Département de Pharmacologie et de Toxicologie

RAPPORT D’ACTIVITÉ 2002-2003

Département de Pharmacologie et de Toxicologie

RAPPORT D’ACTIVITÉ 2002-2003

COVER: Expression of the Hoxb-7 promoter using the Cre-Rosa26 reporter system in

mouse kidney

The activity of Hoxb7-Cre as assayed by analysis of the Rosa reporter. A stained, thick

section of a mouse kidney reveals Hoxb7-Cre activity throughout the cortical and

medullary collecting duct system exclusively (50x magnification), demonstrating the

feasibility of specific gene inactivation in the collecting duct system. This promoter was

used to selectively inactivate the epithelial sodium channel (ENaC) in the collecting duct

and to demonstrate that the expression of ENaC in this nephron segment is not a

prerequisite for achieving sodium and potassium balance in mice. This stresses the

importance of more proximal nephron segments (late distal convoluted tubules or

connecting tubules) to achieve sodium and potassium balance. See reference: Isabelle

Rubera, Johannes Loffing, Lawrence G. Palmer, Gustavo Frindt, Nicole Fowler-Jaeger,

Daniel Sauter, Tom Carroll, Andrew McMahon, Edith Hummler and Bernard C. Rossier.

Collecting duct specific gene inactivation of αENaC in the mouse kidney does not impair

sodium and potassium balance, Journal of Clinical Investigation, 112, p554, 2003.

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TABLE DES MATIÈRES Page

PRÉFACE 3EFFECTIFS ET BUDGET DE L’IPT 5ENSEIGNEMENTS DISPENSÉS PAR L’IPT 10SUBSIDES DE RECHERCHES A L’IPT 2002-2003 12

1 . Homéostasie du sodium et ses implications dans lespathologies cardiovasculaires, pulmonaires et rénalesGroupe B. C. Rossier 17Salt-sensitive hypertension: identification of downstream renal targetsin the aldosterone signaling pathway and their functional role in vivoGroupe D. Firsov 21Genes responsible for the long-term genomic action of vasopressin: functionalcharacterization of identified candidates (II)Groupe E. Hummler 23Functional analysis of the ENaC in vivo.ENaC and its positive regulator, the channel activating protease 1 (CAP1):molecular and functional characterization of ENaC-mediated sodium transportin skin and lungGroupe L. Schild 26Function and regulation of the epithelium Na channel (ENaC)Group S. Kellenberger 28Contribution of acid-sensing ion channels ASICs to electrical signaling in neurons

2 . Molecular control of glucose and energy homeostasis in healthand diseaseGroupe B. Thorens 31

3 . Biosynthèse, régulation et relation structure-fonction desprotéines de transport ioniqueGroupe K. Geering 34Protein-Protein Interactions in the Regulation of Na,K-ATPase Expressionand FunctionGroupe J.-D. Horisberger 37Structure function relationship of the Na,K-ATPaseThe epithelial Na channel (ENaC)Groupe O. Staub 40Role of intracellular protein-protein interaction in ion channel regulationGroup J. Loffing 42Control of electrolyte homeostasis by the distal nephronGroup H. Abriel 44Molecular Physiology and Pathophysiology of the Cardiac Action Potential:Regulation of hNav1.5 and hERG

4 . Récepteurs couplés aux protéines G et mécanismesde signalisation intracellulaireGroupe S. Cotecchia 46The alpha1-adrenergic receptor subtypes: molecular mechanisms of receptorfunction and physiological implicationsGroup D. Diviani 48The AKAP-Lbc signaling complex: molecular characterizationand functional implications

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5 . Toxicologie du NO et génotoxicité des contaminants alimentairesGroupe E. Felley-Bosco 50Cellular biology of inducible nitric oxide synthase (iNOS) in human epithelial cellsGroupe P.-M. Morgenthaler-Leong 52Induction of genetic changes by environmental contaminants

6 . Neurophysiologie de l’olfaction et du goûtGroupe M.-C. Broillet 53Cyclic nucleotide-gated channels: Their roles in neuronal developmentand sensory transduction cascadesInfluence of aging on salt taste perception and its significance for salt inkake

PUBLICATIONS 55

Département de Pharmacologie et de Toxicologie de l’Université de LausanneRue du Bugnon 27, CH-1005 LausanneTél.: 41-21-692 53 50, Fax: 41-21-692 53 55,e-mail: @ipharm.unil.ch, www.unil.ch/ipharm

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PRÉFACE

Les deux missions principales du département de pharmacologie et de toxicologie (DPT)sont l'enseignement et la recherche.

En ce qui concerne l'enseignement (page 10 du rapport), les enseignements de 2ème et de3ème cycles et de formation continue n'ont pas été modifiés de façon significative par rapportà la précédente période d'activité (2000-2001). La création de la nouvelle Faculté deBiologie et de Médecine (FBM), en septembre 2003, va considérablement modifier lecurriculum. De plus, le système de Bologne (batchelor et master) sera introduit dès lesemestre d'hiver 2004. L'organisation de l'enseignement, maintenant sous la responsabilitéde l'Ecole de Biologie et de l'Ecole de Médecine, constitue l'autre élément des changementsque subit actuellement notre Faculté. L'évaluation des enseignements de 2ème cycle, soit enmédecine, soit en biologie, est effectuée chaque année et montre que dans l'ensemble,l'enseignement de la pharmacologie et de la toxicologie est très bien perçu par les étudiants.En ce qui concerne l'enseignement de 3ème cycle et post-gradué, le corps professoral duDPT est fortement impliqué sur le plan local, national et international. La liste desséminaires et des symposiums auxquels participent notre corps professoral le démontreamplement, tant par la quantité que par la qualité des institutions qui ont bénéficié de cesenseignements. Le DPT est également fortement impliqué dans les enseignements de 3ème

cycle, organisés dans le cadre du Département cœur-vaisseaux (ancienne dénominationLUL) et du Département de pharmacologie et de toxicologie. Dans le cadre de la FBM, lefutur de ces départements doit être rediscuté et la possibilité de créer des écoles doctoralessur l'arc lémanique doit être sérieusement envisagée.

En ce qui concerne la recherche, au cours de la dernière période, les thématiques derecherche du Département de pharmacologie et de toxicologie ont continué à se développeret se diversifier selon les axes suivantes:

1. Homéostasie du sodium et ses implications dans les pathologies cardiovasculaires,pulmonaires et rénales.

2. Homéostasie du glucose et ses implications dans les pathologies du métabolismenutritionnel (obésité et diabète).

3. Biosynthèse, régulation et relation structure-fonction des protéines de transportionique.

4. Récepteurs couplés aux protéines G et mécanismes de signalisation intracellulaire.5. Toxicologie du NO et génotoxicité des contaminants alimentaires.6. Neurophysiologie de l’olfaction et du goût.

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Le groupe, dirigé par le Professeur Hugues Abriel, qui étudie la cardiologie moléculaire etl'analyse génétique des troubles du rythme cardiaque, a commencé son travail auDépartement en octobre 2001, bénéficiant d'un subside de professeur boursier du FondsNational. En ce qui concerne la thématique du métabolisme du glucose, le ProfesseurBernard Thorens a été nommé en 2001 professeur ordinaire au Département dePhysiologie, avec affiliation au Département de pharmacologie et de toxicologie, unedouble affiliation non seulement justifiée par de nombreux intérêts communs entre lesdifférentes thématiques du DP et du DPT, mais aussi par le fait que l'ensemble du groupede recherche du Professeur Thorens continuera à travailler dans le bâtiment du Bugnon 27,jusqu'au moment où il pourra emménager dans les nouvelles surfaces qui seront aménagéesau Département de Physiologie au Bugnon 7.

La qualité de la recherche du DPT reste très élevée, comme l'atteste la liste des publicationsjointe à ce rapport d'activité ainsi que les analyses bibliométriques quantitatives des chefsdes groupes de recherche, qui sont pratiquées régulièrement lors des évaluationsquadriennales ou tous les huit ans.

Du point de vue des services, à relever la création d'une plate-forme de transgenèse àlaquelle un des membres du DPT (Edith Hummler) est fortement impliquée, puisque elle aété nommée responsable par la FBM et la mise en route de cette nouvelle plate-forme de laFBM doit se faire au cours du semestre d'hiver 2003-2004.

Du point de vue financement, la situation budgétaire, en particulier le financement de l'Etat,reste toujours très problématique. A cela s'ajoute une situation très défavorable dufinancement fédéral, par l'intermédiaire du Fonds National Suisse de la RechercheScientifique. Comme le montrent les données comparatives (page 9), l'engagement del'Etat, par l'intermédiaire du budget de l'UNIL, a passé de 64% en 1997 à 55% en 1998-1999, à 53% pour la période 2000-2001 et à 55% en 2002-2003, alors que le budget globalde l'Institut passait de Frs 7'155'000 en 1998-1999 à Frs 7'469'396 en 2002-2003,témoignant ainsi de la capacité de l'IPT à obtenir un soutien financier extérieur relativementplus important. Cette situation n'est qu'apparemment positive, car il faut tenir compte d'unfacteur très significatif qui est celui, d'une part, de l'augmentation des coûts courants de larecherche, en particulier en raison de l'introduction de projets de type génomique ou post-génomique et, d'autre part, la nécessité impérieuse d'investir dans l'acquisitiond'équipement lourd ou de son renouvellement.

La création de la FBM est une opportunité unique de créer de nouvelles relations etcollaborations entre la biologie fondamentale et la clinique. Le DPT, avec un intérêt naturelpour le développement d'une recherche translationnelle et des compétences importantes enbiologie fondamentale, devrait pouvoir jouer un rôle important dans ces nouveauxdéveloppements.

Bernard C. RossierDirecteur DPT

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1 . EFFECTIFS 2002-2003 (Situation au 31.12.2003)

° CORPS ENSEIGNANT

H. Abriel Professeur assistant (Professeur boursier FNS)K. Besseghir Privat-docentM.-C. Broillet Maître d’enseignement et de recherche, privatdocentS. Cotecchia Professeure ordinaire, coprésidente de la Section des sciences

fondamentalesJ. Diezi Professeur ordinaireE. Felley-Bosco Professeure assistante remplaçante (décharge rectorale)D. Firsov Maître d’enseignement et de rechercheK. Geering Professeure associéeJ.-D. Horisberger Professeur ordinaire, directeur a.i.E. Hummler Maître d’enseignement et de recherche, privat-docent,

coordinateur de la plate-forme de transgénèse (TAF)P. Iynedjian Privat-docentB. Rossier Professeur ordinaire, directeurL. Schild Professeur associéO. Staub Professeur associéB. Thorens Professeur ordinaire (Département de Physiologie)

° PROFESSEUR(E)S HONORAIRES

G. Peters

° CORPS INTERMÉDIAIRE

D. Claeys Chercheur DocteurD. Diviani Maître assistant remplaçant (boursier Commission de la

recherche de la Faculté de biologie et de médecine)S. Kellenberger Maître assistant (poste-bridge financé par le Rectorat)D. Loffing Maître assistante (relève Confédération)J. Loffing Maître assistant (boursier Cloëtta)P.-M. Morgenthaler-Leong Maître assistante remplaçante (décharge rectorale)

° ASSISTANT(E)S

Postdoctorant(e)s

A. Appert-Collin Bennasroune M. Auberson I. BadyS. Bibert Ch. Boixel A. ChraïbiS. Cottet Parvex G. Crambert M. DallaportaR. Duc M. Flahaut M. ForetzD. Genoux M. Guitard S. HuangD.-I. Karara (campus virtuel) H. Neubauer H. L. OlsenC. Poussin F. Preitner R. L. Stanasila VollmerM. A. Thomas M. Uldry M. van BemmelenG. Vuagniaux M. Xue

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Assistant(e)s médecins

O. Muller M. Nicod (campus virtuel)

Boursier(ère)s

D. Andreasen Th. Jespersen

Doctorant(e)s

M. André L. Baisamy C. BoschatO. Capendeguy V. de Fourmestraux Ch. DebonnevilleS. Y. Flores Urushima F. Fouladkou (Uni Berne) B. GavilletE. Gonzalez-Rodriguez M. Harris S. KlingerC. Leyvraz C. Li N. MartyM. Membrez A. Mercier S. MichligR. Nielsen C. Pélofi O. PoirotM. Rebetez J.-C. Rougier M. VukicevicM. Widmer

Diplômant(e)s

P. Devaud A. Mercier C. S. TauxeF. Terranova

Stagiaires, collaborations scientifiques

C. Da Silva Pinheiro R. Maggio E. ÖgzeC. Planès V. Ponce de Léon I. RuberaM. Sanchez-Sandoval S. S. Wildman

° PERSONNEL TECHNIQUE

Laborantin(e)s

L. Abuin M.-J. Arias (80%) F. ApothélozG. Centeno E. Charrière P. Devaud (80%)W. Dolci (80%) M. Emery N. Fowler Jaeger (50%)H.-P. Gaeggeler I. Gautschi M. GonzalezB. Guisan (70%) P. Hausel (60%) S. Kharoubi Hess (50%)H. Latado A.-M. Mérillat M. Nenniger TosatoC. Pfister Y. Pfister O. RandinS. Roy (50%) D. Schaer (50%) D. Tarussio (50%)

Aides laborantin(e)s

A. C. Da Costa (60%)

Apprenti(e)s laborantin(e)s

S. Chabanel A. C. Da Costa H. LatadoJ.-Y. Schäl

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Etudian(e)ts (jobs d’été)

Th. Besson A. Boesch S. BonvallatD. Cardelli J. Dey A. GaulisS. Nguyen Quac Vinh S. Offner L. PansierS. Rossier D. Thuillard

Stagiaires laborantin(e)s (école, CFC)

S. Brunetti G. Celik E. CharrièreD. Daidié C. Gogniat I. LambercyV. Mauroux E. Vazquez M. Weier

Documentaliste

M. Guidoux (campus virtuel)

° SERVICES GÉNÉRAUX

Secrétariat Comptabilité

I. Rivier Flühmann (60%) Ch. DemontN. Skarda-Coderey (80%) P. Gaudard (80%)M. Laverrière Schultz (30%)

Bibliothèque Atelier

J.-C. Broillet (20%) E. Delacrétaz

Animaleries Achats

L. Fabia (50%) R. GanderY. Guibert (60%)

H. Amoroso, J.-P. Roth (remplaçants)

Apprenti(e)s gardien(ne)s d’animaux

A. Bartolj

Laverie

D. De Gregorio-Mattascio (30%)P. Durgniat (30%)

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5. MEMBRES DU PERSONNEL AYANT QUITTÉ LE DÉPARTEMENT EN2002-2003

CORPS INTERMÉDIAIRE

P.M. Morgenthaler août 2003

° ASSISTANT(E)S

Postdoctorant(e)s

A. Chraïbi juillet 2003S. Cottet Parvex septembre 2003G. Crambert septembre 2003M. Dallaporta août 2003R. Duc juillet 2002M. Flahaut décembre 2002M. Foretz juillet 2003M. Guitard mars 2003H. Neubauer mars 2003F. Preitner août 2003M. A. Thomas février 2003M. Uldry juin 2003G. Vuagniaux février 2002M. Xue juillet 2002

Assistant(e)s médecins

M. Nicod septembre 2003

Doctorant(e)s

Ch. Debonneville décembre 2003R. Nielsen septembre 2003C. Pélofi septembre 2003M. Rebetez décembre 2003

° PERSONNEL TECHNIQUE

Laborantin(e)s

M.-J. Arias novembre 2002P. Devaud août 2003M. Gonzalez octobre 2003

Aides laborantin(e)s

A. C. Da Costa août 2003

° SERVICES GÉNÉRAUX

Comptabilité

P. Gaudard août 2002

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BUDGET 2002-2003 (MOYENNE DE DEUX ANS)

FNRS ET AUTRES FONDS45%

UNIL (ETAT DE VAUD)55%

UNIL (ETAT DE VAUD ) Fr. 4'110'416.00

Ressources extérieures Fr. 3'358'980.00

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DEPARTEMENT DE PHARMACOLOGIE ET DE TOXICOLOGIEENSEIGNEMENTS

2ème cycleEtudiants Nom du cours Nombre d’heures/an Nombre

C TP S APP étudiants

Méd. 3ème Pharmacologie- 75 -- -- 16 ~140toxicologie généraleet spéciale

Séminaires de -- -- 14 -- ~140pharmacologie

Méd. 4ème Ethique biomédicale 14 -- -- 30-80(cours option)

Enseignement informatif sur 12 -- -- -- 10-30les médecines parallèles(cours option)

Pharm. 4ème Pharmacologie-toxicologie 36 -- 14 -- 40-50générale et spéciale

Biologie Module de 110 250 26 16 18-22pharmacologie-toxicologie

Cours complémentaire de 28 -- -- -- 10-15pharmacologie et toxicologie

TOTAL 275 250 54 32

3ème cycle 3ème Cycle romand de biologie 1 semaine/an3ème Cycle pharmacie 1 semaine/an3ème Cycle Département depharmacologie et de toxicologie

1 semaine/an

Formation continue Toxicologie et nutrition 35 participantsCours post-grade d'éthique biomédicale 25 participantsLes pathologies du systèmecardiovasculaire

10-20 participants

Les neurotransmetteurs: du récepteur à lafonction 10-20 participants

C = Cours ex cathedra TP = Travaux Pratiques S = SéminairesAPP = Apprentissage Par Problème

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DEPARTEMENT DE PHARMACOLOGIE ET DE TOXICOLOGIE

ENSEIGNEMENTS

Activités de service et d'expertise

UNIL:Commissions permanentes et ad hoc décanales et rectorales

Confédération:Office Fédéral de la Santé PubliqueSwissmedicToxicologie alimentaireComité d'expertsConseil de l'EuropeCommission Fédérale de l'alimentationConseil National de la Recherche (FNRS)

Commission de biologie, Académie suisse des sciences naturelles

Service d'expertises et de conseils externes(Tribunaux, Conseil en pharmacologie et toxicologie, thèses, etc.)

Rédaction de périodiques d'information médicale (Le Fait Médical, Pharma-flash)

Comités éditoriaux: Arch. Toxicol., Ann. Rev. Physiol., Experimental Nephrology,J. of Gen. Physiol., Am. J. Physiol. (Endocrinol. & Metab.,Renal Physiology), Nephron

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DEPARTEMENT DE PHARMACOLOGIE ET DE TOXICOLOGIE

SUBSIDES DE RECHERCHES 2002-2003(ordre alphabétique des requérants principaux)

1 . H. Abriel

Subside N° 632-66149.01 du Fonds National de la Recherche Scientifique, 1.4.2002-31.3.2006“Molecular Physiology and Pathophysiology of the Cardiac Action Potential:Regulation of hNav1.5 and hERG”

2 . M.-C. Broillet

Fonds National Suisse De La Recherche Scientifique. Subside de recherche, "CyclicNucleotide-Gated Channels: Their roles in neuronal development andsensory transduction cascades", requête 31-63768.00 (2001-2003). Requéranteunique: M.-C Broillet

Fondation Novartis. "Roles of Cyclic Nucleotide-Gated Channels in neuronaldevelopment". Salaire doctorante (2002)

Fondation Roche. "Understanding the bitter taste quality: from a molecular toa physiological approach". Salaire doctorante (2002)

Fonds National Suisse De La Recherche Scientifique. Subside de recherche, "CyclicNucleotide-Gated Channels: Multiple isoforms, multiple roles", requête3100A0-102118/1 (2003-2006). Requérante unique: M.-C Broillet

Fondation Leenaards. "Pheromone information coding in the mammalianvomeronasal system". Requérants: M.-C. Broillet et I. Rodriguez (2003-2006).

3 . S. Cotecchia

Subside N° 31-51043.97 du Fonds National de la Recherche Scientifique, 1.10.97-30.9.02avec extension jusqu’au 31.3.03'The alpha1-adrenergic receptor subtypes: structural basis of receptorfunction and drug action'

Subside N° du Fonds National de la Recherche Scientifique, 1.04.03-31.3.06"Protein-protein interactions at the alpha1-adrenergic receptor subtypes:molecular basis and functional implications"

Subside N° 4047-057572/1 NRP-EPFL Tandem (H. Vogel) du Fonds National de laRecherche Scientifique, 1.4.00-31.3 03

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'Bioassay on a chip: G protein activation on microarrays of G protein-coupled receptors'

CTI projet N° 5636.2 TNS, Tandem avec EPFL-Vogel/Renaud/Pitsch, 1.10.01-30.9.03'Single molecule spectroscopy for probing receptor signalling in vivo andin vitro'

4 . D. Diviani

Subside N° 3100-067955.02 du Fonds National de la Recherche Scientifique, 1.10.2002-30.9.2005‘The AKAP-Lbc signaling complex: molecular characterization andfunctional implications’

5 . E. Felley-Bosco

Swiss National Science Foundation grant, 2002-2003, no. 31-64980.01. "Cellularbiology of inducible nitric oxide synthase (iNOS) in human epithelial cells"

Swiss National Science Foundation grant, NRP50, no. 4050-66591, 2002-2005, co-applicant (EFB): "Endocrine disruption in soil invertebrates: assessingmultigeneration effects and developing a proteomic biomarker approach".5th PCRD key action Quality Of Life And Management Of Living Resources.Proposal Fulltitle : MULTIDISCIPLINARY APPROACH TO AIRBORNE POLLUTANTHEALTH RELATED ISSUES : Modelization with combustion engineexhausts. QLRT2001-02357. University of Lausanne (E.Felley-Bosco),(2002-2005)

6 . D. Firsov

Swiss National Science Foundation, grant 2001-2004, No 3100-65140.01"Genes responsible for the long-term genomic action of vasopressin:functional characterization of identified candidates"

7 . K. Geering

Subside N° 31-64793.01/1 du Fonds National de la Recherche Scientifique, 1.10.01-30.9.04'Protein-protein interactions in the regulation of Na,K-ATPase expressionand function'

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8 . J.-D. Horisberger

Subside N° 31-65441.01 du Fonds National de la Recherche Scientifique, 1.10.01-31.09.04"Mechanism of cation translocation by the Na,K-ATPase and related P -ATPases"

Subside N° RG 0261 de Human Frontier Science Program (HSFP), 1.6.00-31.05.03"Molecular basis for regulation of the epithelial sodium channel (ENaC) byhormones, interacting proteins, ions"

Subside N° 205320-101467/1 du Fonds National de la Recherche Scientifique, 1.11.03-31.10.05" Fluidic rapid exchange microsystem for electrophysiological studies onsingle cells"(Requérant principal : Prof. M- Gijs, EPFL)

9 . E. Hummler

Subside N° 31-63801.00/1 du Fonds National de la Recherche Scientifique, 1.4.01-31.3.03'Molecular physiology of ENaC-mediated sodium transport in skin and lungepithelium'

Subside N° 3100-63801.00/1 du Fonds National de la Recherche Scientifique, 1.10.2003-30.9.2006 "ENaC and its positive regulator, the channel activating protease1(CAP1): molecular and functional characterization in epidermalremodeling, differentiation and physiology"

1 0 . S. Kellenberger

Subside No 3100-065233, 2001-2004, du Fonds National de la Recherche Scientifique"Contribution of acid-sensing ion channels (ASICs) to electrical signalingin neurons"

1 1 . J. Loffing

Subside No 32-61742 du Fonds National de la Recherche Scientifique (2000-4).„Mechanisms regulating sodium transport in the mammalian distal nephron:control of ENaC abundance in the apical cell membrane“

Foundation Prof. Max Cloëtta (2003-8).„Cellular and molecular mechanisms regulating ENaC-mediated sodiumtransport in the distal nephron in-vivo“

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Schweizerische Diabetesstiftung (2003)„Insulin-abhängige Regulation des epithelialen Natriumkanales (ENaC) inder Niere. Mögliche Bedeutung für arterielle Hypertonien beim Typ-2Diabetes mellitus.

1 2 . B. Rossier

Swiss National Science Foundation: Subside 3100-61966.00, 2000-2005 "Salt-sensitive hypertension: identification of downstream renal targets in thealdosterone signaling pathway and the functional role in vivo

Subside NIH-SCOR No 2 P50 HL 55007-06, 23.2.01-30.1.06"Molecular genetics of hypertension and its consequences"

Subside Novartis, 2003: Salt-sensitive hypertension: Identification o fdownstream renal targets in the aldosterone signaling pathway and thefunctional role in vivo

Subside Fondation Emma Muschamp, 2003: "The role of the MAP kinase cascadein controlling amiloride-sensitive transepithelial sodium transport in thekidney"

13. Laurent Schild

Swiss National Science Foundation : Function and regulation of the epithelialsodium channel

Human Frontier Science Program Organisation : Molecular basis for regulation o fthe epithelial sodium channel by hormones, proteins and ions

1 4 . Olivier Staub

Leenards Foundation, Lausanne. “Modulation of channel activity byubiquitination in cystic fibrosis epithelial cells”: (shared with Drs. M. Chansonand N. Demaurex, Geneva). 4/1999-3/04.

Swiss National Science Foundation. “Regulation of surface expression of ionchannels by the ubiquitin-protein ligase Nedd4”: 4/2001-3/04

Ernest Rub Foundation, Lausanne: 2/2002

Roche Research Foundation, Basel. ”Molecular basis of aldosterone dependentregulation of Na+ reabsorption in the kidney”: 7/2003-12/03

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1 5 . Bernard Thorens

Swiss National Sciences Foundation. Grant 4046-058682/1. 2001-2003"Development and preclinical assessment of a bioartificial pancreas"

Swiss national Sciences Foundation. Grant 3100-065219-01. 2001-2004"Glucose transporters and central glucodetection"

Juvenile Diabetes Research Foundation International. Grant 4-2002-461. 2002-2004"Development of a bioartificial pancreas"

Juvenile Diabetes Research Foundation International. Grant 1-2002-366. 2002-2005"Molecular analysis of the sites of hypoglycemia detection controllingglucagons secretion"

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DEPARTEMENT DE PHARMACOLOGIE ET DE TOXICOLOGIE

RECHERCHES

1 . Homéostasie du sodium et ses implications dans les pathologiescardiovasculaires, pulmonaires et rénales

Bernard C. Rossier

“Salt-sensitive hypertension: identification of downstream renal targets in thealdosterone signaling pathway and their functional role in vivo”

Project

Hypertension is the most frequent disease in human populations. Genetic and non-geneticfactors are involved and high salt intake has been proposed as a major risk factor. As ageneral working hypothesis, we have proposed that susceptibility genes could confer salt-sensitivity (or salt-resistance) in a large proportion of the population. A number of candidategenes have been identified through the discovery of mutations in sodium transport systemsexpressed in the distal part of the nephron (TAL and DCT) and in the cortical collecting ducts(CCD). The principal cell of CCD is the target for aldosterone, which tightly controls sodiumreabsorption, allowing sodium balance and control of blood volume and blood pressure.Mutations in the epithelial sodium channel ENaC β/γ subunits have recently been identified asa cause of a monogenic form of salt-sensitive hypertension in humans (pseudoaldosteronismor Liddle’s syndrome). These observations have highlighted the importance of the ENaClocus and demonstrated that a single mutated gene is sufficient to induce large changes inblood pressure.Our main working hypothesis is that genes and pathways involved in this rare form ofhypertension are obvious candidates for harboring more common variances that contribute toless drastic alterations in blood pressure.The general aims of the project are i) to identify downstream renal targets in the aldosteronesignaling pathway by serial analysis of gene expression (SAGE), ii) to characterize candidategenes in vitro with specific emphasis on their role on the aldosterone response on sodiumtransport in CCD principal cells and iii) to express in vivo the candidate gene validated in vitroby gene targeting in the mouse, allowing to understand the pathophysiology of salt-sensitivehypertension and to develop new strategies for preventive medicine and treatment.

Key words

aldosterone, hypertension, salt-sensitivity, epithelial sodium transport, epithelial sodiumchannel (ENaC), transcriptome, serial analysis of gene expression (SAGE), insulin, IGF-1

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Collaborations

R. Boucher, J. Stutts, P. Barker – Div. of Pulmonary Diseases, University of NorthCarolina, Chapel Hill, USA.R.P. Lifton – Yale University, School of Medicine, Boyer Center for Molecular Medicine,(HHI) New Haven, USA.L. Palmer, Cornell, New YorkC. Korbmacher, OxfordK. Chien, San Diego

Selected references

1. Rubera I, Loffing J, Palmer LG, Frindt G, Fowler-Jaeger N, Sauter D, Carroll, T,McMahon A, Hummler E*, Rossier BC (2003). Collecting duct-specific geneinactivation of alphaENaC in the mouse kidney does not impair sodium andpotassium balance. J. Clin. Invest. 112: 554-65.

2. Guipponi, M., Vuagniaux, G., Wattenhofer, M., Shibuya, K., Vazquez, M.,Dougherty, L., Scamuffa, N., Guida, E., Okui, M., Rossier, C., Hancock, M.,Buchet, K., Reymond, A., Hummler, E., Marzella, P.L., Kudoh, J., Shimizu,N., Scott, H.S., Antonarakis, S.E., Rossier, BC. (2002). The transmembraneserine protease (TMPRSS3) mutated in deafness DFNB8/10 activates the epithelialsodium channel (ENaC) in vitro. Hum. Mol. Genet., 11:2829-36.

3. Nicod, M., Michlig, S., Flahaut, M., Salinas, M., Fowler Jaeger, N. ,Horisberger, J.-D., Rossier, B.C., Firsov, D. (2002). A novel vasopressin-induced transcript promotes MAP kinase activation and ENaC downregulation.EMBO J., 21:5109-5117.

4. Vuagniaux, G., Vallet, V., Jaeger, N., Hummler, E., Rossier, B.C. (2002).Synergistic activation of ENaC by three membrane-bound channel-activating serineproteases (mCAP1, mCAP2, and mCAP3) and serum- and glucocorticoid-regulatedkinase (Sgk 1) in Xenopus oocytes. J. Gen. Physiol., 120:191-201.

5. Vallet V, Pfister C, Loffing J, and Rossier B (2002). Cell-surface expression of theChannel Activating Protease xCAP1 is required for activation of ENaC in theXenopus oocyte. J. Am. Soc. Nephrol. 13: 588-94.

Lectures as invited speaker in 2002-2003

2002

- Workshop on "Pathological implications for ion channel dysfunction(channelopathies)", Madrid, March 11-13, 2002. "Epithelial sodium channel: rolein salt-sensitive hypertension".

- Pfluegers Archiv International Workshop on functional genomics, sponsored bySpringer Verlag and by EKRA. "Mouse models for human diseases: a criticalappraisal". Tuebingen, March 15-19, 2002.

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- Meeting of the "Forefronts in Nephrology" series from the International Society ofNephrology. ABC Cassette Proteins in Epithelial Physiology, Ascona, MonteVerita, April 20-25, 2002. Chairman, session CFTR.

- XIV World Congress of Cardiology 2002. Sydney Australia, May 5-9, 2002.Speaker and co-chairman, session Genomics.

- Colloquium on "Frontiers in Molecular Pharmacology and Physiology".Muenchen, May 31 and June 1, 2002. "Regulation of ENaC: aldosterone-dependent and independent signaling pathways".

- Editorial Board "Physiological reviews", Sevilla, June 6-10, 2002.

- Gordon Research Conference, Membrane Transport proteins: Physiological andPathophysiological Implications. New London, Connecticut, July 21-26, 2002.

- 36th Annual Meeting of the European Society for Paediatric Nephrology, Bilbao,Spain, September 20-23, 2002. State of the Art Lecture on "Dysfunction ofepithelial sodium transport: lessons from knock-out mice".

- 13ème colloque, Association Canaux ioniques, Giens, France, September 22-25,2002. Organiser, session: Molecular bases of permeation in ionic channels.

- EMBO Members Workshop on Frontiers of Molecular Biology, Oslo, October 11-15, 2002. "Salt-sensitive hypertension: from monogenic to polygenic".

- Lecture and Professorship, Tulsane University, New Orleans, October 28-29,2002. "Epithelial sodium channel: new insights in the control of blood pressure andlung fluid clearance" and "Epithelial sodium channel: recent advance usingconditional gene targeting".

- ASN Meeting, Philadelphia, October 31 - November 4, 2002. "ENaC: Emerginginsights into regulation of channel function".

- 34th Annual Meeting of the Swiss Society of Nephrology, December 5-6, 2002, St.Gallen, State of the Art Lecture.

2003

- Transatlantic Airway Conference (TAC 2003), Key Biscayne, Florida, USA,January 15-17, 2003. "The epithelial sodium channel (ENaC): Activation bymembrane-bound serine proteases".

- Connaissance 3, Conférence Aigle, 24.1.2003 "Le boutefas et ma pressionsanguine".

- Connaissance 3, Conférence Yverdon, 27.1.2003 "Le boutefas et ma pressionsanguine".

- Leopoldina Symposium, Halle, 19-22.3.2003 "Epithelial transport of ions in healthand disease".

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- Aldosterone meeting, London, 27-30.4.2003. "Epithelial sodium channels: lessonsfrom human diseases and mouse models".

- WCN 2003 / Berlin, 8-12.6.2003. "Dysfunction of the epithelial sodium channel(ENaC) expressed in the kidney of Liddle mice".

- 3rd FEPS Congress, Nice 30.6.-3.7.2003. "Epithelial sodium channels: lessonsfrom human diseases and mouse models".

- APS Conference: Aldosterone and ENaC: From Genetics to Physiology, Banff(Calgary, Canada), 10-14.9.2003.

- Conference and site visit, Heraklion, 22-23.9.2003. "Epithelial sodium channels:lessons from human diseases and mouse models".

- North American CF Conference (2003 NACFC), Anaheim (Los Angeles), 17-18.10.03. "The epithelial sodium channel (ENaC) as limiting factor in the controlof ionic composition of the extracellular fluid".

- Dallas 20-22.10.03 (lectureship, Department of Physiology). "Epithelial sodiumchannels: lessons from human diseases and mouse models".

- 75 th Aarhus Meeting, 24-26.10.03. "Epithelial sodium channels: lessons fromhuman diseases and mouse models".

Honors and Awards

2002 Member: Deutsche Akademie der Naturforscher Leopoldina

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Dmitri Firsov

"Genes responsible for the long-term genomic action of vasopressin:functional characterization of identified candidates (II)"

Vasopressin, a neurohypophyseal peptide hormone, controls the tonicity of extracellularfluids by regulating the rate of water reabsorption in the principal cell of the kidneycollecting duct. Vasopressin is also involved in the regulation of sodium reabsorption bythe principal cell, thus participating in the overall control of sodium balance, maintainingblood volume and thereby of blood pressure. Vasopressin controls the rate of water andsodium reabsorption in the principal cell via (i) a rapid, or transcriptionally independentmechanism, and (ii) a late, or genomic effect during which vasopressin activates a complexnetwork of vasopressin-dependent genes that have been proposed to participate in the long-term regulation of water and sodium reabsorption by the kidney.

Our general working hypothesis states that (i) the vasopressin-dependent gene networkplays an important role in maintaining water and sodium balance, (ii) when mutated, thesegenes may be involved in the pathogenesis of salt and water losing syndromes(nephrogenic diabetes insipidus, PHA type I) and in salt-sensitive hypertension.

We and others have shown that the vasopressin-dependent gene network comprises genescrucial for water and sodium reabsorption in the principal cell (aquaporin-2, ENaCsubunits), as well as genes with a yet not identified function. We propose to study both invitro and in vivo the functional relevance of two vasopressin-induced genes that in a set ofpilot experiments have demonstrated their potential involvement in the regulation of waterand/or sodium transport in the principal cell. These two genes are the vasopressin-inducedtranscript 32 (VIT32) and the regulator of G protein signaling 2 (rgs2).Specific aims:Project A:A-I: to identify the in vitro and in vivo role of VIT32

As a specific working hypothesis, we will test the role of VIT32 as a potentialregulator of MAP kinase cascade in the principal cell and in other cells.

A-II: to identify the role of MAP kinase cascade in sodium and/or water reabsorption inthe principal cell.As a specific working hypothesis, we will test the role of MAPK kinase cascade inbasal and hormone-stimulated activity of principal sodium- and water-transportingproteins in the principal cell (ENaC, Na,K-ATPase, aquaporins).

Project B:B-I: to identify the in vitro and in vivo role of rgs2 in the principal cell.

As a specific working hypothesis, we will test the role of rgs2 in downregulation ofV2 vasopressin receptor signaling the in principal cell.

B-II: to identify the in vitro and in vivo role of rgs2 in other nephron segmentsexpressing rgs2 (MTAL, CTAL, OMCD).As a specific working hypothesis we will test the role of rgs2 in downregulation ofV2 vasopressin receptor and other receptors signaling in these nephron segments.

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Key words

Vasopressin, principal cell of collecting duct, water and sodium reabsorption,transcriptome, serial analysis of gene expression (SAGE), vasopressin-dependent genes,epithelial sodium channel (ENaC), aquaporin-2 water channel (Aqp-2).

Collaborations

X. Jeunemaitre INSERM, ParisA. Doucet CNRS, Paris

Selected references

Flahaut M, Rossier BC, Firsov D. (2002) Respective roles of calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs)in cell surface expression of CRLR/RAMP heterodimeric receptors. J.Biol.Chem,v.277(17), 14731-14737.

Nicod M, Michlig S, Flahaut M, Salinas M, Fowler-Jaeger N, Horisberger JD, RossierBC, Firsov D. (2002) A novel vasopressin-induced transcript promotes MAP kinaseactivation and ENaC downregulation. EMBO J, v.21 (19), 5109-5117.

Muller OG, Parnova RG, Centeno G, Rossier BC, Firsov D, Horisberger JD. (2003)Mineralocorticoid effects in the kidney: correlation between alpha-ENaC, GILZ and Sgk-1mRNA expression and urinary excretion of Na+ and K+. J.Amer.Soc.Nephrol, v. 14 (5),1107-1115.

Flahaut M, Pfister C, Rossier BC, Firsov D. (2003) N-glycosylation and conservedcysteine residues in RAMP3 play a critical role for the functional expression ofCRLR/RAMP3 adrenomedullin receptor. Biochemistry, v.42, 10333-10341.

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Edith Hummler

Projects

Our laboratory is specifically interested in understanding the heterogeneity of stimulating/repressing systems that control activity of the highly amiloride-sensitive epithelial sodiumchannel (ENaC) in distinct organs. This sodium channel is implicated in severalpathological conditions, like hypertension, respiratory distress syndrome and potentiallyalso in skin diseases.

"Functional analysis of the ENaC in vivo"The highly amiloride-sensitive epithelial sodium channel (ENaC) is a membrane constituentof many salt-reabsorbing epithelia which facilitates Na+ movement across tight epithelia.cDNAs encoding the three homologous subunits of this channel (α, β, and γ, or Scnn1a,Scnn1b and Scnn1c) have been characterized in several species, including man and mouse.

In organs, like kidney and colon, ENaC is involved in whole net sodium balance of thebody; this is the classical role of this channel. Using gene targeting experiments, we havegenerated knockout alleles at the ENaC (Scnn1) gene loci and also introduced a singlemutation at the ß-ENaC subunit (Scnn1b) locus. Thereby, we established mouse modelsfor two human diseases, the salt wasting syndrome PHA-1 (pseudohypoaldosteronism)caused by reduced activity of the channel, and Liddle’s syndrome, where the channel isconstitutively active. Additional targeted mutations of ENaC subunits, as tissue-specificgene inactivation in nephron segments should reveal the implication of ENaC inaldosterone-regulated sodium reabsorption. Thus, the collecting duct-specific geneinactivation of ENaC (using the conditional αENaC (Scnn1a) allele) clearly demonstratedthat inactivation in this segment does not impair sodium and potassium balance stressingthe importance of hormone-regulated sodium reabsorption to more proximal nephronsegments. Further analysis of these mouse models should elucidate the causal relationshipbetween dietary salt, dysfunction of genes expressed in kidney, and hypertension.

"ENaC and its positive regulator, the channel activating protease 1(CAP1) : molecular and functional characterization of ENaC-mediatedsodium transport in skin and lung"Inactivation of the α subunit of the epithelial sodium channel (Scnn1a) demonstrated theimportant role of this channel in lung liquid clearance around birth. αENaC -/- mice diewithin 40h after birth due to respiratory failure. It is thus feasible, that ENaC dysfunction isassociated with augmented susceptibility to pulmonary edema in adults. Geneticallymodified mice, where expression of the endogenous Scnn1a gene was substituted for atransgenic one show generally reduced ENaC-mediated sodium transport across alveolarepithelium and developed edema under hypoxia. This suggests an involvement of ENaC inthe pathogenesis of pulmonary edema. In parallel, primary epithelial tracheal cell culturesfrom mice with ENaC dysfunction showed that, in consequence to downregulation ofENaC, the rate of Na+ transport becomes insufficient to maintain fluid balance, and lungedema may result.Serine proteases, like e.g. mCAP1 (channel activating protease 1) can induce ENaC activityvia an extracellular pathway. The mechanism is not yet understood, and may be due toproteolytic cleavage of the channel itself or via an intermediate protein (e.g. serine protease

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inhibitor). CAP1 might regulate ENaC activity in alveolar epithelial cells, and thus maymodulate alveolar fluid absorption primarily driven by active transepithelial sodiumtransport. The study of bronchiolar- and alveolar–specific conditional CAP1 knock-out willallow us to determine the role of these serine proteases in proximal and distal fluidclearance.

Recent evidence indicates that this sodium channel ENaC is implicated in differentiationprocesses, e.g. in the skin; this would be the non-classical role of this channel. Analyses ofαENaC -/- newborn mice showed epidermal hyperplasia, premature lipid secretion,abnormal keratohyalin granules and abnormal expression of skin differentiation markers,like e.g. keratin 1, keratin 10, and involucrin. These data suggest that the epithelial sodiumchannel modulates ionic signaling for specific aspects of epidermal differentiation. Tocircumvent the perinatal lethality of the αENaC -/- mice, we generated a conditionalαENaC (Scnn1a) and a conditional CAP1 (Prss8) knockout mouse. Preliminary functionalstudies of skin-specific αENaC (Scnn1a) knock-outs strongly suggest an involvement ofthe channel in skin barrier function.

Key words

ENaC, Na+ transport, CAP1, serine proteases, kidney, lung, skin, mouse, transgenic,knockout, gene targeting, Cre recombinase, Scnn1a

CollaborationsTom McKee, Sam Rothman, Institute of Pathology, LausanneMichel Burnier, Division of Hypertension, LausanneFriedrich Beermann, ISREC, EpalingesPeter Beard, ISREC, EpalingesMichel Rossier, GenèveNicolette Farman, Faculté Xavier Bichat, Paris, France.Konrad Sandhoff, Kekule-Institut fuer Organische Chemie und BiochemieTheodora Mauro, UCSF, San Francisco, USARic Boucher, Mike Knowles, North Carolina University, USA

Selected references

Rubera, I., Meier, E., Vuagniaux, G., Mérillat, A.-M., Beermann, F., Rossier, B.C.,Hummler, E. (2002). A conditional allele at the mouse channel activating protease 1(Prss8) gene locus. Genesis 32: 173-176.

Hummler, E., Mérillat, A.-M., Rubera, I., Rossier, B.C., Beermann, F. (2002).Conditional gene targeting of the Scnn1a (αENaC) gene locus. Genesis 32: 169-172.

Mauro, T., Guitard M., Oda, Y., Crumrine, D., Komuves, L., Behne, M., Rassner, U. ,Elias, P.M., Hummler, E. (2002). The ENaC channel is required for normal epidermaldifferentiation. J. Investig. Dermatol. 118 : 589-594.

Olivier, R., Scherrer, U., Horisberger, J.-D., Rossier, B.C., Hummler, E. (2002).Limiting sodium transport rate in airway epithelia from αENaC transgenic mice, a modelfor pulmonary edema. J. Appl. Physiol., 93:1881-1887.

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Hoenderop, J.G.J., van Leeuwen, J.P.T.M., van der Eerden, B.C.J., Kersten, F.F.J.,Van der Kemp., A.W.C.M., Mérillat, A.-M., Waarsing, E.J.H., Rossier, B.C., Vallon,V., Hummler, E., Bindels, R.J.M. (2003). Renal Ca2+ wasting, hyperabsorption, andreduced bone thickness in mice lacking TRPV5. J. Clin. Invest., 112: 1906-1914.

Rubera, I., Loffing, J., Palmer, L., Frindt, G., Fowler-Jaeger, N., Sauter, D., Carroll,T., McMahon, A., Hummler, E., Rossier, B.C. (2003). Collecting duct-specific geneinactivation of αENaC in the mouse kidney does not impair sodium and potassium balance.J. Clin. Invest., 112 : 554-565.

Lectures as invited speaker 2002-2003

The Physiology Joint Meeting in Tübingen, DPG, Germany, March 2002. « ENaC andlung liquid clearance ».

Pflügers Archiv International Workshop on functional genomics : Mouse models forhuman diseases : a critical appraisal, Tübingen, Germany, March 2002. « Epithelialsodium channel : novel insights from gene targeting experiments ».

Graduiertenkolleg « Biomedizinische Wirkstoff-Forschung », Konstanz, Germany, July2002. « Analysing the role of ENaC through genetically-engineered mice ».

Congress of Nephrology, Düsseldorf, Germany, September 2002. « Identifizierung vonGenfunktionen durch transgene Mäuse ».

World Congress of Nephrology, WCN, Berlin, June 2003. « Inactivation of alpha ENaCsubunit function in the collecting duct ».

2003 APS Conference : Aldosterone and ENaC : From Genetics to Physiology, Banff,Canada, September 2003. « Animal models for ENaC function in skin ».

26

Laurent Schild

« Function and regulation of the epithelium Na channel (ENaC) »

Project

The epithelial sodium channel is responsible for sodium reabsorption in the distal nephron,and plays a key role in the maintenance of sodium balance and the regulation of bloodpressure. ENaC mutations have been identified in patients with a severe form of salt-sensitive hypertension (Liddle syndrome), leading to a hyperactivity of the channel. Bycontrast loss of function mutations in ENaC have been found in patients with a salt loosingnephropathy and hypotension (pseudohypoaldosteronism type 1). Our interest focuses onthe molecular and cellular mechanisms underlying ENaC channel function, and theircontributions in the pathogenesis of hypertension.

Three main lines of investigations are going on in the laboratory.

1. Molecular basis of channel function. Using site-directed mutagenesis combinedwith functional assays (electrophysiology) we are investigating the structural basis forimportant channel functions such as ion permeation through the channel pore, ionselectivity, and channel openings /closures.We have identified the regions on the channelmolecule involved in ion discrimination, channel inhibition by amiloride, presently we raefocusing on the ion permeation pathway. These studies aim at building structure models tounderstand these basic channel functions at the molecular level.

2. Regulation of channel activity. ENaC is under the hormonal control of aldosteroneor vasopressin. Important informations about the molecular mechanisms underlyingregulation of channel activity can be obtained by the identification of ENaC structures thatparticipate in this regulation. We have demonstrated that a conserved PY motif in the C-terminus of the channel that is mutated in Liddle syndrome, is important for regulation ofthe number of channel molecules at the cell surface. This is achieved by interactions of thePY motif with intracellular regulatory proteins. This mutagenesis approach to understandchannel regulation is pursued in epithelial cells that respond to hormones and vasopressin.

3. ENaC polymorphism in a hypertensive population. We are collaborating withclinicians and geneticists in a project aiming at the determination of the role of ENaC in thepathogenesis of common forms of hypertension. Genetic analysis are performed inhypertensive patients to assess the association of the hypertensive trait with ENaC genesand other candidate genes. The ENaC genes in patients are systematically screened toidentify mutations; these ENaC mutants are tested for functional alterations of channelactivity that could be related to hypertension.

Key words

Epithelial sodium channel, aldosterone, amiloride , hypertension, ionic channel.

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Collaborations

Prof. R.P. Lifton : Department of Genetics, Yale University School of MedicineProf. K. Kontula : Department of Medicine, University of HelsinkiProf. D. Rotin : The Hospital for Sick Children , TorontoProf. M .Burnier : Division d’hypertension et de médecine vasculaire, CHUV LausanneProf. F. Paccaud : Institut Universitaire de Médecine Sociale et Préventive, Lausanne

Lectures as invited speaker

APS Conference – Aldosterone and ENaC : from the genetics to physiology. Banff 2003

Selected references

Auberson M, Hoffmann-Pochon N, Vandewalle A, Kellenberger S and Schild L. (2003).Epithelial Na+ channel mutants causing Liddle's syndrome retain ability to respond toaldosterone and vasopressin. Am. J. Physiol., Renal Physiol 285: F459-F471, 2003.

Henry PC, Kanelis V, O'Brien MC, Kim B, Gautschi I, Forman-Kay J, Schild L andRotin D. (2003). Affinity and specificity of interactions between Nedd4 isoforms and theepithelial Na+ channel. J. Biol. Chem. 278: 20019-20028.

Hiltunen TP, Hannila-Handelberg T, Petajaniemi N, Kantola I, Tikkanen I, Virtamo J ,Gautschi I, Schild L and Kontula K. (2002). Liddle's syndrome associated with a pointmutation in the extracellular domain of the epithelial sodium channel gamma subunit. J .Hypertens. 20: 2383-2390.

Kellenberger S, Gautschi I and Schild L. (2003). Mutations in the epithelial Na+ channelENaC outer pore disrupt amiloride block by increasing its dissociation rate. Mol.Pharmacol. 64: 848-856.

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Stephan Kellenberger

Contribution of acid-sensing ion channels ASICs to electrical signaling inneurons

Project

ASICs are non-voltage-gated Na+ channels that are transiently activated by a rapid drop inextracellular pH. They are members of the Epithelial Na+ channel (ENaC)/degenerin familyof channel proteins. To date four different ASIC genes, ASIC1, 2, 3 and 4 are known, ofwhich ASIC1 and 2 exist as different splice variants. Functional studies suggest that ASICsubunits can assemble to functional homo- or heterotetrameric channels. ASICs areexpressed in the peripheral and the central nervous system. Their physiological roles arenot clear to date. Based on their activation by acidification and their expression pattern ithas been suggested that ASICs in the peripheral nervous system may be involved in painsensation due to inflammation or ischemia and in mechanosensation. Consistent with thishypothesis, it has been shown that inactivation of ASIC3 in mice affected the painresponse. Whereas all known ASICs are found in the peripheral nervous system, onlyASIC1a, 2a and 2b are present in the central nervous system. ASICs in the central nervoussystem may be involved in synaptic functions and may contribute to the neuronal deathassociated with brain ischemia or epilepsy, which are accompanied by extracellularacidification. ASIC1a knockout mice showed a mild defect in spatial learning and fearconditioning, pointing to the proposed synaptic function of ASICs. At the level of theneuron, activation of ASICs is expected to shift the membrane potential towards morepositive potentials and to generate or facilitate action potentials. Our laboratory studiesASIC function at the level of the neuron with the ultimate goal of understanding thephysiological role of ASICs. We investigate whether the functional characteristics ofASICs are compatible with a role in the modulation and generation of action potentials inneurons. We use cell biological and electrophysiological techniques on primary cultures ofneurons and on cells expressing cloned ASICs. The two main projects are:

1. Involvement of ASICs in induction of action potentials by extracellular acidificationIt is known that extracellular acidification can induce action potentials in several types ofneurons. We study in primary culture of hippocampal neurons the involvement of ASICs inthis process. In the course of this work we could show that activation of ASICs is essentialfor the acid-induced action potential generation, because it is prevented by the presence ofthe ASIC inhibitor amiloride or the venom of the spider Psalmopoeus cambridgei, whichselectively inhibits ASIC1a. ASIC-mediated generation and modulation of action potentialscan be induced by extracellular pH changes from 7.4 to pH values slightly below pH 7.Such local extracellular pH values may be reached by the pH fluctuations due to normalneuronal activity. This analysis shows that ASIC activation can affect neuronal signaling.Next we will address the sub-cellular localization of ASICs in hippocampal neuronsespecially with regard to the synapse. ASICs located at the synapse are likely to beactivated by the pH change during neurotransmitter release.

2. ASIC currents in dorsal root ganglion (DRG) neurons ASICs are expressed in the peripheral nervous system and are thought to be involved in thesensation of pain due to tissue acidification which occurs for example during inflammationor ischemia. Channels other than ASICs have been proposed to play a role as acid sensorsin peripheral neurons and to be important for pain sensation. Our goal is to determine thecontribution of ASICs to acid sensing in different types of sensory neurons. As a modelsystem for sensory neurons we use DRG neurons of the rat, which are a veryheterogeneous population of neurons. Our initial analysis showed ASIC-like currents ineither small neurons (diameter < 25 um), which are likely nociceptors and in medium-to-

29

large neurons (diameter > 32 um), which are likely involved in mechanosensation. Basedon the pH dependence of activation and the kinetics of the acid-induced currents we coulddistinguish seven types of ASIC responses. Currently we investigate the potentialmolecular composition of these currents by comparing their pharmacological andbiophysical properties with those of cloned ASICs. In parallel we have started to test theseneurons for the presence of cell-type specific markers to be able to correlate the ASICcurrent type with the type of the neuron.

Keywords

Acid-sensing ion channel, neuronal signaling, action potential, primary neuronal cellculture

Collaborations

Prof. L. Schild, Département de Pharmacologie et de Toxicologie, LausanneProf. H. Vogel, Laboratory of physical chemistry of polymers and membranes, EPFL,LausannePD Dr. Ibtissam Walter Barakat, CHUV, LausanneProf. G. Waeber, CHUV, LausannePD Dr. Isabelle Decosterd, CHUV, Lausanne

Selected references

Kellenberger, S., Gautschi, I. & Schild, L. (2002). An external site controls closing of theepithelial Na+ channel ENaC. J. Physiol. 543.2, 413-424.

Kellenberger, S. & Schild, L. (2002). Epithelial sodium channel/degenerin family of ionchannels: A variety of functions for a shared structure. Physiol. Rev. 82, 735-767.

Schreiter, C. Hovius, R., Costioli, M., Pick, H., Kellenberger S., Schild, L., & Vogel,H. (2003). Characterization of the ligand-binding site of the Serotonin 5-HT3 Receptor:The Role of Glutamate residues 97, 224, and 235. J. Biol. Chem. 278, 22709-22716

Auberson, M., Hoffmann-Pochon, N., Vandewalle, A., Kellenberger, S.,& Schild, L.(2003). Epithelial sodium channel (ENaC) mutants causing Liddle syndrome retain theirability to respond to aldosterone and vasopressin. Am. J. Physiol.- Renal Physiol. 285,F459-F47.

Kellenberger, S., Gautschi, I., & Schild, L. (2003). Mutations in the epithelial Na+

channel ENaC outer pore disrupt amiloride block by increasing its dissociation rate. Mol.Pharmacol. 64, 848-856.

Lectures as invited speaker

30.7.02 Department of Medicinal Chemistry and Molecular Pharmacology, WestLafayette, IN, USA: Watching single channel molecules at work:pharmacology and gating of the epithelial Na channel ENaC.

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25.3.02 Medical Faculty, Lausanne: Molecular analysis of ion conduction and gatingin the epithelial Na channel ENaC.

17.9.03 Faculté de Biologie et de médecine, Lausanne, Leçon d'habilitation pourl'obtention du titre de privat-docent: Canalopathies cardiaques

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2 . Bernard Thorens

“Molecular control of glucose and energy homeostasis in health anddisease”

Project

Our research is aimed at understanding different aspects of the molecular control of glucoseand energy metabolism and the dysfunctions of these mechanisms in the pathogenesis ofobesity and diabetes.

A key aspect of these regulatory events is the monitoring by multiple sensors, located atdifferent anatomical sites, of the body energy status. Our interest is in the molecular andcellular identification of glucose sensors, the signals they generate and the physiologicalfunctions they control. More specifically, we are studying insulin secretion by pancreatic ßcells; the regulation of ß cell function by intestinal hormones released in the blood afterglucose ingestion; the control of insulin release and glucose absorption in muscle and fat byglucose sensors present in the hepatoportal vein; the control of food intake andcounterregulatory response to hypoglycemia by brain glucose sensors.

Glucose sensingWe have demonstrated the role of the glucose transporter GLUT2 in the control of glucosestimulated insulin secretion (GSIS) using GLUT2-null mice. Following transgenicreexpression of GLUT2 in ß cells only, we studied the role of this transporter in the controlof other glucose sensors. We have demonstrated that GLUT2 was required for thefunction of a glucose sensor present in the hepatoportal vein. When activated, this sensorstimulates glucose utilization by muscle in an insulin-independent manner. It also controlsthe first phase of insulin secretion. We have now demonstrated that similar sensors, butlocated in the brainstem and hypothalamus control glucagon secretion by alpha cells. Thesesensors also control food intake through a regulation of orexigenic and anorexigenichypothalamic peptides.We have studied the regulation of glucose-stimulated insulin secretion by the gluco-incretinhormones GLP-1 and GIP using mice with inactivation of both hormone receptor genes.We showed that absence of both receptors led to impaired glucose tolerance and a ß-cellautonomous secretion defect, likely due to dysregulated expression of genes controllinginsulin secretion.

Other hexose and polyols transportersWe have recently characterized novel glucose transporter-like molecules which are nowreferred to as GLUT8, a functional glucose transporter; HMIT, a H+/myo-inositolsymporter; and GLUT9, a protein of so far unidentified function. We have generatedGLUT8 conditional knockout mice and are presently evaluating the physiologicalmodifications of mice with inactivation of this gene in all tissues. We have demonstratedthat HMIT is present in neurons. It is associated with intracellular vesicles that translocateto the cell surface upon nerve stimulation, preferentially at growth cones and synapses, andincrease myo-inositol uptake.

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Keywords

Glucose homeostasis/ glucose transporters/ diabetes/ insulin/ ßcells/ glucose sensors/ transgenic mice

Collaborations

- Professor Christian Boitard, Hôpital Saint-Vincent, Paris.- Professor Claes Wollheim, University of Geneva- Professor Philippe Halban, University of Geneva.- Professor Philippe Froguel, Institut Pasteur, Lille.- Professor Gérard Waeber, CHUV, Lausanne- Dr. Peter Vollenweider, CHUV, Lausanne- Dr. Urs Scherrer, CHUV, Lausanne- Dr. Thierry Pedrazzini, CHUV, Lausanne- Professor Jens Juul Holst, Panum Institute, Copenhagen- Professor Morris J Birnabaum, Philadelphia.- Professor Barbara Kahn, Harvard Medical School- Professor C.Ronal Kahn, Harvard Medical School

Selected References

1. Ibberson, M., Riederer, B.M., Uldry, M., Guhl, B., Roth, J., and Thorens, B.(2002). Immunolocalization of GLUTX1 in testis and to specific brain areas andvasoopressin-containing neurons. Endocrinology, 143: 276-284.

2. Burcelin, R., Crivelli, V., Dacosta, A., Roy-Tirelli, A.,and Thorens, B. (2002)Heterogenous metabolic adaptation of C57BL/6J mice to high fat feeding. Am. J .Physiol. 282: E834-E842.

3. Cottet, S., Dupraz, P., Hamburger, F., Dolci, W., Jaquet, M., and Thorens, B.(2002) c-FLIP protein prevents tumor necrosis factor-α-mediated induction ofcaspase-8-dependent apoptosis in insulin-secreting ßTc-Tet cells. Diabetes, 51:1805-1814.

4. Thorens, B. (2003) A gene knockout approach in mice to identify glucose sensorscontrolling glucose homeostasis. Pflugers Arch – Eur. J. Physiol. 445: 482-490..

5. Burcelin, R., Crivelli, V., Perrin, P., DaCosta, A., Mu, J., Kahn, B.B.,Birnbaum, M.J., Kahn, C.R., Vollenweider, P., and Thorens, B. (2003) GLUT4,AMP-kinase, but not the insulin receptor are required for hepatoportal glucosesensor-stimulated muscle glucose utilization. J. Clin. Invest. 111:155-1562.

Invited speaker to international meetings :

2002: • Ernst Klenk Foundation Meeting, Köln. February 17-19, 2002.• EKRA Workshop, Tuebingen March 19th, 2002.• 19th International Conference on Biological Membranes, Troina, Sicily, June 9-12,

2002.

2003: • Academia Europaea- Obesity and Diabetes Meeting. Heidelberg, March 6-8, 2003.• Forum Engelberg, 14th Annual Conference. “Stem Cells: Hopes and Ethical

Conflicts”. Engelberg, March 26-28, 2003.

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• Endocrine Society Meeting, Philadelphia, USA. June 19-22, 2003.• Federation of the European Physiological Societies. Nice, June 28-July 3, 2003.• FASEB Summer Research Conference. Glucose Transporter Biology. Snowmass,

CO, August 9-14, 2003.• Genomics of Diabetes and Associated Diseases in the Post Genome Era. Lille,

August 22-24, 2003.• International Diabetes Federation Meeting. Paris, August 24-29, 2003.

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3 . Biosynthèse, régulation et relation structure-fonction desprotéines de transport ionique

Käthi Geering

"Protein-Protein Interactions in the Regulation of Na,K-ATPase Expressionand Function"

Project

Na,K-ATPase (NKA) plays an important physiological and pathophysiological and is thepharmacological target for cardiac glycosides. To better understand the mechanismslinking altered NKA function to diseases, we study several aspects of NKA regulationwhich results from interaction with other proteins.Project A: The Role of β Assembly in the Expression and Function of NKA: The mostfundamental process, which regulates membrane protein expression and function, takesplace in the ER and depends on the correct membrane insertion and folding of the protein.Misfolded proteins are retained in the ER and are ultimately degraded by mechanisms of theER quality control sytem. There are now hundreds of exemples of proteins with pointmutations that impede correct folding and in consequence are determining for the etiologyof many diseases. By using the oligomeric NKA as a model protein, we analyze severalaspects of the regulation of protein maturation in the ER including the structural andfunctional consequences of partner subunit assembly in oligomeric proteins and the role ofsugar moieties and chaperone interactions in the degradation of glycoproteins. Acombination of immunological, biochemical and electrophysiological techniques will permitto analyze the structural and functional properties of wild type or mutant NKA subunitsexpressed in Xenopus oocytes. The results of these studies should provide importantinformation on mechanistic details of the processes involved in the correct folding ofmembrane proteins, or the degradation of misfolded proteins, and may ultimately help toidentify new pharmacological targets to favor correct folding, or interfere with degradationof misfolded proteins.Project B: Regulation of Na,K-ATPase by small proteins of the FXYD family. The so-called γ subunit, expressed in certain segments of renal tubules, specifically associates withand regulates the transport properties of the basolaterally expressed NKA. A pointmutation in the γ subunit gene has recently been identified which leads to defective cellularrouting of NKA and is linked to a genetic human disease (dominant renalhypomagnesaemia). The γ subunit belongs to the recently defined FXYD family of smalltype I proteins including CHIF, phospholemman, Mat-8, RIC and two new membersFXYD6 and FXYD7, all with unknown function. FXYD proteins exhibit a specific tissuedistribution, with the exception of the ubiquitously expressed FXYD6. Since not only theγ subunit, but CHIF and FXYD7 as well, are able to associate with NKA and to modulateits activity in different ways, we study whether most, if not all, of the FXYD proteins maybe selective, tissue- and/or isozyme-specific modulators of NKA. In view of thephysiological and pathophysiological importance of NKA, the identification of newmodulators and the characterization of their mechanism of action is of great interest andmay lead to the design of new therapeutic agents that may permit to modulate NKA activityspecifically e.g. in excitable tissues where particular NKA isozymes have a fundamentalrole in the control of an equilibrated, cellular activity.Project C: The identification of new NKA isoform-specific regulatory proteins: The NKAα2-β isozyme exhibits a distinct tissue and cellular distribution as well as particulartransport and pharmacological properties which emphasize its specific role in skeletal andheart muscle physiology and in the pharmacological action of cardiac glycosides. In thisproject, we intend to analyze tissue-specific regulatory pathways linked to α2-β isozymeswhich are mediated by protein-protein interactions and which may be involved in theregulation of its cell surface expression and its function, its correct cellular targeting tofunctional microdomains of the plasma membrane and possibly in the transmission of

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signals from the NKA to intracellular transduction pathways and gene transcription. Toidentify proteins which transiently interact with intracellular domains of α2 isoforms, wescreen heart libraries with the yeast two-hybrid assay and perform pull-down experimentswith crude extracts of healthy and diseased human hearts. Identification of such proteins isof considerable interest to better understand physiological processes such as muscle fatigueor heart contraction or pathophysiological conditions such as digitalis-induced cardiachypertrophy and ultimately may lead to the discovery of new putative pharmacologicaltargets for the treatment of heart failure.

Key wordsP-type ATPase, Na,K-ATPase, ER quality control system, protein degradation,biosynthesis and membrane insertion of membrane proteins, assembly of oligomericproteins, Xenopus oocyte, cardiac glycosides

Collaborations

Nikolai Modyanov (Medical College of Ohio, USA)Haim Garty (Weizmann Institute, Rehovot, Israel)George Sachs (CURE, V.A. Med Center, Los Angeles, USA)Steve Karlish (Weizmann Institute, Rehovot, Israel)Jean-Daniel Horisberger (Institut de Pharmacologie et Toxicologie, UNIL)Olivier Staub (Institut de Pharmacologie et Toxicologie, UNIL)Florianne Monnet-Tschudi, Paul Honegger (Institut de Physiologie, UNIL)

Selected References

Crambert, G., Béguin, P., Pestov, N. B., Modyanov, N. N., and Geering, K. (2002).βm, a structural member of the X,K-ATPase β subunit family, resides in the ER and doesnot associate with any known X,K-ATPase α subunit. Biochemistry 41, 6723-6733.

Crambert, G., Horisberger, J-D., Modyanov, N. N., and Geering K. (2002). Humannongastric H+,K+-ATPase: transport properties of ATP1al1 assembled with different β-subunits. Am. J. Physiol. 283, C 305-C314.

Béguin, P., Crambert, G., Monnet-Tschudi, F., Uldry, M., Horisberger, J-D., Garty. H. ,and Geering K. (2002). FXYD7 is a brain-specific regulator of Na,K-ATPase α1-βisozymes. EMBO J. 21, 3264-3273.

Crambert, G., Füzesi, M., Garty, H., Karlish, S., and Geering, K. (2002).Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transportproperties. Proc. Natl. Acad. Sci. USA 99, 11476-11481.

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Lectures as invited speaker 2002-2003

Gordon Research Conference on ‘Membrane Transport Proteins’, New London,Connecticut, 21-26 July 2002. ‘Role of FXYD proteins in the regulation of Na,K-ATPase’.

10th International Conference on Na,K-ATPase and related cation pumps, Elsinore,Denmark, 8-14 August, 2002. ‘FXYD proteins : New tissue- and isoform-specificregulators of Na,K-ATPase’.

World Congress of Nephrology, Berlin 8-12 June, 2003. ‘Differential regulation of Na,K-ATPase by FXYD proteins in the kidney’

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Jean-Daniel Horisberger

Projects

The group is following two lines of research concerning the function of membrane proteinsthat perform the transport of sodium ions across epithelial cells. Both these proteins, thesodium pump or Na,K-ATPase and the epithelial Na channel, are involved in humanarterial hypertension:.

"Structure function relationship of the Na,K-ATPase"Na,K-ATPase is a cation transport system present in all animals cells with basichousekeeping functions and essential roles in epithelial solute and fluid transport, and inexcitable cell physiology. It has been implicated in many human diseases, particularlyarterial hypertension. We have developed or adapted a range of electrophysiologicaltechniques that allow us to characterize several detailed aspects of the cation transport byNa,K-pumps such as sided cation affinities and their voltage dependence, kinetics of singlesteps of the transport cycle and « channel-like » properties of the Na,K-pump. We arepresently working on the following specific projects : 1) Structure of the external cationaccess channel using Cysteine Scanning Mutagenesis, an approach that has been usedsuccessfully to determine the structure of the pore of ion channels. 2) Structure of thecation occlusion site using Na,K / H,K ATPase chimera and site directed mutagenesis. 3)measurement of geometric distances between transmembrane helices in the E1 and E2conformation by expression of double cystein mutants and induction of disulfide bridgeformation by oxidant. These studies provide essential information concerning theorientation and the relation between the α helices and possibly other structural elements thatconstitute the transmembrane cation translocation mechanism in Na,K-ATPase and relatedP-ATPases. These results are interpreted using a molecular model of the Na,K-ATPasebuid by homology with the recently published structure of the calcium pump SERCA. 4)Recently 4 mutations in the gene coding for the Na,K-ATPase α2 isoform have beenassociated with a hereditary form of migraine, Familial Hemiplegic Migraine, that ischaracterized by hemiplegic symptoms during the aura. We are studying the effect of thesemutations on the expression and function of the Na,K-ATPase to get some insight in thepathogenic process leading to migraine.

The epithelial Na channel (ENaC), a multimeric protein made of three homologoussubunits α, β and γ, is the rate limiting element in the reabsorption of sodium by epitheliain the distal part of the nephron, distal colon and airway epithelia. ENaC has an essentialrole in the control of sodium balance and blood pressure. ENaC is also the target of the K-sparing diuretics amiloride and triamterene. The physiological regulation of its expressionand activity is complex, involving several types of hormones (mineralo- andglucocorticoid, vasopressin, prostaglandins), intracellular (cAMP, calcium, pH) andextracellular (Na, pH) factors, but the molecular mechanisms of these regulations are notwell understood. ENaC is also the. Our objective is to understand the molecular basis ofthe physiological control of the function of ENaC and of its modulation by pharmacologicalmeans. We use electrophysiological studies of ENaC (wild type rat or Xenopus isoforms,chimera or mutants) expressed in Xenopus oocyte, and in epithelial cells (A6 cells ormammalian CCD-deriver cell lines). The physiological regulation of ENaC is investigated

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in Xenopus oocytes by second messenger/protein kinase stimulation and coexpression ofENaC with potentially involved G-protein coupled receptors. We have recently observedthe role of an extracellular protease in the regulation of ENaC, demonstrating a novelmechanism of ion channel regulation. We are presently investigating the mechanism ofaction of several agent that regulate ENaC activity by interaction with the extracellulardomain of this protein. Together these approaches aim at an integrated understanding of theregulation of the amiloride-sensitive Na channel.

Microfluidic device: In a collaborative work with a group of the EPFL (Prof. M. Gijswe are pursuing the development of microfluidic device allowing the study ion channelactivity with high time resolution, low solution and a high potential for automation. Thedevices are designed and build in the EPFL and then they are tested with various types ofcells in our laboratory. This system will be initially applied to the study of one of ourproteins of interest, ENaC in order to investigate the control of gating and inactivation thatoccurs with a fast time course.

Key words

Na,K-ATPase, H,K-ATPase, epithelial Na channel, amiloride, site directed mutagenesis, structure-function relationship, cysteine scanning mutagenesis, cation transport, cationbinding site, palytoxin, protein 3-D structure, homology modelling.

Collaborations

Martin .A.M. Gijs, EPFL, Institute of Microsystems, CH-1015 Lausanne, Switzerland

Dr Gordon Stewart, Department of Medicine, University College London,Rayne Institute,London

Dr. Stefan Gründer, Department of Physiology II, Tübingen, Germany.

Selected references

1. Burnay,M., Crambert,G., Kharoubi-Hess,S., Geering,K., and Horisberger,J.-D. (2003).Electrogenicity of Na,K- and H,K-ATPase activity and presence of a positively chargedamino acid in the fifth transmembrane segment. J. Biol. Chem., 278:19237

2. Horisberger,J.-D. (2003). ENaC-CFTR interactions: the role of electrical couplingexplored in an epithelial cell model. Pflügers Arch., 445:522-528.

3. Muller,O.G., Parnova,R.G., Centeno,G., Rossier,B.C., Firsov,D., and Horisberger,J.-D. (2003). Mineralocorticoid effects in the kidney: Correlation between a ENaC, GILZ,and Sgk-1 mRNA expression and urinary excretion of Na+ and K+. J. Am. Soc.Nephrol., 14:1107-1115.

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4. Chraïbi,A. and Horisberger,J.-D. (2002). Na self inhibition of human Epithelial NaChannel:Temperature dependence and effect of extracellular proteases. J. Gen. Physiol.,120:133-145.

5. Horisberger,J.-D. and Kharoubi-Hess,S. (2002). Functional differences between αsubunit isoforms of the rat Na,K-ATPase expressed in Xenopus oocytes.J.Physiol.(London), 539:669-680.

6.

Lectures as invited speaker in 2002-2003

- Postgraduate Kurs für Exp. Medizin und Biologie. Universität-Spital, Zürich, 2002 and2003. "The epithelial sodium channel and its role in human disease".

- Defect of secretion in Cystic Fibrosis and Other Diseases. 7-8 n^Novembre 2003, EMBL,Heidelberg, Germany. CFTR, ENaC and apical K+ channels : a mathematical modelapproach to understand their interactions

- Aldosterone and ENaC: From Genetics to Physiology September 10-14, 2003,Extracellular Mechanisms of ENaC self- inhibition.

- Les cellules rénales et les interactions cellulaires dans les épithéliums : du gène à laphysiopathologie. Faculté de Médecine Necker. Paris 20-21 juin 2002. What makes theNa, K-pump electrogenic?

- 10th International Conference on Na,K-ATPase and related Cation Pumps. 8-14 August2002, Elsinore, Danemark. Cation stoichiometry and cation pathway in the Na,K-ATPaseand non-gastric H,K-ATPase

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Olivier Staub

“Role of intracellular protein-protein interaction in ion channel regulation”

Projects

Our research focuses on the regulation of ion channels by intracellular regulatory proteins.Thereby we are interested to know what proteins interact with these channels, how theyaffect their function and if they are playing a physiological role. We have particularly beeninterested in the role of Nedd4/Nedd4-like ubiquitin-protein ligases, which we have shownto directly interact with channel proteins. Ubiquitin-protein ligases are at the end point of anenzymatic cascade, which covalently attaches ubiquitin-moieties on target proteins. Proteinsincluded in this cascade are E1 (ubiquitin-activating enzyme), E2s (ubiquitin-conjugatingenzymes) and E3s (ubiquitin-protein ligases). In recent years this process has gainedconsiderable interest as a mechanism that controls the cell surface expression of plasmamembrane proteins. We are interested into two different model proteins:

The epithelial Na+ channel ENaCENaC represents the prototype for the regulation of ion channels by Nedd4-like proteins.This protein complex, composed of three homologous subunits (αβγ), which is localizedto the aldosterone-sensitive distal nephron (ASDN), plays a major role in regulating Na+

balance and blood. It is genetically linked to Liddle’s syndrome, an inherited form ofhuman hypertension. The disease is caused by mutations, which delete/alter PY motifs inthe C-termini of β or γENaC, resulting in increased Na+ channel activity due to elevatedchannel number and open probability. We have shown that these PY-motifs act as bindingsites for the ubiquitin-protein ligase Nedd4-2, that ENaC becomes ubiquitinated and thatNedd4-2 suppresses ENaC activity by controlling channel number at the cell surface,providing an explanation for the regulatory defect in Liddle's syndrome. Now, we haveidentified the ubiquitin-conjugating enzyme UBE2E3 that acts in concert with Nedd4-2 inthe regulation of ENaC. Moreover, we found that the aldosterone induced Sgk1 kinasephosphorylates Nedd4-2 in Xenopus laevis oocytes, and that such phosphorylation reducesENaC-Nedd4-2 interaction, resulting in decreased ENaC ubiquitination and accumulation atthe plasma membrane. This suggests for the first time a complete signaling pathwaybetween aldosterone and one of its effector molecules, ENaC. Indeed, in mpkCCDcl4 cells,a murine cell model of the transepithelial Na+ transport in the cortical collecting duct, we areable to demonstrate that aldosterone induces the phosphorylation of Nedd4-2. Incollaboration with the group of Dr. Brigitte Frey in Bern, we have searched in patients andhealthy controls for polymorphisms in the gene encoding Nedd4-2. Indeed, we haveidentified several mutations, which we are currently analyzing with respect to ENaCregulation.

The gap junction proteins connexin 43 and 45The gap junctions are intracellular channels which allow passage of ions and smallmolecules between cells. They are composed of members of the connexin protein family.Although two members of this family, connexin 43 and 45 contain also PY-motifs, theyappear not to be regulated by Nedd4/Nedd4-like ubiquitin-protein ligases. In contrast, theirstability and turnover seems to be controlled by a tyrosine-base sorting motif that overlapswith the PY-motif. We have identified several other proteins that interact with connexin 43and 45, which we currently analyze.

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Key words

Ubiquitin-protein ligase, Nedd4, PY-motif, protein-protein interaction, Na+-homeostasis,hypertension, kinase, ENaC, gap junction, phosphorylation

Collaborations

Dr. Marc Chanson, Department of Pediatrics, University of GenevaDr. Nicolas Demaurex, Department of Physiology, University of GenevaDr. Brigitte Frey, Division of Nephrology/Hypertension, Inselspital, University of BernDr. René Bindels, Dept. of Cell Biology, University of Nijmegen, The NetherlandsDr. Claudine Pique, CNRS UMR 8104 et INSERM U567, Institut Cochin, Paris, FranceDr. David Pearce, Cellular and Molecular Pharmacology, UCSF, San Francisco, USADr. J. Howard Pratt, Endocrinology & Hypertension, Indiana University, Indianapolis,USADr. A. Vandewalle, INSERM, Faculté de Médecine Xavier Bichat, Paris Cédex, France

Selected References

1. Kamynina,E., C. Debonneville, M. Bens, A. Vandewalle, and O. Staub. A novelmouse Nedd4 protein regulates the epithelial Na+ channel. FASEB J., 15:204-214,2001.

2. Debonneville, C., S. Y. Flores, E. Kamynina, P. J. Plant, C. Tauxe, M.A. Thomas,C. Münster, A. Chraïbi, J. H. Pratt, J.-D. Horisberger, D. Pearce, J. Loffing, and O.Staub. Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na+ channel cellsurface expression. EMBO J., 20:7052-7059, 2001.

3. Kamynina, E., and O. Staub. Concerted action of ENaC, Nedd4-2 and Sgk1 intransepithelial Na+ transport. Am.J.Physiol./Renal Physiol. 283:F377-F387, 2002

4. Thomas, M.A., N. Zosso, I. Scerri, N. Demaurex, M. Chanson, and O. Staub. Atyrosine-based sorting signal is involved in connexin43 stability and gap junctionturnover. J.Cell Sci., 116:2213-2222, 2003

Oral presentations at in ternational meetings

22/10/02 28th Meeting of the Federation of European Biochemical Societies (20– 25/10/02), Istanbul, Turkey.Characterization of the enzyme cascade that regulates the epithelial Na+

channel via ubiquitination.

06/7/03 2003 FASEB Summer Research Conferences, Perspectives inTransport Biology (5-10/7/2003), Tuscon, Arizona, USARegulation by phosphorylation and ubiquitination of the epithelial Na+

channel ENaC.

11/9/03 APS Conference, Aldosterone and ENaC: From Genetics toPhysiology (10-14/9/2003), Banff, Alberta, CanadaRegulation of the epithelial sodium channel ENaC by ubiquitinationand phosphorylation.

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Johannes Loffing

„Control of electrolyte homeostasis by the distal nephron“

Regulation of electrolyte transport in the kidney is crucial for maintenance of extracellularion and fluid homeostasis. The fine-tuning of renal ion excretion takes place in the distalnephron which is composed by several, morphologically and functionally distinctsubsegments. In these segments, ion transport is under complex control of varioushormones (e.g. aldosterone, angiotensin II, insulin), the tubular fluid composition (e.g. ionconcentration, pH) and the flow-rate. We study pathways and mechanisms that control theabundance and activity of the ion transport proteins (e.g. NCC, ENaC, ROMK) along thedistal nephron in vivo under various physiological or pathophysiological conditions (e.g.altered dietary sodium/potassium intake). The specific role of given ion transportingproteins is also investigated in genetically modified mice, representing murine models forhuman heriditary tubulopathies (e.g. Bartter’s and Gitelman’s syndrome). The studies areaimed to get further insights into cellular and molecular mechanisms by which the distalnephron maintains ion homeostasis and whereby dysregulations of these processes maylead to renal electrolyte transport disorders.

Key wordsdistal nephron, epithelial sodium channel - ENaC, thiazide-sensitive sodium chloridecotransporter - NCC, renal outer medulla potassium channel – ROMK, proteintrafficking.

Collaborations

- Kaissling B, Verrey F, Wagner C – Institutes of Anatomy and Physiology,University of Zurich

- Firsov D, Hummler E, Rossier B, Staub O – DPT, University of Lausanne- Meneton P – INSERM, Paris- Hebert S – Yale University, New Haven- Bindels R, Hoenderop J – Department of Cell Physiology, University of Nijmegen- Vallon V – Dept. of Medicine and Pharmacology, University of California San

Diego

Selected references

1. Loffing J, Kaissling B: Sodium and calcium transport pathways along themammalian distal nephron: from rabbit to human. Am. J. Physiol. – Renal284:F628-43 (2003).

2. Rubera I, Loffing J, Palmer LG, Frindt G, Fowler-Jaeger N, Sauter D, Carroll T,McMahon A, Hummler E*, Rossier BC: Collecting duct-specific gene inactivationof alphaENaC in the mouse kidney does not impair sodium and potassium balance.J Clin Invest. 112: 554-65 (2003)

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Lectures as invited speaker

- APS Conference "Aldosterone and ENaC: from Genetics to Physiology", Banff,Canada, September 10-14; 2003 “Localization and Regulation of ENaC in theKidney”

- World Congress of Nephrology 2003, Berlin, Germany, June 8-12; 2003.“Genetic Mouse Models for Bartter’s and Gitelman’s Syndromes”

- 19th International Conference on Biological Membranes, Troina, Italy, June 9-12;2002. „Regulation of Sodium Balance by the Distal Nephron“

- Nephrologisches Grundlagenkolloquium der Universität Zürich, February 4; 2002.„The TSC/NCC knockout mouse as model for Gitelman’s syndrome “

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Hugues Abriel

“Molecular Physiology and Pathophysiology of the Cardiac Action Potential:Regulation of hNav1.5 and hERG”

Projects

The main focus of our group is to elucidate novel molecular and cellular mechanismsunderlying cardiac arrhythmias causing sudden death. To this end, we are investigatingmutations of ion channel genes found in patients and families presenting with genetic typesof lethal arrhythmias such as the congenital long QT syndrome and Brugada syndrome. Inaddition, we are studying new forms of regulation of cardiac ion channels relevant to thearrhythmogenic mechanisms.We established our group in early 2002, and thus far, we mainly focussed on investigatingthe regulation of the principal voltage-gated sodium channel expressed in the heart: Nav1.5.On one hand, we obtained strong evidence that the PY-motif of Nav1.5 (similar to ENaCPY-motif) is a binding site for Nedd4-like protein-ubiquitin ligases. We found that Nedd4-2 can ubiquitinate Nav1.5, and, consequently, regulate its surface density in HEK cells.We are now pursuing these experiments using animal and human cardiomyocytes, whichwill allow us to address the physiological relevance of these findings.On the other hand, in collaboration with the service of Cardiology, CHUV, we found intwo Brugada syndrome patients with clinical manifestations exacerbated by fever, two newmutations in the gene encoding Nav1.5. We studied the alterations of the Nav1.5 functioncaused by these mutations, and found that both were “loss-of-function” mutations. We arecurrently investigating the detailed mechanisms by which these mutations decrease thesodium current, and how the specific thermo-dependant phenotype can be explained.

Key words

Voltage-gate sodium channels, Voltage-gates potassium channels, Cardiac action potential,congenital Long QT syndrome, drug-induced long QT syndrome, Brugada syndrome,Cardiac electrophysiology, Ubiquitination, Nedd4, Molecular pharmacology.

Collaborations

Dr. Olivier Staub, Institute of Pharmacology and Toxicology, Uni. Lausanne,Switzerland

Dr. Robert S. Kass, Dpt. of Pharmacology, Columbia University, New York

Dr. Silvia G. Priori, Molecular Cardiology, Pavia, Italy

Dr. Isabelle Decosterd, Department of Anesthesiology, CHUV, University ofLausanne

Division of Cardiovascular Diseases, Research Center Pierre Fabre, Castres,FranceDr. Stephan Rohr, Physiology, University of BernDr. Jan Kucera, Physiology, University of Bern

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Selected references

Liu, H., Tateyama, M., Abriel, H., and Kass, R.S. (2002) Channel openings arenecessary but not sufficient for use-dependent block of cardiac Na+ channels by flecainide:evidence from the analysis of disease-linked mutations. Journal of General Physiology,Jul;120(1):39-51.

Lectures as invited speaker

Abriel, H., Rivolta, I., Tateyama, M., Liu H., Memmi, M., Vardas, P., Napolitano, C.,Priori, S.G., and Kass R.S. Brugada and Congenital Long QT Syndromes Caused byMutations of a Single Amino-Acid Residue of the Cardiac Sodium Channel. Meeting of theSwiss Society of Cardiology, Bern, 13 June 2002.

Abriel, H. and Staub, O., Regulation of the Cardiac Voltage-Gated Sodium Channel by aProtein-Ubiquitin Ligase, Montpellier, France, 14 September 2002, Meeting of theEuropean Working Group of Cardiac Cellular Electrophysiology.

Abriel, H., Physiologie Moléculaire de Nav1.5, Castres, France, 16 September 2002,Pierre-Fabre Company.

Abriel, H., Rôle du C-terminus de Nav1.5: nouvelles données, Nantes, France, 20Septembre 2002, Inserm, invited by Dr. D. Escande.

Abriel, H., Molecular basis of the Congenital and Drug-Induced forms of the Long QTSyndrome, Mini-symposium on drug-induced long QT syndrome, CHUV, Lausanne, 11October 2002.

Abriel, H., Molecular Physiology of Cardiac Ion Channels, First Training Day inElectrophysiology of the Swiss Cardiovascular Network of Research and Education,EPFL, Lausanne, 11 December 2002.

Abriel, H., Regulation of Cardiac Sodium Channels by the Ubiquitin-Protein LigaseNedd4-2, Institute of Physiology, University of Bern, 27 June 2003.

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4 . Récepteurs couplés aux protéines G et mécanismes designalisation intracellulaire

Susanna Cotecchia

"The alpha1-adrenergic receptor subtypes: molecular mechanisms ofreceptor function and physiological implications"

Projects

Our research activity concerns the molecular mechanisms underlying the function andregulation of the adrenergic receptor subtypes in vitro and in vivo.The adrenergic receptors (ARs) mediate the effects of adrenaline and noradrenaline inseveral organs including heart, vessels, brain and liver. They belong to the family of theheptahelical G protein-coupled receptors which transduce a large number of signals acrossthe cell membrane and are the targets for the majority of clinically used drugs. The ARfamily includes nine gene products divided in three groups, three alpha1 (alpha1a, alpha1band alpha1d), three alpha2 (alpha2A, alpha2B and alpha2C) and three beta (beta1, beta2and beta3). Our investigation is predominatly focussed on the alpha1b-AR subtype coupledto the Gq/phospholipase C signaling pathway.

The main research projects ongoing in our laboratory are the following:

A) Molecular basis of receptor function and regulation

We combine site-directed mutagenesis and molecular modelling of receptors and G proteinsto investigate the molecular basis of receptor activation and G protein coupling. We havegenerated a large number of either inactive or constitutively active receptor mutants andstudied their theoretical structures by molecular dynamics analysis (in collaboration withDr. F. Fanelli, Italy). We use the yeast 2-hybrid system and other proteomic approaches tofind new proteins interacting with the alpha1-AR subtypes. In particular, we are interestedin exploring the role in receptor regulation of the µ2 subunit of the AP2 complex and ezrin,that bind to the C-tail of the alpha1b-AR subtype. In addition, in collaboration with Prof.H. Vogel, EPFL, we measure FRET between receptors tagged with different fluorescenttags to monitor receptor oligomerization and protein-protein interactions.

B) Molecular basis of drug action and selectivity.

We investigate the pharmacological behaviour, i.e. agonism, inverse agonism and neutralantagonism, of a large number of drugs at different adrenergic receptor subtypes. Our goalis to elucidate the mechanisms of action of adrenergic drugs at a molecular level as well asto provide further insight into their action in vivo.

C) Study of the physiological role of the alpha1-AR subtypes using knock out mice.

We have created a knock out mouse model lacking the alpha1b-AR. The knock out micedisplay changes in their blood pressure response, in glucose metabolism as well as in somebehavioural parameters. A number of collaborations have been established with groups ofthe University of Lausanne as well as with other institutions to further explore thephenotype of the alpha1b-AR knock out mice and of those crossed with knock out micelacking other AR subtypes.

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Key words

Receptors - G proteins - Desensitization - Molecular dynamics - Inverse agonism -Constitutive activity- knock out mice

Collaborations

Dr. Francesca Fanelli and Prof. P.G. DeBenedetti, Universitá di Modena, Italy.Dr. Tommaso Costa, Istituto Superiore di Sanitá, Rome, Italy.Dr. Federico Mayor, Jr. Universidad Autonoma, Madrid, Spain.Prof. Martin Lohse, Department of Pharmacology, University of Wurzburg, Germany.Prof. Graeme Milligan, Department of Biochemistry, University of Glasgow, UK.Prof. Horst Vogel, Ecole Polythecnique Fédérale, Lausanne, Switzerland.Dr Jean-Pol Tassin, Collège de France, Paris.

Selected references

1. Greasley P., Fanelli F. Rossier O., Abuin L. and Cotecchia S. (2002).Mutagenesis and modelling of the alpha1b-adrenergic receptor highlights the role ofthe helix3/helix 6 interface in receptor activation. Molecular Pharmacol. 61, 1025-1032.

2. Drouin C., Darracq L., Trovero F., Blanc G., Brochet D., Glowinski J., CotecchiaS. and Tassin J-P. (2002). Alpha1b-adrenergic receptors control locomotor andrewarding effects of psychostimulants and opiates. J. Neuroscience, 22, 2873-2884.

3. Diviani, D., Lattion, A.-L., Abuin, L., Staub. O., and Cotecchia, S. (2003). Theadaptor complex 2 directly interacts with the alpha-1b-adrenergic receptor and playsa role in receptor endocytosis. J.Biol.Chem. 278:19331-19340.

4. Stanasila L., Perez J.B., Vogel H. and Cotecchia S. (2003). Oligomerization of thealpha1a- and alpha1b-adrenergic receptor subytpes: potential implications inreceptor internalization. J. Biol. Chem. 278, 40239-40251.

Lectures as invited speaker in 2002-2003

- 2nd annual ASPET G-protein coupled receptor Symposium, of the IUPHAR,“Pharmacology of Adrenoceptors”, DoubleTree Sonoma Valley, California, Juillet2002

- Leiden/Amsterdam center for Drug Research – Fall Symposium “New tools andconcepts for the medicinal chemistry”, Amsterdam, 21-22 Novembre, 2002

- ELSO meeting, Dresden, Germany, 20-24 Septembre, 2003

- Workshop "G protein-Coupled Receptors: New Insights into the Signaling andRegulation" (Université Pierre et Marie Curie-CNRS-INSERM) Paris, Octobre 23-24, 2003

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Dario Diviani

‘The AKAP-Lbc signaling complex: molecular characterization and functionalimplications’

A-kinase anchoring proteins (AKAPs) are a family of functionally related proteins thatcoordinate cAMP-responsive events by targeting the cAMP-dependent protein kinase(PKA) at defined subcellular compartments towards preferred substrates. We have recentlyidentified a novel anchoring protein, called AKAP-Lbc, that functions as a PKA-targetingprotein as well as guanine nucleotide exchange factor (GEF) for the small GTP bindingprotein Rho. This suggested that the cAMP and Rho signaling pathways arecompartmentalized within the AKAP-Lbc signaling complex. Importantly, our functionalstudies indicated that the Rho-GEF activity of AKAP-Lbc is selectively stimulated by thealpha subunit of the heterotrimeric G protein G12 as well as by G protein-coupled receptors(GPCRs) that couple to G12. AKAP-Lbc is predominantly expressed in the heart, whereits function is yet to be elucidated.Although it is well established that Rho is a key mediator of cardiac hypertrophy, the exactmechanisms through which hypertrophic stimuli activate Rho are poorly characterized.Therefore, we consider that elucidating the signaling properties of AKAP-Lbc is afundamental step in the understanding of how Rho function is regulated in the heart. Theresearch ongoing in our laboratory is directed at elucidating the molecular mechanismsinvolved in the activation of AKAP-Lbc by GPCRs (project1), the functional relevance ofthe AKAP-Lbc/PKA interaction in the heart (project 2) and the functional implications ofAKAP-Lbc in the heart (project 3). The molecular analysis of the AKAP-Lbc signalingcomplex will be carried out in recombinant systems whereas the potential role of AKAP-Lbc in cardiac hypertrophy will be explored in primary cultures of rat ventricular myocytes.In addition to expanding our knowledge on Rho signaling, our research aims to provide ageneral contribution to the field of AKAPs.

Project 1 : Investigation of the molecular mechanisms involved in theactivation of AKAP-Lbc by Gαααα12.

We have demonstrated that AKAP-Lbc has a very low basal Rho-GEF activity. Thisactivity can be dramatically enhanced by the overexpression of constitutively active Gα12or the incubation of cells with agonists that stimulate G12 signaling. Furthermore, activatedGα12 can form a complex with AKAP-Lbc. Our working hypothesis is that, in the absenceof upstream activating signals, AKAP-Lbc is maintained in inactive autoinhibitedconformation. Upon stimulation of G12-coupled seven transmembrane receptors, Gα12 isrecruited to the AKAP-Lbc signaling complex, releases the autoinhibition and enhances theRho-GEF activity. To address this point we are determining the molecular determinants thatmaintain AKAP-Lbc in an inactive state under basal unstimulated conditions and assessinghow activated Gα12 interacts with AKAP-Lbc.

Project 2 : To determine the functional relevance of the AKAP-Lbc/PKAinteraction.

While the association between PKA and AKAP-Lbc is well characterized, its functionalrelevance remains to be elucidated. One possibility is that the catalytic activity of AKAP-Lbc might be regulated by the PKA holoenzyme associated with the anchoring protein.This hypothesis is investigated by determining the functional impact of mutating the PKAanchoring domain of AKAP-Lbc on its Rho-GEF activity. Another possibility is that

49

AKAP-Lbc might interact with other signaling proteins that are regulated by PKA. Toaddress this point, we are identifying novel proteins interacting with AKAP-Lbc either byusing the yeast-two-hybrid approach or by mass spectrometry.

Project 3 : To determine the biological function of AKAP-Lbc in the heart andwhether it plays a role in cardiac hypertrophy.

To determine whether AKAP-Lbc can mediate Rho activation and hypertrophic responseswe will overexpress wild type AKAP-Lbc in primary cultures of rat neonatal ventricularmyocytes using the lentivirus expression system and determine if it can enhance Rhoactivation, hypertrophic gene transcription and myofibrillar organization in the absence orpresence of hypertrophic stimuli such as angiotensin II, norepinephrine.

Key words :

A-kinase anchoring protein (AKAP) - PKA - G protein-coupled receptors GPCR) - Rho -cardiomyocytes

Collaborations

Dr. Thierry Pedrazzini (Division of Hypertension, CHUV, Lausanne)Dr. Vassalli (Cardiology division, CHUV, Lausanne)

Lectures as invited speaker in 2002-2003

2003

35th Annual Meeting of the Swiss Societies for Experimental Biology. March 19-21, 2003.Davos, Switzerland. Modulation of the function of the small GTP binding protein Rho byan A-kinase anchoring protein signaling complex.

Selected reference

Diviani D., Abuin L., Lattion A.L., Cotecchia S. (2003) The µ2 subunit of the AP2complex interacts with the α1b-adrenergic receptor and modulates its internalization J.Biol.Chem. 278: 19331-40.

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5 . Toxicologie du NO et génotoxicité des contaminantsalimentaires

Emanuela Felley-Bosco

"Cellular biology of inducible nitric oxide synthase (iNOS) in humanepithelial cells"

Project

The activity of the inducible nitric oxide synthase (iNOS) is elevated in several pathologicalsituations, in particular colon adenomas, early stages of carcinomas; inflammatory boweldiseases. Although iNOS expression is found in epithelial cells, the physiological role ofiNOS in these cells is poorly characterized. The objective of this project is to obtain a betterunderstanding of the cellular biology of iNOS in human epithelial cells.

The profile of proteins induced by cytokines together with iNOS was defined by aproteomic approach. Arginosuccinate synthase, tryptophanyl-tRNA synthetase andindoleamine-2, 3-dioxygenase were found induced with expression kinetics similar toiNOS, highlighting the importance of both arginine and tryptophan metabolism in inflamedepithelium. Analysis of clinical samples validated the in vitro characterization.

By controlled expression of iNOS in intestinal cells we found that iNOS activity increasedsynergistically the expression of heme oxygenase-1, a biomarker of oxidative stress, in thepresence of glutathione depletion, while iNOS expression alone had no effect. Therefore,cellular redox environment is able to modify NO effects.

Key words

Toxicology, nitric oxide, inducible nitric oxide synthase, colon epithelial cell

Collaborations

Dr. M. Reymond, University of Magdeburg, GermanyDr. C. Servis, Biochemistry Institute, Epalinges, SwitzerlandDr. L.L. Keefer, National Cancer Institute, Frederick Cancer Research and DevelopmentCenter, Frederick, MD 21702, USA

Selected references

Barcelo-Batllori, S., André, M., Servis, C., Lévy, N., Takikawa, O, Michetti, P. ,Reymond, M and Felley-Bosco E. Proteomic analysis of cytokine-induced proteins inhuman intestinal epithelial cells: implications for inflammatory bowel diseases. Proteomics,2: 551-560, 2002.

Felley, C.P., Pignatelli, B., Van Melle, G.D., Crabtree, J.E., Stolte, M., Diezi, J . ,Corthésy-Theulaz, I., Michetti, P., Bancel, B., Patricot, L.M., Ohshima, H., Felley-Bosco, E. Oxidative stress in gastric mucosa of asymptomatic humans infected withhelicobacter pylori: effect of bacterial eradication. Helicobacter, 7: 342-348, 2002.

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André, M., Felley-Bosco, E. Heme oxygenase-1 induction by endogenous nitric oxide:influence of intracellular glutathione. FEBS Letters, 546: 223-227, 2003

Terrand, J., Felley-Bosco, E., Courjault-Gautier, F., Rochat, A.-C., Kucera, P., andRaddatz, E. Effects of endogenous NO and NO donor deta-NONOate on postanoxicrecovery of the developing heart. Mol. Cell. Biochem. 252: 53-63 2003

Reviews

Felley-Bosco, E., Bender, F. and Quest A. Caveolin-1 mediated post-transcriptionalregulation of inducible nitric oxide synthase in colon carcinoma cells. Biological Research,35:169-176, 2002

Lectures as invited speaker in 2002-2003

- Minisymposium of the Swiss Society of Free Radical Research, 28.06.02Gottlieben, "Tyrosine Phosphorylation of the Human Inducible Nitric OxideSynthase in Cultured Intestinal Epithelial Cells: Effects on Enzyme Specific Activityand Subcellular Localization".

- Symposium on "Supramolecular complex formation in signaling and disease", 26-28 September 2002, Santiago, CHILE: "Caveolin-1 mediated down-regulation ofiNOS in colon carcinoma cells".

Autumn meeting 2002 of the Toxicology Section of the Swiss Society ofPharmacology and Toxicology and XERR 3rd annual meeting 2002, "Health impactof xenobiotics from environmental and occupational exposure", Zurich 8-9November 2002, " Inducible nitric oxide synthase: a role in the intestinal mucosa-bacteria interaction"

- Symposium "Inflammation, degeneration and regeneration: from basic mechanismsto clinical manifestations", 16-18 October 2003, Magdeburg, "Human induciblenitric oxide synthase: friend or foe?"

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Phaik-Mooi Morgenthaler-Leong

« Induction of genetic changes by environmental contaminants »

Project

The focus of this group is the study of the gene-environment interactions that lead to thegeneration of genetic changes (mutations) in humans. We apply cellular, molecular andbiochemical methods to study the pathway from lesions on the DNA resulting fromexogenous and endogenous insults to the formation of mutations in the genome. One of ourgoals is to elucidate the mutagenic pathway of heterocyclic aromatic amines (HCAs), aclass of compounds that are formed during cooking of meats. We are evaluating theinfluence of DNA repair on the effects (apoptosis and mutagenesis) of HCAs. Because oftheir potential ubiquitous presence in our diet, there is concern that HCAs may have asignificant effect on human health, in particular, on the incidence of colon cancer. Detailedinformation on the mechanism of action of these compounds and appropriate biomarkerswill permit us to assess the health risks posed by HCAs.We are also involved in the development of novel methods for the detection of mutations inmixed populations and the direct measurement of mutations in humans.

Key words

Mutations, food contaminants, DNA repair, molecular toxicology, SNP detection

Collaborations

Prof. W.G. Thilly, Mass. Inst. of Technology, USA.

Lectures as invited speaker in 2002-2003

- The search for individual susceptibilities to environmental pollutants through singlenucleotide polymorphisms (SNPs) detection. International conference of riskmanagement for preventive medicine. March 27-28, 2003 Tokyo, Japan

- Inhibition of the repair of G:T mispairs by PhIP-DNA adducts, 1st Swiss Meetingon Genomes Stability: DNA Dynamics and Epigenetics. October 1-3, 2003Uetendorf, Switzerland

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6 . Neurophysiologie de l’olfaction et du goût

Marie-Christine Broillet

Project I

Cyclic nucleotide-gated channels: Their roles in neuronaldevelopment and sensory transduction cascades

Cyclic nucleotide-gated (CNG) channels are critical for signal transduction in the vertebratevisual and olfactory systems. These channels are activated by the binding of cAMP orcGMP to a particular domain of the protein. We have shown that nitric oxide (NO) can alsodirectly activate CNG channels independently from cyclic nucleotides and on a distinctbinding site. Three types of subunits have been identified for the olfactory CNG channel,the A2, the A4 and the B1b subunit. Through the direct gating of NO, we have shown thatΑ4 subunits can form functional homomeric channels in heterologous expression systems.We also found a conductance having the characteristics of the Α4 channel in the neurons ofthe vomeronasal organ. Recently, olfactory CNG channel subunits have been found inother tissues and in the central nervous system, in particular in hippocampal neurons. Theirroles in these cells have not been identified yet.We believe that depending on the type of tissue, native “olfactory” CNG channels may,beside the combination of A2, Α4 and Β1b subunits, consist of different homo andheteromeric combinations of the three subunits. Furthermore, various combinations of A2,Α4 and Β1b subunits may be expressed differentially on microdomains of the same cell.We also hypothesize that the subunit composition of the CNG channels plays a critical rolein ligand sensitivity (cyclic nucleotides or NO) and channel functions.

We are now focusing on the roles of CNG channels in growth cone behavior during neuraldevelopment and regeneration of olfactory neurons as well as on the roles of CNGchannels in yet unidentified sensory transduction cascades (pheromone transduction, tastetransduction).

Keywords

cyclic nucleotide-gated channel, olfaction, taste, nitric oxide, neuronal development andregeneration, patch-clamp, immunohistochemistry, calcium imaging.

Collaborations

Dr. Stuart Firestein, Columbia University, New York, USA.Drs. U. Benjamin Kaupp and Stephan Frings, Forschungszentrum, Jülich, Germany.Dr. I. Rodriguez, Dep. of Zoology and Animal Biology, University of Geneva,SwitzerlandDr. P. Mombaerts, Rockefeller University, New York, USA

54

Project II

"Influence of aging on salt taste perception and its significance for saltintake"

Taste sensitivity serves as a primary directive of food selection and has often been reportedto decline with increasing age. Salt taste perception among the elderly is of particularinterest because this population is at increased risk for hypertension (striking over 20% ofthe population over 60 years old) and sodium intake is recognized as a major risk factor forhypertension.We are applying a combination of molecular, behavioral and electrophysiologicalapproaches to test the influence of aging on salt taste perception. Recently, the epithelialsodium channel (ENaC) has been implicated in the entry of sodium into taste receptor cellsand we concentrate on studying the changes with aging in the expression of the differentsubunits (α, β, and γ) of this channel in the different taste papillae of mice. Preliminaryfindings using semi-quantitative RT-PCR have already shown us that the expression ofthese subunits (and with this, the Na entry) seems differentially regulated during aging. Atthe same time, we are addressing the basic mechanisms underlying salt taste perception andthe changes that may occur during aging using electrophysiology. Findings from thisapproach may allow us to prevent or counteract changes in taste perception occurring atolder age or/and lead to new methods to enhance taste perception (e.g. for low-salt diets).

Key words

taste, ion channel, sensory transduction , salt, aging, ENaC

Collaborations

Dr. Bernd Lindemann, Department of Physiology, Saar University, Homburg, Germany

Selected references

Chatton, J.-Y., Broillet, M.-C., (2002). Detection of nitric oxide production by fluorescentindicators. In: Nitric Oxide, a volume of Methods in Enzymology, edited by Cadenas, E.and Packer, L., Academic Press 359:134-148

Boschat C., Pélofi C., Randin O., Roppolo D., Luscher C., Broillet M.-C., Rodriguez I.(2002). Pheromone detection mediated by a V1r vomeronasal receptor. NatureNeuroscience 5(12):1261-2

Lectures as invited speaker in 2002-2003

13ème Colloque "Canaux ioniques", Presqu'île de Giens, France, Septembre 22-25, 2002Firmenich; Symposium "Chemosensory perception", Genève, Suisse, Mars, 17-18, 2003Prix Leenaards pour la recherche 2003, Lausanne Palace Hotel, Suisse, Avril 3, 2003Nestlé Research Center, Minisymposium on olfaction, Lausanne, Suisse, Juin 16, 2003.

Distinction

Prix Leenaards 2003 pour la promotion de la recherche scientifique. "Pheromoneinformation coding in the mammalian vomeronasal system".

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DEPARTEMENT DE PHARMACOLOGIE ET DE TOXICOLOGIE

PUBLICATIONSListe A

2002-2003

André, M., Felley-Bosco, E. (2003). Heme oxygenase-1 induction by endogenous nitricoxide: influence of intracellular glutathione. FEBS Letters, 546: 223-227.

Auberson, M., Hoffmann-Pochon, N., Vandewalle, A., Kellenberger, S., & Schild, L.(2003). Epithelial sodium channel (ENaC) mutants causing Liddle syndrome retain theirability to respond to aldosterone and vasopressin. Am. J. Physiol.- Renal Physiol. 285,F459-471.

Auclair A., Cotecchia S., Glowinski J. and Tassin J-P. (2002). D-Amphetamine fails toincrease extracellular dopamine levels in mice lacking alpha1b-adrenergic receptors:relationship between functional and nonfunctional dopamine release. J. Neuroscience, 22,9150-9154.

Bacic D, Capuano P, Gisler SM, Pribanic S, Christensen EI, Biber J, Loffing J, KaisslingB, Wagner CA, Murer H (2003). Impaired PTH-induced endocytotic down-regulation ofthe renal type IIa Na+/Pi-cotransporter in RAP-deficient mice with reduced megalinexpression. Pflugers Arch. 446: 475-84.

Barcelo-Batllori, S., André, M., Servis, C., Lévy, N., Takikawa, O, Michetti, P. ,Reymond, M and Felley-Bosco E. (2002). Proteomic analysis of cytokine-induced proteinsin human intestinal epithelial cells: implications for inflammatory bowel diseases.Proteomics, 2: 551-560.

Béguin, P., Crambert, G., Monnet-Tschudi, F., Uldry, M., Horisberger, J-D., Garty. H. ,and Geering K. (2002). FXYD7 is a brain-specific regulator of Na,K-ATPase α1-βisozymes. EMBO J. 21, 3264-3273.

Björklöf K., Lundström K., Abuin L., Greasley P. and Cotecchia S. (2002). Co- andPost-translational modifications of the alpha1b-adrenergic receptor: effects on receptorexpression and function. Biochemistry, 41, 4281-4291.

Bonny, O., Knoers, N., Monnens, L., Rossier, B.C. (2002). A novel mutation of theepithelial Na+ channel causes type 1 pseudohypoaldosteronism. Pediatr. Nephrol., 17:804-808.

Boschat C., Pélofi C., Randin O., Roppolo D., Luscher C., Broillet M.-C., Rodriguez I.(2002). Pheromone detection mediated by a V1r vomeronasal receptor. NatureNeuroscience 5(12):1261-2

Burcelin, R., Crivelli, V., Dacosta, A., Roy-Tirelli, A.,and Thorens, B. (2002)Heterogenous metabolic adaptation of C57BL/6J mice to high fat feeding. Am. J. Physiol.282: E834-E842.

Burcelin, R., Crivelli, V., Perrin, P., DaCosta, A., Mu, J., Kahn, B.B., Birnbaum, M.J.,Kahn, C.R., Vollenweider, P., and Thorens, B. (2003) GLUT4, AMP-kinase, but not the

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insulin receptor are required for hepatoportal glucose sensor-stimulated muscle glucoseutilization. J. Clin. Invest. 111:155-1562.

Burcelin R, Thorens B, Glauser M, Gaillard RC, and Pralong FP. (2003) GnRH secretionfrom hypothalamic neurons: stimulation by insulin and potentiation by leptin. Endocrinol.144 : 4484-4491.

Burnay, M., Crambert, G., Kharoubi-Hess, S., Geering, K., and Horisberger J.-D.(2003). Electrogenicity of Na,K- and H,K-ATPase activity and presence of a positivelycharged amino acid in the fifth transmembrane segment. J. Biol. Chem. 278, 19237-19244.

Cattan, P., Rottembourg,D., Cottet, S., Tardivel, I., Dupraz, P., Thorens, B., Boitard,C., Carel, J.C. (2003) Destruction of conditional insulinoma cell lines in NOD mice: A roleof autoimmunity. Diabetologia 46: 504-510.

Cheng J, Moyer BD, Milewski M, Loffing J, Ikeda M, Mickle JE, Cutting GG, Li M,Stanton BA, Guggino WB. (2002). A GOLGI associated PDZ domain protein modulatescystic fibrosis transmembrane regulator plasma membrane expression. J Biol Chem. 277:3520-9.

Chraïbi,A. and Horisberger,J.-D. (2003). Dual effect of temperature on the humanepithelial Na+ channel . Pflügers Arch., 447:316-320.

Chraïbi,A. and Horisberger,J.-D. (2002). Na self inhibition of human Epithelial NaChannel:Temperature dependence and effect of extracellular proteases. J. Gen. Physiol.,120:133-145.

Cook S, Hugli O, Egli M, Vollenweider P, Burcelin R, Nicod P, Thorens B, Scherrer U.(2003) Clustering of cardiovascular risk factors mimicking the human metabolic syndromeX in eNOS null mice. Swiss Med Wkly. 133:360-363.

Cottet, S., Dupraz, P., Hamburger, F., Dolci, W., Jaquet, M., and Thorens, B. (2002) c-FLIP protein prevents tumor necrosis factor-α-mediated induction of caspase-8-dependentapoptosis in insulin-secreting ßTc-Tet cells. Diabetes, 51: 1805-1814.

Crambert, G., Béguin, P., Pestov, N. B., Modyanov, N. N., and Geering, K. (2002).βm, a structural member of the X,K-ATPase β subunit family, resides in the ER and doesnot associate with any known X,K-ATPase α subunit. Biochemistry 41, 6723-6733.

Crambert, G., Horisberger, J-D., Modyanov, N. N., and Geering K. (2002). Humannongastric H+,K+-ATPase: transport properties of ATP1al1 assembled with different β-subunits. Am. J. Physiol. 283, C 305-C314.

Crambert, G., Füzesi, M., Garty, H., Karlish, S., and Geering, K. (2002).Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transportproperties. Proc. Natl. Acad. Sci. USA 99, 11476-11481.

Crambert G., Schaer D., Roy S., and Geering K. (2004). New molecular determinantscontroling the accessibility of ouabain to its binding site in human Na,K-ATPase αisoforms. Mol. Pharmacol., 65:335-341.

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Dahlmann, A. Pradervand, S., Hummler, E., Rossier, B.C., Frindt, G., Palmer, L.G.(2003). Mineralocorticoid regulation of epithelial Na+ channels is maintained in a mousemodel of Liddle's syndrome. Am. J. Physiol. Renal Physiol.285:F310-F318.

Del Carmine R., Caterina Ambrosio C., Sbraccia, M., Cotecchia S. Ijzerman A.P. andCosta T. (2002). Mutations inducing divergent shifts of constitutive activity reveal differentmodes of binding among catecholamine analogues to the beta2-adrenergic receptor. BritishJ. of Pharmacol. 135, 1715-1722.

Diviani, D., Lattion, A.-L., Abuin, L., Staub. O., and Cotecchia, S. (2003). The adaptorcomplex 2 directly interacts with the alpha-1b-adrenergic receptor and plays a role inreceptor endocytosis. J.Biol.Chem. 278:19331-19340.

Drouin C., Darracq L., Trovero F., Blanc G., Brochet D., Glowinski J., Cotecchia S. andTassin J-P. (2002). Alpha1b-adrenergic receptors control locomotor and rewarding effectsof psychostimulants and opiates. J. Neuroscience, 22, 2873-2884.

Felley, C.P., Pignatelli, B., Van Melle, G.D., Crabtree, J.E., Stolte, M., Diezi, J . ,Corthésy-Theulaz, I., Michetti, P., Bancel, B., Patricot, L.M., Ohshima, H., Felley-Bosco, E. (2002). Oxidative stress in gastric mucosa of asymptomatic humans infectedwith helicobacter pylori: effect of bacterial eradication. Helicobacter, 7: 342-348.

Flahaut, M., Rossier, B.C., Firsov, D. (2002). Respective roles of calcitonin receptor-likereceptor (CRLR) and receptor activity modifying proteins (RAMPs) in cell surfaceexpression of CRLR/RAMP heterodimeric receptors. J. Biol. Chem., 277:14731-7.

Flahaut, M., Pfister, C., Rossier, B.C., Firsov, D. (2003). N-Glycosylation andconserved cysteine residues in RAMP3 play a critical role for the functional expression ofCRLR/RAMP3 adrenomedullin receptor. Biochemistry 42:10333-10341.

Galbiati. F., Polastri, L., Thorens, B., Dupraz, P., Fiorina, P., Cavallaro, U. ,Christofori, G., Davalli, A.M. (2003) Molecular pathways involved in the antineoplasticeffects of calcitriol on insulinoma cells. Endocrinology 144:1832-41.

Gouyon F, Caillaud L, Carriere V, Klein C, Dalet V, Citadelle D, Kellett GL, Thorens B,Leturque A, Brot-Laroche E. (2003) Simple-sugar meals target GLUT2 at enterocyte apicalmembranes to improve sugar absorption: A study in GLUT2-null mice. J Physiol 552:823-32.

Greasley P., Fanelli F. Rossier O., Abuin L. and Cotecchia S. (2002). Mutagenesis andmodelling of the alpha1b-adrenergic receptor highlights the role of the helix3/helix 6interface in receptor activation. Molecular Pharmacol. 61, 1025-1032.

Guennoun,S. and Horisberger,J.-D. (2002). Cysteine-scanning mutagenesis study of thesixth transmembrane segment of the Na,K-ATPase α subunit. FEBS Lett., 513:277-281.

Guipponi, M., Vuagniaux, G., Wattenhofer, M., Shibuya, K., Vazquez, M., Dougherty,L., Scamuffa, N., Guida, E., Okui, M., Rossier, C., Hancock, M., Buchet, K. ,Reymond, A., Hummler, E., Marzella, P.L., Kudoh, J., Shimizu, N., Scott, H.S.,Antonarakis, S.E., Rossier, BC. (2002). The transmembrane serine protease (TMPRSS3)mutated in deafness DFNB8/10 activates the epithelial sodium channel (ENaC) in vitro.Hum. Mol. Genet., 11:2829-36.

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Haefliger, J.-A., Tawadros, T., Meylan, L., Le Gurun, S., Roehrich, M.-E. Martin,D.,Thorens, B., and Waeber, G. (2003) The scaffold protein IB1/JIP-1 is a critical mediatorof cytokine-induced apoptosis in pancreatic ß cells. J. Cell Sci. 116:1463-1469.

Henry PC, Kanelis V, O'Brien MC, Kim B, Gautschi I, Forman-Kay J, Schild L andRotin D. (2003). Affinity and specificity of interactions between Nedd4 isoforms and theepithelial Na+ channel. J. Biol. Chem. 278: 20019-20028.

Hiltunen TP, Hannila-Handelberg T, Petajaniemi N, Kantola I, Tikkanen I, Virtamo J ,Gautschi I, Schild L and Kontula K. (2002). Liddle's syndrome associated with a pointmutation in the extracellular domain of the epithelial sodium channel gamma subunit. J .Hypertens. 20: 2383-2390.

Hoenderop, J.G.J., van Leeuwen, J.P.T.M., van der Eerden, B.C.J., Kersten, F.F.J.,Van der Kemp., A.W.C.M., Mérillat, A.-M., Waarsing, E.J.H., Rossier, B.C., Vallon,V., Hummler, E., Bindels, R.J.M. (2003). Renal Ca2+ wasting, hyperabsorption, andreduced bone thickness in mice lacking TRPV5. J. Clin. Invest., 112: 1906-1914.

Horisberger,J.-D. and Kharoubi-Hess,S. (2002). Functional differences between αsubunit isoforms of the rat Na,K-ATPase expressed in Xenopus oocytes.J.Physiol.(London), 539:669-680.

Horisberger,J.-D. (2003). ENaC-CFTR interactions: the role of electrical couplingexplored in an epithelial cell model. Pflügers Arch., 445:522-528.

Hosokawa, M., and Thorens, B. Glucose release from GLUT2-null epatocytes is througha major membrane traffic pathway and minor pathway nvolving diffusion across the plasmamembrane. Am. J. Physiol. 282:E794-01.

Hummler, E., Mérillat, A.-M., Rubera, I., Rossier, B.C., Beermann, F. (2002).Conditional gene targeting of the Scnn1a (αENaC) gene locus. Genesis, 32:169-172.

Ibberson, M., Riederer, B.M., Uldry, M., Guhl, B., Roth, J., and Thorens, B. (2002).Immunolocalization of GLUTX1 in testis and to specific brain areas and vasoopressin-containing neurons. Endocrinology, 143: 276-284.

Joost,H.G., Bell, G.I., Best, J.D., Birnbaum, M.J., Charron, M.J., Chen, Y.T., Doege,H,, James, D.E., Lodish, H.F., Moley, K.H., Moley, J.F., Mueckler, M., Rogers, S. ,Schurmann, A., Seino, S., and Thorens B. (2002) Nomenclature of the GLUT/SLC2Afamily of sugar/polyol transport facilitators. Am. J. Physiol. 282:E974-E976

Kellenberger, S., Gautschi, I. & Schild, L. (2002). An external site controls closing of theepithelial Na+ channel ENaC. J. Physiol. 543.2, 413-424.

Kellenberger, S., Gautschi, I., & Schild, L. (2003). Mutations in the epithelial Na+

channel ENaC outer pore disrupt amiloride block by increasing its dissociation rate. Mol.Pharmacol. 64, 848-856.

Lagger Biner H, Arpin-Bott MP, Loffing J, Wang X, Knepper M, Hebert SC, Kaissling B(2002). The human cortical distal nephron: distribution of electrolyte and water transportpathways. J. Am. Soc. Nephrol. 13: 836-47.

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Liu, H., Tateyama, M., Abriel, H., and Kass, R.S. (2002) Channel openings arenecessary but not sufficient for use-dependent block of cardiac Na+ channels by flecainide:evidence from the analysis of disease-linked mutations. J. Gen. Physiol., Jul;120(1):39-51.

Mauro, T., Guitard M., Oda, Y., Crumrine, D., Komuves, L., Behne, M., Rassner, U. ,Elias, P.M., Hummler, E. (2002). The ENaC channel is required for normal epidermaldifferentiation. J. Invest. Dermatol., 118 : 589-594.

Mayo,K.E., Miller,L.J., Bataille, D., Dalle,S., Göke, B., Thorens, B., and Drucker, D.J.(2003) International Union of Pharmacology. XXXV. The Glucagon Receptor Family.Pharmacol. Rev. 55: 167-194.

Muller, O.G., Parnova, R.G., Centeno, G., Rossier, B.C., Firsov, D., Horisberger, J.D.(2003). Mineralocorticoid effects in the kidney: correlation between αENaC, GILZ, andSgk-1 mRNA expression and urinary excretion of Na+ and K+. J. Am. Soc. Nephrol.,14:1107-1115.

Nicod, M., Michlig, S., Flahaut, M., Salinas, M., Fowler Jaeger, N., Horisberger, J.-D.,Rossier, B.C., Firsov, D. (2002). A novel vasopressin-induced transcript promotes MAPkinase activation and ENaC downregulation. EMBO J., 21:5109-5117.

Nijenhuis T, Hoenderop JG, Loffing J, van der Kemp AW, van Os CH, Bindels RJ(2003). Thiazide-induced hypocalciuria is accompanied by a decreased expression of Ca2+

transport proteins in kidney. Kidney Int. 64:555-64.

Nikulina, M.A., Sandhu, N., Shamim, Z., Andersen, N.A., Oberson, A., Dupraz, P. ,Thorens, B., Karlsen, A.E., Bonny, C., Mandrup-Poulsen, T. (2003) The JNK bindingdomain of islet-brain 1 inhibits IL-1 induced JNK activity and apoptosis but not thetranscription of key proapoptotic or protective genes in insulin-secreting cell lines.Cytokine 24:13-24

O'Connell T.D., Ishizaka S., Nakamura A., Swigart P.M., Rodrigo M.C., SimpsonG.L., Cotecchia S., Rokosh D.G., Grossman W., Foster E. and Simpson P.C. (2003).Alpha1A/C- and alpha1B adrenergic receptors are required for physiological cardiachypertrophy in the double knockout mice. J. Clinical Investig. 111, 1783-1791.

Olivier, R., Scherrer, U., Horisberger, J.D., Rossier, B.C., Hummler, E. (2002).Selected contribution: limiting Na(+) transport rate in airway epithelia from alpha-ENaCtransgenic mice: a model for pulmonary edema. J. Appl. Physiol., 93:1881-7.

Pericin, M., Althage,A., Freiganng,S., Hengartner, H., Rolland, E., Dupraz, P. ,Thorens, B., Aebischer, P., Zinkernagel, R.M. (2002) Allogeneic ß-islet cells correctdiabetes and resist immune rejection. Proc. Natl. Acad. Sci. USA 99: 8203-8206.

Pradervand, S., Vandewalle, A., Bens, M., Gautschi, I., Loffing, J., Hummler, E.,Schild, L., Rossier, B.C. (2003). Dysfunction of the epithelial sodium channel (ENaC)expressed in the kidney of a mouse model for Liddle's syndrome. J. Am. Soc. Nephrol.,14:2219-2228.

Rubera I, Loffing J, Palmer LG, Frindt G, Fowler-Jaeger N, Sauter D, Carroll, T,McMahon A, Hummler E*, Rossier BC (2003). Collecting duct-specific gene inactivation

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of alphaENaC in the mouse kidney does not impair sodium and potassium balance. J. Clin.Invest. 112: 554-65.

Rubera, I., Meier, E., Vuagniaux, G., Merillat, A.-M., Beermann, F., Rossier, B.C.,Hummler, E. (2002). A conditional allele at the mouse channel activating protease 1(Prss8) gene locus. Genesis,32:173-176.

Schreiter, C. Hovius, R., Costioli, M., Pick, H., Kellenberger S., Schild, L., & Vogel,H. (2003). Characterization of the ligand-binding site of the Serotonin 5-HT3 Receptor:The Role of Glutamate residues 97, 224, and 235. J. Biol. Chem. 278, 22709-22716.

Sevillano, L. G., Melero, C. P., Caballero, E., Tomé F., Lelièvre L. G., Geering, K. ,Crambert, G., Carron, R., Medarde, M., and San Feliciano, A. (2002). Inotropic activityof hydroindene amidinohydrazones. J. Med. Chem. 45, 127-136.

Stanasila L., Perez J.B., Vogel H. and Cotecchia S. (2003). Oligomerization of thealpha1a- and alpha1b-adrenergic receptor subytpes: potential implications in receptorinternalization. J. Biol. Chem. 278, 40239-40251.

Sugimoto Y., Fujisawa R., Tanimura R., Lattion A.L., Cotecchia S., Tsujimoto G. ,Nagao T. and Kurose H. (2002). Beta1-selective agonist (-)-1-(3,4-dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)RO363] differentially interactswith key amino acids responsible for beta1-selective binding in resting and active states. J .Pharmacol. Experim. Ther. 30, 51-58.

Terrand, J., Felley-Bosco, E., Courjault-Gautier, F., Rochat, A.-C., Kucera, P., andRaddatz, E. (2003). Effects of endogenous NO and NO donor deta-NONOate onpostanoxic recovery of the developing heart. Mol. Cell. Biochem. 252: 53-63.

Thomas, M.A., N. Zosso, I. Scerri, N. Demaurex, M. Chanson*, and O. Staub. (2003).A tyrosine-based sorting signal is involved in connexin43 stability and gap junctionturnover. J.Cell Sci., 116:2213-2222.

Uldry, M., Ibberson, M., Hosokawa, M., and Thorens, B. (2002) GLUT2 is a highaffinity glucosamine transporter. FEBS letters 524: 199-203.

Vallet V, Pfister C, Loffing J, and Rossier B (2002). Cell-surface expression of theChannel Activating Protease xCAP1 is required for activation of ENaC in the Xenopusoocyte. J. Am. Soc. Nephrol. 13: 588-94.

van de Graaf, S.F.J., J.G.J Hoenderop, D. Gkika, D. Larners, J. Prenen, U. Rescher,V. Gerke, O. Staub, B. Nilius, and R.J.M. Bindels. (2003). Functional expression of theepithelial Ca 2+ channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex. EMBO J., 22:1478-1487.

Vecchione C., Fratta L., Rizzoni D., Notte A., Poulet R., Porteri E., Frati G., Guelfi D.,Trimarco V., Mulvany M.J., Agabiti-Rosei E., Trimarco B., Cotecchia S., Lembo G.(2002). Cardiovascular influences of alpha1b-adrenergic receptor defect in mice.Circulation 105, 1700-1707.

Vuagniaux, G., Vallet, V., Jaeger, N., Hummler, E., Rossier, B.C. (2002). Synergisticactivation of ENaC by three membrane-bound channel-activating serine proteases (mCAP1,mCAP2, and mCAP3) and serum- and glucocorticoid-regulated kinase (Sgk 1) in Xenopusoocytes. J. Gen. Physiol., 120:191-201.

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Wulff P, Vallon V, Huang DY, Volkl H, Yu F, Richter K, Jansen M, Schulz M, KlingelK, Loffing J, Kauselmann G, Bosl MR, Lang F, Kuhl D (2002). Impaired renal Na(+)

retention in the sgk1-knockout mouse. J. Clin. Invest. 110: 1263-1268.

Xue MZ, Bonny O, Morgenthaler S, Bochud M, Mooser V, Thilly WG, Schild L andLeong-Morgenthaler PM. (2002). Use of constant denaturant capillary electrophoresis ofpooled blood samples to identify single-nucleotide polymorphisms in the genes (Scnn1aand Scnn1b) encoding the alpha and beta subunits of the epithelial sodium channel. Clin.Chem. 48: 718-728.

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Books, book chapters, reviews 2002-2003 Liste B

Bonny, O., Rossier, B.C. (2002). Disturbances of Na/K balance: pseudoaldosteronismrevisited. J. Am. Soc. Nephrol. 13:2399-2414.

Broillet, M.-C., (2002). Cysteine-NO interaction and olfactory function. In: Redox CellBiology & Genetics a volume of Methods in Enzymology, edited by Abelson, J.N. andSimon, M. I. ,Academic Press 353 (19): 209-219

Broillet, M.-C, (2003). Cyclic nucleotide-gated channels: Multiple isoforms, Multipleroles. In: Molecular Insights into Ion Channel Biology in Health and Disease a volume ofPrinciples of Medical Biology, edited by Maue, R. Elsevier Science, Amsterdam.

Chatton, J.-Y., Broillet, M.-C., (2002). Detection of nitric oxide production by fluorescentindicators. In: Nitric Oxide, a volume of Methods in Enzymology, edited by Cadenas, E.and Packer, L., Academic Press 359:134-148

Cotecchia S, Bjorklof K, Rossier O, Stanasila L, Greasley P, Fanelli F. (2002). Thealpha1b-adrenergic receptor subtype: molecular properties and physiological implications.J Recept Signal Transduct Res. 22(1-4):1-16.

Crambert, G., Béguin, P., Hasler, U. and Geering, K. (2002) Functional consequences ofsubunit interactions in Na,K- and H,K-ATPases. In : Mechanisms and Consequences ofProton Transport, Kluwer Academic Publishers, Norwell, Massachustts, T.Urushidani, J .G. Forte, G. Sachs (eds), pp 33-42.

Crambert, G. and Geering, K. (2003) FXYD proteins: new tissue-specific regulators of theubiquitous Na,K-ATPase. Sci STKE. 2003 Jan 21;2003(166):RE1.

Crambert, G., Beguin, P., Uldry, M., Monnet-Tschudi, F., Horisberger, J.D., Garty, H.and Geering, K. (2003) FXYD7, the first brain- and isoform-specific regulator of Na,K-ATPase: Biosynthesis and function of its posttranslational modifications. Ann NY Acad Sci986, 444-448.

Felley-Bosco, E., Bender, F. and Quest A. (2002). Caveolin-1 mediated post-transcriptional regulation of inducible nitric oxide synthase in colon carcinoma cells.Biological Research, 35:169-176.

Firsov D, Muller OG. (2003) Aldosterone action in the heart. Pflugers Arch-Eur.J.Physiol,v.446, 328-333

Firsov D, Muller OG. (2003) Aldosterone action in the heart. Pflugers Arch-Eur.J.Physiol,v.446, 328-333

Firsov D. (2004) Revisiting sodium and water reabsorption with functional genomic tools.Current Opinion in Nephrology and Hypertension, v. 13, 59-65.

Flores, S.Y., Debonneville, C., and Staub, O. (2003). The role of Nedd4/Nedd4-likedependant ubiquitylation in epithelial transport processes. Pflügers Archiv., 446:328-333.

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Geering, K., Beguin, P., Garty, H., Karlish, S., Fuzesi, M., Horisberger, J.D. andCrambert, G. (2003) FXYD proteins: New tissue- and isoform-specific regulators ofNa,K-ATPase. Ann NY Acad Sci 986, 388-394.

Horisberger, J.D., Guennoun, S., Burnay, M., and Geering, K. (2003) Cationstoichiometry and cation pathway in the Na,K-ATPase and nongastric H,K-ATPase. AnnNY Acad Sci 986, 127-132.

Hummler, E. (2003). Epithelial sodium channel, salt-intake and hypertension. CurrentHypertension Reports, 5, 11-18.

Kamynina, E., and Staub, O. (2002). Concerted action of ENaC, Nedd4-2 and Sgk1 intransepithelial Na+ transport. Am.J.Physiol./Renal Physiol. 283:F377-F387.

Kellenberger, S. & Schild, L. (2002). Epithelial sodium channel/degenerin family of ionchannels: A variety of functions for a shared structure. Physiological Reviews 82, 735-767.

Li, C., Crambert, G., Hasler, U. and Geering, K. (2003) Na,K-ATPase α-β Subunitinteractions in the transmembrane domain. Ann NY Acad Sci 986, 226-228.

Loffing J, Kaissling B (2003). Sodium and calcium transport pathways along themammalian distal nephron: from rabbit to human. Am. J. Physiol. – Renal 284:F628-43.

Modyanov, N., Pestov, N., Adams, G., Crambert, G., Tillekeratne, M., Zhao, H. ,Korneenko, T., Shakhparonov, M. and Geering, K. (2003) Nongastric H,K-ATPase:Structure and functional properties. Ann NY Acad Sci 986, 183-187.

Pestov N.B., Crambert, G., Zhao, H., Korneenko, T.V., Shakhparonov, M.I., Geering,K. and Modyanov, N.N. (2003) The muscle-specific βm protein is functionally differentfrom other members of the X,K-ATPase β-subunit family. Ann NY Acad Sci 986, 304-305.

Rossier, B.C., Pradervand, S., Schild, L., Hummler, E. (2002). Epithelial sodiumchannel and the control of sodium balance: interaction between genetic and environmentalfactors. Annual Review of Physiology, 64:877-897.

Rossier, B. (2003). "Back to the future..." a word from the editor. Pflugers Arch.445:455.

Rossier, B.C. (2003). Negative regulators of sodium transport in the kidney: key factors inunderstanding salt-sensitive hypertension? J. Clin. Invest., 111:947-950.

Rossier, B.C. (2003). The epithelial sodium channel (ENaC): new insights into ENaCgating. Pflügers Arch., 446: 314-316.

Rossier, B.C. (2002). Hormonal regulation of the epithelial sodium channel ENaC: N orPo? J. Gen. Physiol. 120:67-70.

Rubera, I., Rossier, B.C., Hummler, E. (2003). Inactivation of sodium-transportingproteins in the kidney. Pflugers Arch. 445:463-469.

Stone EA, Cotecchia S, Lin Y, Quartermain D. (2002). Role of brain alpha 1B-adrenoceptors in modafinil-induced behavioral activity. Synapse. 46(4):269-70.

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Thomas, M.A., Huang, S., Cokoja, A., Riccio, O., Staub, O., Suter, S. and Chanson, M.(2002). Interaction of connexins with protein partners in the control of channel turnoverand gating. Biology of the Cell 94:445-456.

Thorens, B. (2003) A gene knockout approach in mice to identify glucose sensorscontrolling glucose homeostasis. Pflugers Arch – Eur. J. Physiol. 445: 482-490..

Thorens, B.(2003) Incrétines, sécrétion d’insuline et diabète. Médecine/Sciences 8-9: 860-863.

Uldry M, Thorens B, (2003) The SLC2 family of facilitated hexose and polyol transportersPflügers’ Arch./Eur. J. Physiol. Epub May 16.

Verrey F, Loffing J, Zecevic M, Heitzmann D, Staub O (2003). SGK1: Aldosterone-induced relay of Na+ transport regulation in distal nephron kidney cells. Cell PhysiolBiochem 13:021-028.