (CNRS)-Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
kidney, chloride channel, Xenopus, [SDV]Life Sciences [q-bio], Mutant, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, medicine.disease_cause, Bartter syndrome, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, CLCNKB, Cell Line, 03 medical and health sciences, Chloride Channels, Genetics, medicine, Animals, Humans, Genetics (clinical), ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Mutation, Binding Sites, biology, Endoplasmic reticulum, 030305 genetics & heredity, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, medicine.disease, biology.organism_classification, ClC-Kb, 3. Good health, Cell biology, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Protein Transport, Chloride channel, biology.protein, Oocytes, Calcium, Protein Multimerization, mutation, Intracellular, Protein Binding
Abstract

International audience; Pathological missense mutations in CLCNKB gene give a wide spectrum of clinical phenotypes in Bartter syndrome type III patients. Molecular analysis of the mutated ClC-Kb channels can be helpful to classify the mutations according to their functional alteration. We investigated the functional consequences of nine mutations in the CLCNKB gene causing Bartter syndrome. We first established that all tested mutations lead to decreased ClC-Kb currents. Combining electrophysiological and biochemical methods in Xenopus laevis oocytes and in MDCKII cells, we identified three classes of mutations. One class is characterized by altered channel trafficking. p.A210V, p.P216L, p.G424R, and p.G437R are totally or partially retained in the endoplasmic reticulum. p.S218N is characterized by reduced channel insertion at the plasma membrane and altered pH-sensitivity; thus, it falls in the second class of mutations. Finally, we found a novel class of functionally inactivated mutants normally present at the plasma membrane. Indeed, we found that p.A204T alters the pH-sensitivity, p.A254V abolishes the calcium-sensitivity. p.G219C and p.G465R are probably partially inactive at the plasma membrane. In conclusion, most pathogenic mutants accumulate partly or totally in intracellular compartments, but some mutants are normally present at the membrane surface and simultaneously show a large range of altered channel gating properties.

Published
2019
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3. ClC-K chloride channels: emerging pathophysiology of Bartter syndrome type 3

Author

Mathilde Keck, Jacques Teulon, Stéphane Lourdel, Olga Andrini, Rodolfo Briones, Rosa Vargas-Poussou, Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft, Service de Génétique [CHU HEGP], Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Centre de Recherche des Cordeliers (CRC), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Service de Génétique [AP-HP Hôpital Européen Georges Pompidou, Paris], and Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)

Subjects
chloride channel, Physiology, [SDV]Life Sciences [q-bio], Anion Transport Proteins, DNA Mutational Analysis, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Bartter syndrome, [SDV.MHEP.UN]Life Sciences [q-bio]/Human health and pathology/Urology and Nephrology, Chloride, Type (biology), Chloride Channels, medicine, Animals, Humans, Genetic Predisposition to Disease, ComputingMilieux_MISCELLANEOUS, Genetics, CLCNKB gene, medicine.disease, Pathophysiology, 3. Good health, Mutation, Chloride channel, CLC family of chloride transporters and channels, medicine.drug
Abstract

International audience; The mutations in the CLCNKB gene encoding the ClC-Kb chloride channel are responsible for Bartter syndrome type 3, one of the four variants of Bartter syndrome in the genetically based nomenclature. All forms of Bartter syndrome are characterized by hypokalemia, metabolic alkalosis, and secondary hyperaldosteronism, but Bartter syndrome type 3 has the most heterogeneous presentation, extending from severe to very mild. A relatively large number of CLCNKB mutations have been reported, including gene deletions and nonsense or missense mutations. However, only 20 CLCNKB mutations have been functionally analyzed, due to technical difficulties regarding ClC-Kb functional expression in heterologous systems. This review provides an overview of recent progress in the functional consequences of CLCNKB mutations on ClC-Kb chloride channel activity. It has been observed that 1) all ClC-Kb mutants have an impaired expression at the membrane; and 2) a minority of the mutants combines reduced membrane expression with altered pH-dependent channel gating. Although further investigation is needed to fully characterize disease pathogenesis, Bartter syndrome type 3 probably belongs to the large family of conformational diseases, in which the mutations destabilize channel structure, inducing ClC-Kb retention in the endoplasmic reticulum and accelerated channel degradation.

Published
2015
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4. Clinical and Genetic Spectrum of Bartter Syndrome Type 3

Author

Maria Mileva, Sandrine Lemoine, Christiane Mousson, Elsa Seys, Rosa Vargas-Poussou, Mathilde Cailliez, Gwenaëlle Roussey-Kesler, Aurélia Bertholet-Thomas, Paul Cozette, Theresa Kwon, Anne Blanchard, Guylhène Bourdat-Michel, Pauline Krug, Jean Sébastien Borde, Jacques Teulon, Christophe Simian, Laurence Dubourg, Robert Novo, Isabelle Vrillon, Bertrand Knebelmann, Brigitte Llanas, Djamal Djeddi, Dominique Chaveau, Stephen B. Walsh, Olga Andrini, François Nobili, Jean Daniel Delbet, Marc Fila, Georges Deschênes, Mathilde Keck, Ferielle Louillet, Lamisse Mansour-Hendili, Elodie Merieau, Stéphane Decramer, Noémie Jourde-Chiche, Marie-Pierre Lavocat, Pierre-Yves Courand, Luisa Mota-Vieira, Guillaume Bobrie, Pediatric Nephrology Unit, American Memorial Hospital, Reims University Hospital, Centre de Recherche des Cordeliers (CRC), Université Pierre et Marie Curie - Paris 6 (UPMC)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Paris Descartes - Paris 5 (UPD5)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Faculté de Médecine (UPD5 Médecine), Université Paris Descartes - Paris 5 (UPD5), Service de Génétique [CHU HEGP], Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Centre d'Investigation Clinique - Epidemiologie Clinique/essais Cliniques [CHU HEGP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM), Service de Cardiologie [Hôpital Croix- Rousse, HCL] (Centre d'excellence de la Société européenne d'hypertension artérielle), Hospices Civils de Lyon (HCL)-Hôpital de la Croix-Rousse [CHU - HCL], Hospices Civils de Lyon (HCL), Centre de Recherche en Acquisition et Traitement de l'Image pour la Santé (CREATIS), Université Jean Monnet [Saint-Étienne] (UJM)-Hospices Civils de Lyon (HCL)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Service Génétique, Union nationale des coopératives d’élevage et d’insémination animale (UNCEIA), Unité de Néphrologie Pédiatrique, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Robert Debré, Centre de Référence des Maladies Rénales Héréditaires de l'Enfant et de l'Adulte (MARHEA), CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre de Référence des Maladies Rénales Rares Néphrogones, Hôpital Femme Mère Enfant [CHU - HCL] (HFME), Hospices Civils de Lyon (HCL)-Hospices Civils de Lyon (HCL), Nephrology Unit, Clinique du Vert Galant, Tremblay-en-France, Nephrology Unit, Centre hospitalier de Saintonge, Saintes, Centre Hospitalo-Universitaire de Grenoble (CHU de Grenoble), CHU Grenoble, Service de pédiatrie multidisciplinaire [Hôpital de la Timone Enfants - APHM], Hôpital de la Timone [CHU - APHM] (TIMONE), Assistance Publique - Hôpitaux de Marseille (APHM), Pediatric Nephrology Unit, Centre Hospitalier du Pays d'Aix, Nephrology Unit, Centre Hospitalier du Pays d'Aix, CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Métabolomique et maladies métaboliques, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Référence du Sud Ouest des Maladies Rénales Rares, CHU Toulouse [Toulouse]-Hôpital des Enfants, CHU Toulouse [Toulouse], Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Vascular research center of Marseille (VRCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Service de néphrologie pédiatrique [CHU Necker], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre Hospitalier Universitaire de Saint-Etienne (CHU de Saint-Etienne), Cardiovasculaire, métabolisme, diabétologie et nutrition (CarMeN), Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National de la Recherche Agronomique (INRA), Pédiatrie médicale et médecine de l'adolescent [Amiens], CHU Amiens-Picardie, Service de pédiatrie néonatale et réanimation - neuropédiatrie [CHU Rouen], CHU Rouen, Normandie Université (NU)-Normandie Université (NU)-Hôpital Charles Nicolle [Rouen]-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Department of Pediatrics, Centre Hospitalier Pierre Oudot de Bourgoin-Jallieu, Bourgoin-Jallieu, Molecular Genetics and Pathology Unit, Hospital of Divino Espirito Santo of Ponta Delgada, Service de néphrologie (CHU de Dijon), Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon), Service de néphrologie pédiatrique [CHRU Besançon], Centre Hospitalier Régional Universitaire de Besançon (CHRU Besançon), Hôpital Jeanne de Flandres, Université de Lille, Droit et Santé-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Centre hospitalier universitaire de Nantes (CHU Nantes), Centre Hospitalier Régional Universitaire de Nancy (CHRU Nancy), Department of Aeronautical and Automotive Engineering, Loughborough University, Paris-Centre de Recherche Cardiovasculaire (PARCC - UMR-S U970), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Santé Lyon Est - Louis Léopold Ollier, Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Union Nationale des Coopératives Agricoles d'Elevage et d'Insémination Animale, Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpital Robert Debré, Unité d'hypertension artérielle, Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), CHU Necker - Enfants Malades [AP-HP]-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hospices Civils de Lyon (HCL), Service de nephrologie pédiatrique, Centre Hospitalier Régional Universitaire [Besançon] (CHRU Besançon), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP), Université de Paris - Faculté de Médecine Paris Centre (UP Médecine Paris Centre), Université de Paris (UP), Service de Génétique [AP-HP Hôpital Européen Georges Pompidou, Paris], Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-Université Jean Monnet [Saint-Étienne] (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre hospitalier universitaire de Grenoble (CHU de Grenoble), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université, Centre de néphrologie et transplantation rénale [Hôpital de la Conception - APHM], Assistance Publique - Hôpitaux de Marseille (APHM)-Hôpital de la Conception [CHU - APHM] (LA CONCEPTION), Service de pédiatrie néonatale et réanimation - neuropédiatrie [Rouen], Hôpital Charles Nicolle [Rouen]-CHU Rouen, Normandie Université (NU)-Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Université de Lyon, Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie - Paris 6 (UPMC)-École Pratique des Hautes Études (EPHE), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de Référence des Maladies Rénales Héréditaires de l'Enfant et de l'Adulte [CHU-Necker] (MARHEA), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Centre Hospitalier Universitaire de Saint-Etienne [CHU Saint-Etienne] (CHU ST-E), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Charles Nicolle [Rouen], and Normandie Université (NU)-Normandie Université (NU)-CHU Rouen

Subjects
0301 basic medicine, Adult, Male, medicine.medical_specialty, Adolescent, [SDV]Life Sciences [q-bio], Nonsense mutation, 030232 urology & nephrology, Bartter syndrome, urologic and male genital diseases, Hypocalciuria, 03 medical and health sciences, Young Adult, 0302 clinical medicine, Tubulopathy, Clinical Research, Internal medicine, Genotype, medicine, Humans, Child, Genetic Association Studies, ComputingMilieux_MISCELLANEOUS, Retrospective Studies, CLCNKB, biology, business.industry, Bartter Syndrome, Infant, General Medicine, Gitelman syndrome, medicine.disease, 3. Good health, Bartter's syndrome, 030104 developmental biology, Endocrinology, Nephrology, Child, Preschool, Mutation, biology.protein, Female, medicine.symptom, business
Abstract

Bartter syndrome type 3 is a clinically heterogeneous hereditary salt-losing tubulopathy caused by mutations of the chloride voltage-gated channel Kb gene (CLCNKB), which encodes the ClC-Kb chloride channel involved in NaCl reabsorption in the renal tubule. To study phenotype/genotype correlations, we performed genetic analyses by direct sequencing and multiplex ligation-dependent probe amplification and retrospectively analyzed medical charts for 115 patients with CLCNKB mutations. Functional analyses were performed in Xenopus laevis oocytes for eight missense and two nonsense mutations. We detected 60 mutations, including 27 previously unreported mutations. Among patients, 29.5% had a phenotype of ante/neonatal Bartter syndrome (polyhydramnios or diagnosis in the first month of life), 44.5% had classic Bartter syndrome (diagnosis during childhood, hypercalciuria, and/or polyuria), and 26.0% had Gitelman-like syndrome (fortuitous discovery of hypokalemia with hypomagnesemia and/or hypocalciuria in childhood or adulthood). Nine of the ten mutations expressed in vitro decreased or abolished chloride conductance. Severe (large deletions, frameshift, nonsense, and essential splicing) and missense mutations resulting in poor residual conductance were associated with younger age at diagnosis. Electrolyte supplements and indomethacin were used frequently to induce catch-up growth, with few adverse effects. After a median follow-up of 8 (range, 1-41) years in 77 patients, chronic renal failure was detected in 19 patients (25%): one required hemodialysis and four underwent renal transplant. In summary, we report a genotype/phenotype correlation for Bartter syndrome type 3: complete loss-of-function mutations associated with younger age at diagnosis, and CKD was observed in all phenotypes.

Published
2017
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5. Characterization of the mouse ClC-K1/Barttin chloride channel

Author

Alexei Diakov, Stéphane Lourdel, Jacques Teulon, Teddy Grand, Olga Andrini, Mathilde Keck, Sébastien L'Hoste, Christoph Korbmacher, Laurent Beck, Laurent Pinelli, Marc Paulais, Mathieu Genete, Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche des Cordeliers (CRC), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Université de Nantes - UFR Odontologie, Université de Nantes (UN), Université Paris Descartes - Paris 5 (UPD5)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ERL 7226, Centre National de la Recherche Scientifique (CNRS), Institut für Zelluläre und Molekulare Physiologie [Erlanger], Lourdel, Stéphane, Laboratoire de physiologie, physiopathologie et génomique rénales, Centre National de la Recherche Scientifique (CNRS)-Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité)-École Pratique des Hautes Études (EPHE), and Université de Nantes - UFR Odontologie (UFR Odonto)

Subjects
Patch-Clamp Techniques, [SDV]Life Sciences [q-bio], Xenopus, Biophysics, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Kidney, Biochemistry, Xenopus laevis, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Chloride Channels, ClC, medicine, Animals, Humans, Patch clamp, Distal convoluted tubule, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, ClC-K1, 0303 health sciences, biology, urogenital system, Depolarization, Cell Biology, biology.organism_classification, Recombinant Proteins, Connecting tubule, [SDV] Life Sciences [q-bio], Electrophysiology, HEK293 Cells, Kidney Tubules, medicine.anatomical_structure, chemistry, DIDS, Chloride channel, Patch-clamp, 030217 neurology & neurosurgery
Abstract

International audience; Several Cl(-) channels have been described in the native renal tubule, but their correspondence with ClC-K1 and ClC-K2 channels (orthologs of human ClC-Ka and ClC-Kb), which play a major role in transcellular Cl(-) absorption in the kidney, has yet to be established. This is partly because investigation of heterologous expression has involved rat or human ClC-K models, whereas characterization of the native renal tubule has been done in mice. Here, we investigate the electrophysiological properties of mouse ClC-K1 channels heterologously expressed in Xenopus laevis oocytes and in HEK293 cells with or without their accessory Barttin subunit. Current amplitudes and plasma membrane insertion of mouse ClC-K1 were enhanced by Barttin. External basic pH or elevated calcium stimulated currents followed the anion permeability sequence Cl(-)>Br(-)>NO3(-)>I(-). Single-channel recordings revealed a unit conductance of ~40pS. Channel activity in cell-attached patches increased with membrane depolarization (voltage for half-maximal activation: ~-65mV). Insertion of the V166E mutation, which introduces a glutamate in mouse ClC-K1, which is crucial for channel gating, reduced the unit conductance to ~20pS. This mutation shifted the depolarizing voltage for half-maximal channel activation to ~+25mV. The unit conductance and voltage dependence of wild-type and V166E ClC-K1 were not affected by Barttin. Owing to their strikingly similar properties, we propose that the ClC-K1/Barttin complex is the molecular substrate of a chloride channel previously detected in the mouse thick ascending limb (Paulais et al., J Membr. Biol, 1990, 113:253-260).

Published
2013
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6. Emergence of Orai3 activity during cardiac hypertrophy

Author

Youakim Saliba, Olivier Cazorla, Jean-Sébastien Hulot, Alain Lacampagne, Nassim Fares, Mathilde Keck, Anne Marie Lompré, Aude Ouillé, Mohamed Trebak, Fabrice Atassi, Jérémy Fauconnier, Alexandre Marchand, Catherine Pavoine, MORNET, Dominique, Laboratoire de Recherche en Physiologie et Physiopathologie, Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Research Unit on Cardiovascular and Metabolic Diseases (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut de Cardiométabolisme et Nutrition = Institute of Cardiometabolism and Nutrition [CHU Pitié Salpêtrière] (IHU ICAN), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046] (PhyMedExp), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Center for Cardiovascular Sciences, Albany Medical College, Icahn School of Medicine at Mount Sinai [New York] (MSSM), Université Saint-Joseph de Beyrouth (USJ), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Institute of cardiometabolism and nutrition (ICAN), Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [APHP], Université Pierre et Marie Curie - Paris 6 (UPMC), Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Signalisation et physiopathologie des cellules épithéliales, Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Mondor de recherche biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Physiopathologie, génétique et pharmacologie du remodelage cardiovasculaire, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR14-Institut National de la Santé et de la Recherche Médicale (INSERM), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), and Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Male, inorganic chemicals, medicine.medical_specialty, Small interfering RNA, Time Factors, ORAI1 Protein, Fura-2, Physiology, [SDV]Life Sciences [q-bio], Biology, Transfection, Membrane Potentials, Muscle hypertrophy, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Physiology (medical), Internal medicine, medicine, Animals, Myocytes, Cardiac, Calcium Signaling, Stromal Interaction Molecule 1, Rats, Wistar, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Calcium signaling, 0303 health sciences, Arachidonic Acid, Membrane Glycoproteins, Voltage-dependent calcium channel, ORAI1, Cardiac myocyte, STIM1, [SDV] Life Sciences [q-bio], Calcium Channel Agonists, Disease Models, Animal, Endocrinology, chemistry, Hypertrophy, Left Ventricular, RNA Interference, Calcium Channels, Cardiology and Cardiovascular Medicine, 030217 neurology & neurosurgery, Protein Binding
Abstract

Aims Stromal interaction molecule 1 (STIM1) has been shown to control a calcium (Ca2+) influx pathway that emerges during the hypertrophic remodeling of cardiomyocytes. Our aim was to determine the interaction of Orai1 and Orai3 with STIM1 and their role in the constitutive store-independent and the store-operated, STIM1-dependent, Ca2+ influx in cardiomyocytes. Methods and Results We characterized the expression profile of Orai proteins and their interaction with STIM1 in both normal and hypertrophied adult rat ventricular cardiomyocytes. Orai1 and 3 protein levels were unaltered during the hypertrophic process and both proteins co-immunoprecipitated with STIM1. The main partner of STIM1 in control cardiomyocytes was Orai1, but the abundance of Orai3 in the STIM1/Orai1/Orai3 macromolecular complex was markedly enhanced in hypertrophied cardiomyocytes. We then used a non-viral method to deliver Cy3-tagged siRNAs in vivo to adult ventricular cardiomyocytes and silence Orai channel candidates. Cardiomyocytes were subsequently isolated then the voltage-independent i.e. store-independent and store-operated Ca2+ entries were measured on Fura-2 AM loaded Cy3-labelled and control isolated cardiomyocytes. The whole cell patch-clamp technique was used to measure Orai-mediated currents. Specific Orai1 and Orai3 knockdown established Orai3, but not Orai1, as the critical partner of STIM1 carrying these voltage-independent Ca2+ entries in the adult hypertrophied cardiomyocytes. Orai3 also drove an arachidonic acid-activated inward current. Conclusion Cardiac Orai3 is the essential partner of STIM1 and drives voltage-independent Ca2+ entries in adult cardiomyocytes. Arachidonic acid-activated currents, which are supported by Orai3, are present in adult cardiomyocytes and increased during hypertrophy.

Published
2015
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7. Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

Author

Patricia Serradas, Armelle Leturque, Raphael Scharfmann, Sébastien L'Hoste, Jacques Teulon, Maude Le Gall, Aurélien Michau, Mathilde Keck, A. Grosfeld, E. Brot-Laroche, Ghislaine Guillemain, Pascale de Lonlay, Danielle Chateau, Sandrine Vuillaumier-Barrot, Teddy Grand, Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), U845 Department of Physiology, CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Service de biochimie hormonale, métabolique et génétique, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-AP-HP - Hôpital Bichat - Claude Bernard [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Diderot - Paris 7 (UPD7), Centre de référence pour les maladies métaboliques héréditaires [CHU Necker Enfants Malades - AP-HP], Université Paris Descartes - Paris 5 (UPD5)-CHU Necker - Enfants Malades [AP-HP], Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Research Unit on Cardiovascular and Metabolic Diseases (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut de Cardiométabolisme et Nutrition = Institute of Cardiometabolism and Nutrition [CHU Pitié Salpêtrière] (IHU ICAN), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Institute of cardiometabolism and nutrition (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Institut de Génomique Fonctionnelle (IGF), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche des Cordeliers (CRC), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Nutrition, métabolisme, obésité: aspect cellulaire et moléculaire, Institut National de la Santé et de la Recherche Médicale (INSERM), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-AP-HP - Hôpital Bichat - Claude Bernard [Paris]-Université Paris Diderot - Paris 7 (UPD7), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Pancréas endocrine et diabète de l'enfant, Centre de recherche sur l'Inflammation (CRI (UMR_S_1149 / ERL_8252 / U1149)), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)

Subjects
medicine.medical_treatment, Cellular differentiation, [SDV]Life Sciences [q-bio], Mutant, Mutation, Missense, Biological Transport, Active, Biology, medicine.disease_cause, Biochemistry, Polymorphism, Single Nucleotide, 03 medical and health sciences, Mice, Xenopus laevis, 0302 clinical medicine, Cell Line, Tumor, Insulin-Secreting Cells, Insulin Secretion, medicine, Animals, Humans, Insulin, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Glucose Transporter Type 2, 0303 health sciences, Mutation, Cell growth, Glucose transporter, Cell Differentiation, Cell Biology, Rats, Glucose, Metabolism, Amino Acid Substitution, biology.protein, GLUT2, Signal transduction, 030217 neurology & neurosurgery, Signal Transduction
Abstract

The structure-function relationships of sugar transporter-receptor hGLUT2 coded by SLC2A2 and their impact on insulin secretion and β cell differentiation were investigated through the detailed characterization of a panel of mutations along the protein. We studied naturally occurring SLC2A2 variants or mutants: two single-nucleotide polymorphisms and four proposed inactivating mutations associated to Fanconi-Bickel syndrome. We also engineered mutations based on sequence alignment and conserved amino acids in selected domains. The single-nucleotide polymorphisms P68L and T110I did not impact on sugar transport as assayed in Xenopus oocytes. All the Fanconi-Bickel syndrome-associated mutations invalidated glucose transport by hGLUT2 either through absence of protein at the plasma membrane (G20D and S242R) or through loss of transport capacity despite membrane targeting (P417L and W444R), pointing out crucial amino acids for hGLUT2 transport function. In contrast, engineered mutants were located at the plasma membrane and able to transport sugar, albeit with modified kinetic parameters. Notably, these mutations resulted in gain of function. G20S and L368P mutations increased insulin secretion in the absence of glucose. In addition, these mutants increased insulin-positive cell differentiation when expressed in cultured rat embryonic pancreas. F295Y mutation induced β cell differentiation even in the absence of glucose, suggesting that mutated GLUT2, as a sugar receptor, triggers a signaling pathway independently of glucose transport and metabolism. Our results describe the first gain of function mutations for hGLUT2, revealing the importance of its receptor versus transporter function in pancreatic β cell development and insulin secretion.

Published
2013
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8. Novel CLCNKB Mutations Causing Bartter Syndrome Affect Channel Surface Expression

Author

Sébastien L‘Hoste, L. Pablo Cid, Jacques Teulon, Olivier Lahuna, Francisco V. Sepúlveda, Johanna Burgos, Olga Andrini, Anne Blanchard, Mathilde Keck, Rosa Vargas-Poussou, Stéphane Lourdel, Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Paris Descartes - Paris 5 (UPD5)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centro de Estudios Científicos (CECs), Centre d'Investigation Clinique - Epidemiologie Clinique/essais Cliniques [CHU HEGP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Paris - UFR Médecine Paris Centre [Santé] (UP Médecine Paris Centre), Université de Paris (UP), Service de génétique [CHU HEGP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche des Cordeliers (CRC), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Faculté de Médecine (UPD5 Médecine), Université Paris Descartes - Paris 5 (UPD5), Service de Génétique [AP-HP Hôpital Européen Georges Pompidou, Paris], Hôpital Européen Georges Pompidou [APHP] (HEGP), Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), UFR Médecine [Santé] - Université Paris Cité (UFR Médecine UPCité), and Université Paris Cité (UPCité)

Subjects
Adult, Male, Adolescent, [SDV]Life Sciences [q-bio], Mutant, Anion Transport Proteins, Xenopus, Bartter syndrome, 03 medical and health sciences, Xenopus laevis, Young Adult, 0302 clinical medicine, Western blot, Tubulopathy, Chloride Channels, Genetics, medicine, Animals, Humans, Point Mutation, Genetics (clinical), ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, CLCNKB, medicine.diagnostic_test, biology, urogenital system, HEK 293 cells, Bartter Syndrome, Infant, medicine.disease, biology.organism_classification, Molecular biology, HEK293 Cells, Biochemistry, Chloride channel, biology.protein, Oocytes, Female, 030217 neurology & neurosurgery
Abstract

Mutations in the CLCNKB gene encoding the ClC-Kb Cl(-) channel cause Bartter syndrome, which is a salt-losing renal tubulopathy. Here, we investigate the functional consequences of seven mutations. When expressed in Xenopus laevis oocytes, four mutants carried no current (c.736G>C, p.Gly246Arg; c.1271G>A, p.Gly424Glu; c.1313G>A, p.Arg438His; c.1316T>C, p.Leu439Pro), whereas others displayed a 30%-60% reduction in conductance as compared with wild-type ClC-Kb (c.242T>C, p.Leu81Pro; c.274C>T, p.Arg92Trp; c.1052G>C, p.Arg351Pro). Anion selectivity and sensitivity to external Ca(2+) and H(+), typical of the ClC-Kb channel, were not modified in the partially active mutants. In oocytes, we found that all the mutations reduced surface expression with a profile similar to that observed for currents. In HEK293 cells, the currents in the mutants had similar profiles to those obtained in oocytes, except for p.Leu81Pro, which produced no current. Furthermore, p.Arg92Trp and p.Arg351Pro mutations did not modify the unit-conductance of closely related ClC-K1. Western blot analysis in HEK293 cells showed that ClC-Kb protein abundance was lower for the nonconducting mutants but similar to wild-type for other mutants. Overall, two classes of mutants can be distinguished: nonconducting mutants associated with low total protein expression, and partially conducting mutants with unaltered channel properties and ClC-Kb protein abundance.

Published
2013
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9. Abstract 313: STIM1 Silencing Reverses Pressure-overload Induced Cardiac Hypertrophy In Mice

Author

Ludovic Benard, Mathilde Keck, Daniel S Matasic, Roger J. Hajjar, Jean-Sébastien Hulot, and Anne-Marie Lompré

Subjects
Pressure overload, medicine.medical_specialty, Pathology, Physiology, Hemodynamics, STIM1, Biology, medicine.disease, Left ventricular hypertrophy, Muscle hypertrophy, Endocrinology, Fibrosis, Internal medicine, Heart failure, medicine, Myocyte, Cardiology and Cardiovascular Medicine
Abstract

STromal Interaction Molecule 1 (STIM1), a membrane protein of the sarcoplasmic reticulum, has recently been proposed as a positive regulator of cardiomyocyte growth by promoting Ca2+ entry through the plasma membrane and the activation of Ca2+-mediated signaling pathways. We demonstrated that STIM1 silencing prevented the development of left ventricular hypertrophy (LVH) in rats after abdominal aortic banding. Our aim was to study the role of STIM1 during the transition from LVH to heart failure (HF). For experimental timeline, see figure. Transverse Aortic Constriction (TAC) was performed in C57Bl/6 mice. In vivo gene silencing was performed using recombinant Associated AdenoVirus 9 (AAV9). Mice were injected with saline or with AAV9 expressing shRNA control or against STIM1 (shSTIM1) (dose: 1e+11 viral genome), which decreased STIM1 cardiac expression by 70% compared to control. While cardiac parameters were similar between the TAC groups at weeks 3 and 6, shSTIM1 animals displayed a progressive and total reversion of LVH with LV walls thickness returning to values observed in sham mice at week 8. This reversion was associated with the development of significant LV dilation and severe contractile dysfunction, as assessed by echography. Hemodynamic analysis confirmed the altered contractile function and dilation of shSTIM1 animals. Immunohistochemistry showed a trend to more fibrosis. Despite hypertrophic stimuli, there was a significant reduction in cardiac myocytes cross-section area in shSTIM1-treated animals as compared to other TAC mice. This study showed that STIM1 is essential to maintain compensatory LVH and that its silencing accelerates the transition to HF.

Published
2013
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11. Renal phenotype in mice lacking the Kir5.1 (Kcnj16) K+ channel subunit contrasts with that observed in SeSAME/EAST syndrome

Author

May Bloch-Faure, Marc Paulais, Stéphane Lourdel, Mathilde Keck, Suresh Krishna Ramakrishnan, Pascal Houillier, Jacques Teulon, Dominique Eladari, Thibaut Jacques, Nicolas Picard, Fabien Sohet, Stephen J. Tucker, Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Clarendon Laboratory, Department of Physics, University of Oxford [Oxford], Laboratoire de physiologie, physiopathologie et génomique rénales, Centre National de la Recherche Scientifique (CNRS)-Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Université Paris Descartes - Faculté de Médecine (UPD5 Médecine), Université Paris Descartes - Paris 5 (UPD5), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Clarendon Laboratory [Oxford], University of Oxford, This work was supported by Agence Nationale de la Recherche (ANR) Grant Physio 2007-RPVO7084. M.K. holds a PhD fellowship from the French Ministère de la Recherche. S.K.R. holds a PhD fellowship from the Erasmus Mundus External Cooperation Window Lot 15 India European scholarship program. M.P. is an Institut National de la Santé et de la Recherche Médicale researcher. S.J.T. was supported by the Royal Society., Paulais, Marc, Laboratoire de physiologie génomique et de physiopathologie rénale (CNRS ERL 7226), Centre National de la Recherche Scientifique (CNRS)-Centre de Recherche des Cordeliers, École Pratique des Hautes Études (EPHE), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité)-École Pratique des Hautes Études (EPHE)

Subjects
medicine.medical_specialty, Patch-Clamp Techniques, Intracellular pH, [SDV]Life Sciences [q-bio], 030232 urology & nephrology, Hypokalemia, KCNJ10, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.MHEP.UN]Life Sciences [q-bio]/Human health and pathology/Urology and Nephrology, Amiloride, Mice, 03 medical and health sciences, 0302 clinical medicine, Sodium Potassium Chloride Symporter Inhibitors, Tubulopathy, Furosemide, Internal medicine, EAST syndrome, medicine, Animals, Humans, Hypercalciuria, Distal convoluted tubule, Potassium Channels, Inwardly Rectifying, Diuretics, Ion transporter, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Mice, Knockout, 0303 health sciences, Multidisciplinary, biology, Chemistry, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Syndrome, Biological Sciences, medicine.disease, [SDV.MHEP.UN] Life Sciences [q-bio]/Human health and pathology/Urology and Nephrology, Hydrochlorothiazide, Kidney Tubules, Phenotype, Endocrinology, medicine.anatomical_structure, biology.protein, medicine.symptom, Acidosis, Sodium Channel Blockers
Abstract

The heteromeric inwardly rectifying Kir4.1/Kir5.1 K + channel underlies the basolateral K + conductance in the distal nephron and is extremely sensitive to inhibition by intracellular pH. The functional importance of Kir4.1/Kir5.1 in renal ion transport has recently been highlighted by mutations in the human Kir4.1 gene ( KCNJ10 ) that result in seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME)/epilepsy, ataxia, sensorineural deafness, and renal tubulopathy (EAST) syndrome, a complex disorder that includes salt wasting and hypokalemic alkalosis. Here, we investigated the role of the Kir5.1 subunit in mice with a targeted disruption of the Kir5.1 gene ( Kcnj16 ). The Kir5.1 −/− mice displayed hypokalemic, hyperchloremic metabolic acidosis with hypercalciuria. The short-term responses to hydrochlorothiazide, an inhibitor of ion transport in the distal convoluted tubule (DCT), were also exaggerated, indicating excessive renal Na + absorption in this segment. Furthermore, chronic treatment with hydrochlorothiazide normalized urinary excretion of Na + and Ca 2+ , and abolished acidosis in Kir5.1 −/− mice. Finally, in contrast to WT mice, electrophysiological recording of K + channels in the DCT basolateral membrane of Kir5.1 −/− mice revealed that, even though Kir5.1 is absent, there is an increased K + conductance caused by the decreased pH sensitivity of the remaining homomeric Kir4.1 channels. In conclusion, disruption of Kcnj16 induces a severe renal phenotype that, apart from hypokalemia, is the opposite of the phenotype seen in SeSAME/EAST syndrome. These results highlight the important role that Kir5.1 plays as a pH-sensitive regulator of salt transport in the DCT, and the implication of these results for the correct genetic diagnosis of renal tubulopathies is discussed.

Published
2011
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12. Functional analysis of novel CLCNKB mutations in patients with Bartter syndrome

Author

Mathilde Keck, Anne Blanchard, Mathieu Genete, Teddy Grand, Jacques Teulon, Rosa Vargas-Poussou, Sébastien L'Hoste, and Stéphane Lourdel

Subjects
Genetics, CLCNKB, Functional analysis, biology, urogenital system, business.industry, Metabolic alkalosis, food and beverages, urologic and male genital diseases, medicine.disease, Bartter syndrome, Biochemistry, Tubulopathy, biology.protein, Medicine, In patient, business, Molecular Biology, Biotechnology
Abstract

Bartter’s syndrome is a hereditary renal tubulopathy characterized by salt-wasting and hypokalaemic metabolic alkalosis, which can be due to loss-of-function mutations in genes encoding ClC-Kb / Ba...

Published
2011
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13. Heterogeneity in the processing of ClC-5 mutants related to Dent disease

Author

Nadia Defontaine, Mathieu Genete, Mathilde Keck, David Mordasini, Kamel Laghmani, Jacques Teulon, Teddy Grand, Sébastien L'Hoste, Thomas Pennaforte, Stéphane Lourdel, Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ERL 7226, Centre National de la Recherche Scientifique (CNRS), Laboratoire de physiologie, physiopathologie et génomique rénales, Centre National de la Recherche Scientifique (CNRS)-Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité)-École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Centre de Recherche des Cordeliers ( CRC (UMR_S 872) ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Centre National de la Recherche Scientifique ( CNRS ), Université Paris Descartes - Paris 5 (UPD5)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Sorbonne Université (SU)

Subjects
[SDV]Life Sciences [q-bio], Mutant, Xenopus, Dent Disease, ClC-5, Kidney Tubules, Proximal, Xenopus laevis, 0302 clinical medicine, Jacques, Genetics (clinical), ComputingMilieux_MISCELLANEOUS, Cells, Cultured, INSERM, 0303 health sciences, biology, Life Sciences, Cell biology, Complete List of Authors: Grand, David, Biochemistry, ERL 7226 Key Words: Dent's disease, ERL 7226 Mordasini, ERL 7226 Keck, Endosome, CNRS, Protein subunit, Nadia, Molecular Sequence Data, ERL 7226 Lourdel, Chloride/proton exchanger, Kamel, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Processing, ERL 7226 L'Hoste, Mathilde, 03 medical and health sciences, Chloride Channels, Genetics, Stéphane, Animals, Humans, Amino Acid Sequence, Sébastien, 030304 developmental biology, UPMC Univ Paris 06, ERL 7226 Pennaforte, Teddy, Mathieu, urogenital system, CLCN5, Endoplasmic reticulum, HEK 293 cells, ERL 7226 Teulon, biology.organism_classification, Thomas, HEK293 Cells, Mutation, biology.protein, Oocytes, ERL 7226 Defontaine, UMR_S 872, Sequence Alignment, 030217 neurology & neurosurgery, ERL 7226 Laghmani, ERL 7226 Genete
Abstract

International audience; Mutations 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.

Published
2011
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