Legend

Scanning electron micrograph of a mouse Grueneberg ganglion cell. The cell body has been post-colorized in green, the axon in red and the sensory cilia, putative site of alarm pheromone recognition, in blue (Brechbühl et al. 2008). Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

TABLE DES MATIERES

PREFACE 2

RECHERCHE 2 ENSEIGNEMENT 3 BUDGET 4 PERSONNEL 5 ENSEIGNEMENT 11 RECHERCHE 12 SODIUM AND CALCIUM HOMEOSTASIS AND THE RENAL SYSTEM 13 FIRSOV DMITRI 13 Maintaining salt and water balance by the kidney: role of circadian timing system 13 OLIVIER BONNY 15 Renal calcium reabsorption 15 EDITH HUMMLER 17 Molecular and functional characterization of the epithelial Na channel and its regulators in vivo 17 KÄTHI GEERING 22 FXYD proteins: New regulators of Na,K-ATPase 22 JEAN-DANIEL HORISBERGER 25 Structure-function relation of Na,K-ATPAse, regulation of the epithelial Na channel 25 BERNARD C. ROSSIER 27 Activation of the epithelial Na channel by membrane-bound proteases 27 OLIVIER STAUB 30 Role of intracellular protein-protein interaction in ion channel regulation 30 LAURENT SCHILD 34 Structure and function of the epithelial sodium channel 34 CARDIOVASCULAR SYSTEM AND ASSOCIATED PATHOLOGIES 37 DARIO DIVIANI 37 The Role of the A-kinase anchoring proteins signaling complexes in G protein-coupled receptor- induced cardiac remodeling 37 HUGUES ABRIEL 40 Molecular bases of cardiac arrhythmias 40 NEURONAL ION CHANNELS AND G-PROTEIN COUPLED RECEPTORS 43 SUSANNA COTECCHIA 43 Adrenergic receptors: molecular mechanisms of receptor function and physiological implications 43 STEPHAN KELLENBERGER 46 Acid-sensing ion channels (ASICs) : function in sensory neurons and structure-function relationship 46 MARIE-CHRISTINE BROILLET 49 Chemosensory systems: Ion channels and receptors involved in odor and pheromone transductions 49 PUBLICATIONS 52

Page 1 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Préface

La pharmacologie occupe une place centrale dans la recherche biomédicale, compte tenu des nombreux efforts consentis actuellement dans le développement de nouveaux médicaments ; en thérapeutique médicale son rôle est également primordial, au vu de l’introduction et de l’utilisation constante de nouvelles entités pharmacologiques. Le Département de pharmacologie et toxicologie (DPT) de l’Université regroupe ses compétences autour de la pharmacologie fondamentale, tandis que l’aspect clinique de cette discipline dépend de la Division de pharmacologie et toxicologie clinique du Département de Médecine du CHUV. Les deux missions principales du DPT sont d’une part l’enseignement de la pharmacologie fondamentale aux étudiants en biologie et en médecine, d’autre part le développement d’une recherche relevante dans le domaine de la pharmacologie et de la thérapeutique en général. La période de 2006 à 2008 a vu de nombreux changements au sein du DPT avec pas moins de trois directeurs qui se sont succédés à la tête du Département. Le Professeur Bernard Rossier, nommé directeur en 1991, a pris sa retraite août 2006 ; la Professeure Susanna Cotecchia lui a succédé de 2006-2007. J’ai repris la direction du Département en 2007. Il convient également de relever le départ à la retraite de la Professeure K. Geering en 2008 ; la Prof. K. Geering, tout comme le Prof. B. Rossier, gardent des activités de recherche au sein du DPT, en tant que professeurs honoraires.

Recherche

La recherche en pharmacologie fondamentale a pour but de mieux comprendre les mécanismes d’actions des médicaments, leurs effets moléculaires et cellulaires, afin d’en améliorer leur usage. La recherche dans notre département s’intéresse essentiellement à la fonction et à la régulation de récepteurs pharmacologiques tels que les récepteurs liés aux protéines G, récepteurs nucléaires, les canaux et transporteurs ioniques ; cette recherche s’articule autour des thématiques suivantes:

. L’homéostasie électrolytique du sodium potassium et calcium et ses implications dans les pathologies vasculaires, cardiaques, pulmonaires, cutanées, osseuses . La structure, les fonctions moléculaires, les mécanismes de signalisation des récepteurs liés aux protéines G, en particulier cardiaques . Les troubles de l’excitabilité cardiaque et arythmies . La neurophysiologie et la neuropharmacologie des récepteurs aux phéromones et des canaux senseurs de pH

La qualité de la recherche du DPT est élevée comme l’attestent des publications parues dans les meilleurs journaux scientifiques à politique éditoriale tels que Science, The Proceedings of the National Academy of Sciences, EMBO Journal.

En 2007, le DPT s’est vu décerner un important financement (6 millions € sur cinq ans) pour un programme de recherche transatlantique sur l’hypertension artérielle et sur son lien étroit avec l’homéostasie du sodium. Ce projet de recherche est financé par la Fondation Leducq et le coordinateur européen de ce projet est le Prof. B. Rossier. Les différents partenaires et collaborateurs de ce projet regroupent des chercheurs des Universités de Yale, de Mexico, du Collège de France, ainsi que plusieurs groupes du DPT impliqués dans une recherche sur la physiologie, la physiopathologie et la génétique de l’hypertension artérielle et des transports électrolytiques.

Le DPT a accueilli en 2008 un nouveau groupe de recherche avec la venue du Dr O. Bonny, qui partage ses activités entre le Service de néphrologie de CHUV et son laboratoire de recherche au DPT. Il s’agit d’un premier engagement conjoint entre le CHUV et l’UNIL permettant à de jeunes médecins, destinés à une carrière académique, de garder une activité clinique conjointement à une activité de recherche

Page 2 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Le DPT participe activement aux activités de recherche sur le site du Bugnon 27 grâce à ses collaborations avec le Département de Génétique Médicale (DGM), avec les groupes de recherche du CHUV (Prof. T. Pedrazzini, Prof. M. Burnier) et la plate-forme de transgenèse (TAF) de la FBM. En 2008, la plate-forme de phénotypage des rongeurs dans le domaine cardiovasculaire (CAF : Cardiovascular Assessment Facility) a débuté ses activités au Bugnon 27 sous la responsabilité du Prof. T. Pedrazzini.

Enseignement

Le DPT est fortement impliqué dans l’enseignement de la pharmacologie aux étudiants de l’Ecole de médecine (Faculté de Biologie et Médecine). Cet enseignement, centré sur la pharmacologie fondamentale, est donné en étroite collaboration avec la Division de Pharmacologie et Toxicologie cliniques du CHUV. Dans le cadre du Master en Biologie Médicale de l’Ecole de biologie, le DPT est responsable de l’enseignement de la pharmacologie.

En ce qui concerne l’enseignement aux étudiants en médecine (Ecole de médecine), la période 2006-2008 a vu l’implantation du nouveau curriculum d’étude basé sur le modèle de ‘Bologne’ avec un baccalauréat (bachelor) de trois ans puis une maîtrise (Master) en médecine. La mise en place du baccalauréat avec ses trois années s’est terminée au cours de l’année académique 2007-2008. L’implantation du Master a débuté avec l’année académique 2008-09 et se terminera en 2011. Cette réforme a nécessité la mise en place d’un nouveau programme d’enseignement de la pharmacologie qui débute actuellement en 2e année et se poursuit jusqu’au Master. En outre le DPT est en charge de l’organisation d’un module (B2.6) pour les étudiants de 2e année du baccalauréat qui a pour thématique le rein et l’homéostasie hydro-électrolytique ; dans ce module, le DPT couvre l’enseignement de la pharmacologie et de la physiologie rénale.

L’enseignement de la pharmacologie donné aux étudiants en biologie, dans le cadre de l’école de biologie, a été intégré dans la réforme des études de biologie, qui visait à l’introduction d’un bachelor pour la période 2006 – 2008. Ces travaux ont commencé en 2006 et ont débouché sur une mise en place graduelle des trois années réformées (1ère année en 2007, 2e en 2008, et la 3e en 2009). Plusieurs membres du DPT étaient activement impliqués dans cette réforme (Käthi Geering, Olivier Staub, Marie-Christine Broillet, Jean-Daniel Horisberger). Durant les années 2006 à 2009, le DPT a contribué à l’enseignement du bachelor en organisant le bloc option « Structures et fonctions cellulaires» qui est suivi par une quarantaine d’étudiants chaque année, et qui est très apprécié.

Le DPT joue un rôle très actif dans le Master en biologie médicale (BM), qui était dirigé par Käthi Geering de 2004 à 2007 et depuis 2007 par Olivier Staub. Dans le cadre de ce Master, le DPT est en charge de la filière « Sciences pharmacologiques » (2e/3e semestre du Master, responsable Marie-Christine Broillet) pour les étudiants qui veulent se destiner à la recherche en pharmacologie dans un milieu académique ou industriel.

Le DPT offre un cours de pharmacologie générale dans le cadre des programmes des Masters en « Bioingénierie et Biotechnologie » et « Sciences et technologie du vivant » à l’EPFL, ainsi qu’un cours d’introduction à la biologie cellulaire pour les étudiants en chimie (EPFL) et étudiants de sciences forensiques (UNIL) pendant la première année Bachelor.

Le DPT vit actuellement une période de profonds changements tant dans ses activités d’enseignement que de recherche. L’enseignement de la pharmacologie aux étudiants en médecine et en biologie doit être repensé quant à ses objectifs de formation. Pour ce qui est de la recherche, le DPT est actuellement dans une phase de transition ; ces prochaines années seront marquées par l’arrivée de nouveaux groupes de recherche et de nouvelles compétences au sein du département, ceci dans le but d’une part, de renforcer et de développer la pharmacologie sur l’arc lémanique, d’autre part de maintenir l’excellence et la visibilité internationale de sa recherche.

Laurent Schild

Page 3 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Budget

Partenaires financiers pour la période 2006- Montants alloués pour la 2008 recherche durant la période 2006-2008

Fonds National 3'590'667.00

UNIL 233'885.00 Projet interdisciplinaire Bourse de relève académique

Fondations 1'362'760.00 Fondation Muschamp Fondation Désirée et Niels Yde Fondation Suisse du Rein Fondation Leducq Fondation Suisse de Cardiologie Synapsis Foundation Association Française des Myopathies

Industrie 379'635.00 Novartis UCB Beiersdorf

Page 4 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Personnel

1. PERSONNEL (SITUATION DECEMBRE 2008)

Personnel rétribué par l’Etat de Vaud: 40.9 EPT (équivalent plein temps) * Personnel rétribué par des fonds privés: 14.6 EPT **Personnel rétribué par des ressources externes au DPT: 4.1 EPT

• CORPS ENSEIGNANT Corps professoral H. Abriel Professeur associé K. Besseghir Privat-docent externe M.-C. Broillet Maître d’enseignement et de recherche, privat-docente S. Cotecchia Professeure associée J. Diezi Professeur honoraire D. Diviani Professeur assistant E. Felley-Bosco Privat-docente externe D. Firsov Maître d’enseignement et de recherche, privat-docent K. Geering Professeure honoraire J.-D. Horisberger Professeur ordinaire, vice-doyen de la recherche E. Hummler ** Maître d’enseignement et de recherche, privat-docente, coordinatrice de la plateforme de transgénèse (TAF) S. Kellenberger Maître d’enseignement et de recherche, privat-docent B. Rossier Professeur honoraire L. Schild Professeur ordinaire, directeur O. Staub Professeur associé Corps intermédiaire O. Bonny Maître assistant Chargé(e)s de recherche S. Bibert * M. van Bemmelen * Assistant(e)s Postdoctorant(e)s M. Auberson ** V. Bize R.-Ph. Charles N. Faresse * H. Fodstad K. Gusev * R. Perrier * C. Ronzaud * Doctorant(e)s M. Albesa B. Bargeton M. Blanchard A. Boillat J. Brechbühl L. Cariolato S. Cavin A. Debonneville S. Frateschi C. Laedermann ** D. Lagnaz A. Maquelin S. Nikolaeva I. Perez Lopez S. Petitprez D. Pouly L. Sintra Grilo ** Ph. Suarez A. Takeda J.-J. Vitagliano V. Zavadova A.-F. Zmoos

Page 5 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

• ETUDIANTS Etudiant(e)s Master ès sciences en biologie médicale (18.02.08 au 17.02.09) D. Huser L. A. Liechti

Etudiant(e)s 7ème semestre Master ès sciences en biologie médicale (23.09.08 au 19.12.08) V. Bonvin Th. Bonduban M. Di Chiara A. Dousse B. Genoud F. Gerster T. Menendez G. Tschopp

Stagiaires universitaires S. R. Malsure (01.12.08 au 28.02.09) *

Civilistes M. Bardelli (01.09.08 au 06.03.09) *

• ADMINISTRATION GENERALE ET PLATES-FORMES TECHNIQUES ET DE LOGISTIQUE Laboratoires Laborantin(e)s G. Centeno * N. Fowler Jaeger (60%) * H.-P. Gaeggeler I. Gautschi P. Hausel (60% dont 10%*) S. Kharoubi Hess (60%) * A.-M. Mérillat M. Nenniger Tosato (30%) S. Roy (50%) D. Ruffieux-Daidié * Apprenti(e)s laborantin(e)s A. Calame Stagiaires laborantin(e)s (ESsanté, CFC, autres) A. Durret (ESsanté) (01.12.08 au 13.03.09) Ch. Pahud (CFC Changins) (01.10.08 au 31.03.09) N. Rainoldi (ESsanté) (01.12.08 au 13.03.09) Stagiaires (stages préalables pour intégrer la Haute école valaisanne à Sion) S. Rieser (15.09.08 au 30.06.09) *

Administration générale Secrétariat Ch. Demont S. Kittel (70%) I. Rivier Flühmann (60%) Informatique A. Brun (support informatique) S. Meier (50%) * ** Bibliothèque J.-C. Broillet (20%) *

Service technique E. Delacrétaz

Page 6 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Achats R. Gander (50%)

Service des animaleries du Bugnon 27 et laverie Animaleries D. De Gregorio (70%) L. Fabia (50%) R. Gander (50%) Ch. Gremay (90%) * A.-L. Huber M. Laeser (50%) ** M. Nenniger Tosato (70%) N. Pineau-Gremay (temporaire) M. Virot (80%) * Apprenti(e)s gardien(ne)s d’animaux S. Koenig Laverie D. De Gregorio (30%) P. Durgniat (30%) * N. Pineau-Gremay (temporaire) *

2. DEPARTS 2006-2008 • CORPS ENSEIGNANT Corps professoral E. Felley-Bosco Professeure assistante suppléante (03.2006) P. Iynedjian Privat-docent externe (08.2008)

Chargé(e)s de recherche E. Gonzalez Rodriguez (05.2006) M. Membrez (08.2006) R. L. Stanasila Vollmer (01.2008)

Hôtes sabbatiques G. Garruti (06.2006)

Assistant(e)s Postdoctorant(e)s D. Andreasen (02.2006) A. Appert-Collin (06.2006) S. Boulkroun (08.2006) O. Capendeguy (08.2007) R.-P. Charles (12.2008) Ch. Debonneville (06.2007) B. Delprat (11.2006) B. Gavillet (07.2007) M. Harris (09.2007) A. Mercier Zuber (10.2007) O. Poirot (02.2007) J.-S. Rougier (12.2006) Boursier(ère)s A. Aggeli (10.2007) B. Christensen (08.2007) Th. Jespersen (04.2006) C. Lindholm (05.2006) E. Zaklyaziminskaya (08.2008) Doctorant(e)s L. Baisamy (03.2007) S. Bron (01.2008) L. Cardinaux (11.2007) L. Maquelin (08.2008) Ch. Moghini (12.2006) I. Rybicki (12.2006) S. B. Sayar (12.2006) S. Vollery (10.2007)

Page 7 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Stagiaires doctorant(e)s hors Unil S. El Haou (02.07.07 au 03.08.07) P. San Cristobal Zepeda (01.09.08 au 10.10.08)

• ETUDIANTS Etudiant(e)s Master ès sciences en biologie médicale 13.03.06 au 31.01.07 R. Béhar A. Boillat S. Bron L. Cardinaux S. Cavin D. Lagnaz M. Mbefo Kamdem D. Pouly 05.03.07 au 31.01.08 M. Klaey A. Maquelin A.-F. Zmoos

Etudiant(e)s 7ème semestre Master ès sciences en biologie médicale 31.10.05 au 28.02.06 J. Aebischer N. Ausderau 30.10.06 au 09.02.07 F. Desgranges M. Klaey A. Maquelin 24.09.07 au 21.12.07 D. Aebischer V. Nesca J. Quebatte A. Righetti

Autres étudiant(e)s Master en biologie hors Unil S. Jaques (23.10.06 au 09.02.07)

Stagiaires étudiant(e)s en pharmacie C. Bacca (13.03.06 au 16.06.06) Ch. Greiner (01.11.06 au 30.04.07)

Stagiaires étudiant(e)s en médecine (stage de pharmacologie/toxicologie expérimentales) A. Ansorge (01.04.07 au 31.05.07) J. Zaldivar-Jolissaint (01.05.08 au 30.06.08)

Stagiaires étudiant(e)s en médecine N. Faller (13.08.07 au 28.09.07)

Stagiaires universitaires A. Bezprozvanny (01.06.08 au 18.07.08) V. Bonvin (07.07.08 au 15.08.08) C. D. Del Vescovo (01.03.08 au 30.11.08) A. Lambert (12.06.07 au 11.09.07)

Civilistes B. Pochon (02.06.08 au 23.10.08) D. Pouly (27.10.07 au 02.04.08)

Page 8 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

• ADMINISTRATION GENERALE ET PLATEFORMES TECHNIQUES ET DE LOGISTIQUE Laboratoires Laborantin(e)s L. Abuin (09.2007) H. Dias Abelairas Latado (08.2006) M. Klaey (90%) (03.2008) O. Randin (12.2007) D. Schaer (50%) (07.2007) S. Sutter (08.2007) Apprenti(e)s laborantin(e)s S. Chabanel (07.2006) R. Groux (07.2008) J.-Y. Schaël (07.2006) Stagiaires laborantin(e)s (ESsanté, CFC, autres) A. Comand (ESsanté) (21.04.08 au 08.08.08) A. Gloor (ESsanté) (24.04.06 au 11.08.06) J. Guiu (CFC Changins) (02.10.06 au 30.03.07) C. Liardon (ESsanté) (03.12.07 au 21.03.08) S. Mermoud (CFC Argot Lab) (03.11.06 au S. Perroset (ESsanté) (28.11.05 au 17.03.06) 30.04.07) A. Pichon (CFC Changins) (01.10.07 au N. Ramirez Cobos (ESsanté) (23.04.07 au 10.08.07) 31.03.08) Y Rappaz (stage 1 semaine 19.11.07 au 23.11.07) P. Riond (ESsanté) (04.12.06 au 23.03.07) S. Verdon (stage 1 semaine 03.12.07 au 07.12.07) Stagiaires (stages préalables pour intégrer la Haute école valaisanne à Sion) S. Egli (01.10.06 au 31.08.07) A. Rieder (03.09.07 au 29.08.08) Etudiant(e)s (job d’été) D. Aebischer (09.07.07 au 24.08.07) D. Jacot-Descombes (16.07.07 au 24.08.07)

Administration générale Secrétariat N. Skarda-Coderey (80%) (Retraite 08.2008)

Service des animaleries du Bugnon 27 et laverie Animaleries Ch. Gremay (90%) (12.2008) Y. Guibert (60%) (04.2006) C. Matthey (04.2007) M. Vuillet-Gremion (20%) (02.2008) Stagiaires animaleries C. Gremay (10.03.08 au 14.03.08) S. Koenig (09.06.08 au 13.06.08) Laverie P. Durgniat (30%) (Retraite 12.2008)

Page 9 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

3. Organigrammes

Page 10 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Enseignement

Ecole de médecine Cours de Pharmacologie, semestres 2008 – 2009 Bachelor 2ème année Cours TP S B2.1 Cellule organe système 12 8 4 B2.3 Neurosciences 6 B2.4 Circulation / Respiration 3 B2.5 Digestion Métabolisme 3 B2.6 Urogénital et homéostasie 14 4 Bachelor 3ème année B3.1 Cœur poumon 6 B3.2 Douleurs abdominales 4 B3.3 Inflammation du 3 B3.4 Cerveau et fontion supérieure 3 B3.5 Croissance poids 2 Master 4ème année M1.1 Maladies infectieuses et onco-hémato 4 M2.3 Douleurs articulaires 3 Ecole de Biologie Cours pharmacologie / Physiologie, Semestre 2008 – 2009 Bachelor 4ème semestre Unité de Physiologie Système urinaire, homéostasie intérieure 14 2 Bachelor 6ème semestre Cours à option Structure - Fonction Cel. Homéostasie intracellulaire 18 144 18 Master Biologie Médicale / 7ème semestre Tronc commun Biologie cellulaire 12 4 Signalisation I et II 15 3 PPP cardio-vasculaire 6 2 PPP epithelia 14 2 Master en Biologie Médicale, 8ème semestre Cours commun Médecine / Biostat. 2 Filière pharmacologie 64 24 Filière neurosciences 8 Filière métabolisme 4 2 EPFL Master 8ème semestre Pharmacologie générale (cours à option) 28 Nombre d'heures total 248 154 63

Page 11 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Recherche

Research domains

Sodium and calcium homeostasis and the renal system

 Maintaining salt and water balance by the kidney: role of circadian timing system (group Firsov) p. 13  Renal calcium reabsorption (group Bonny) p. 15  Molecular and functional characterization of the epithelial Na channel and its regulators in vivo (group Hummler) p. 17  FXYD proteins: New regulators of Na,K-ATPase (group Geering) p. 22  Structure-function relation of Na,K-ATPAse, regulation of the epithelial Na channel (groups Horisberger) p. 25  Activation of the epithelial Na channel by membrane-bound proteases (group Rossier) p. 27  Role of intracellular protein-protein interaction in ion channel regulation (group Staub) p. 30  Structure and function of the epithelial sodium channel (group Schild) p. 34

Cardiovascular system and associated pathologies

 The Role of A-kinase anchoring proteins signaling complexes in G protein-coupled receptor-induced cardiac remodeling (groupe Diviani) p. 37  Molecular bases of cardiac arrhythmias (groupe Abriel) p. 40

Neuronal ion channels and G-protein coupled Receptors

 Adrenergic receptors: molecular mechanisms of receptor function and physiological implications (group Cotecchia) p. 43  Acid-sensing ion channels (ASICs): function in sensory neurons and structure-fonction relationship (group Kellenberger) p. 46  Chemosensory systems: Ion channels and receptors involved in odor and pheromone transductions (group Broillet) p. 49

Page 12 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Sodium and calcium homeostasis and the renal system

Maintaining salt and water balance by the kidney: role of circadian timing system

The kidney maintains homeostasis of the extracellular environment by selectively excreting or retaining water and solutes, according to the organism’s needs. It is well established that under basal conditions, water + + - - 2+ 2+ and major solutes (Na , K , Cl , PO4 , Ca , Mg , urea, ..) are excreted in the urine with a regular (circadian) rhythmicity. It has also been shown that this rhythmicity is driven by circadian changes in both glomerular filtration and tubular reabsorption/secretion. Circadian changes in renal Firsov Dmitri filtration/reabsorption/secretion are thought to be mediated by interplay between the humoral circadian stimuli (hormones, food components, food Dmitri Firsov received his PhD in 1992 from the Russian metabolites) and self-sustained/self-autonomous renal circadian timing Academy of Sciences system. Functionally, this circadian timing system is thought to provide (research on vasopressin and the kidney with an anticipatory advantage of the daily environmental vasopressin receptor changes (food and water intake, activity, posture, etc.). physiology). From 1993 to 1995, first postdoctoral Whilst, the hormonal regulation of renal function has been extensively training in France (College de France, Paris and CEA, investigated, the studies of intrinsic renal circadian timing system are just Saclay). From 1995 to 1997, beginning. According to a current model, the circadian timing system is second postdoctoral training in driven by transcriptional/translational feedback loops (core oscillators). the Department of These core oscillators confer circadian rhythmicity on a set of output Pharmacology, University of Lausanne, Switzerland. From genes underlying the tissue-specific functional rhythms. Which of the 1997 to present, group leader elements of circadian core oscillators and output genes underlie the in the same Department. diurnal rhythmicity of specific renal functions remains largely unknown. The assessment of the role of circadian timing system in the kidney is the main project of my research group.

Membres du groupe (période 2006-2008) Collaborations Technicien de laboratoire: - Gabriel Centeno Dr D. Weaver, Department of Neurobiology, University of Massachusetts Doctorants: Medical School, Massachusetts, USA - Léonard Cardinaux - Lionel Maquelin Drs. J. Loffing and Jurg Biber, Institutes of Anatomy (JL) and Physiology - Svetlana Nikolaeva (JB), University of Zurich, Zurich, Switzerland - Annie Zuber Mercier

Stagiaire universitaire: - Arseny Bezprozvanny

light/dark neurohypophysis vasopressin cycle adrenal glands aldosterone other hormones resetting circadian activity/expression of oscillator in the channels / transporters SCN expressed in the distal distal nephron cells nephron

activity/feeding food components phase food metabolites circadian rhythm of

urinary excretion

Legend. Regulation of urinary excretion rhythms by the circadian timing system

Page 13 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Selected references

Zuber AM, Singer D, Penninger JM, Rossier BC, Firsov D. Increased renal responsiveness to vasopressin and enhanced V2 receptor signaling in RGS2-/- mice. J Am Soc Nephrol. (2007) 18(6): 1672-8 Muller O, Pradervand S, Berger S, Centeno G, Milet A, Nicod P, Pedrazzini T, Tronche F, Schütz G, Chien K, Rossier BC, Firsov D. Identification of corticosteroid-regulated genes in cardiomyocytes by serial analysis of gene expression. Genomics (2007) 89(3): 370-7

Harris M, Firsov D, Vuagniaux G, Stutts MJ, Rossier BC. A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes. J Biol Chem. (2007) 282(1): 58-64 Key words Harris M, Garcia-Caballero A, Stutts MJ, Firsov D, Rossier BC. Circadian timing system, Preferential assembly of epithelial sodium channel (ENaC) subunits in kidney, distal nephron, water and sodium homeostasis, Xenopus oocytes: role of furin-mediated endogenous proteolysis. J Biol clock genes, knockout mice. Chem. 2008 283(12): 7455-63

Lectures as invited speaker

October 2007, Aldosterone and ENaC Meeting, Zermatt, Switzerland February 2008, UPMC University of Paris 06, Unité Mixte de Recherche Scientifique UMRS 872, and INSERM, UMRS 872, Paris France March 2008, Institute of Anatomy, University of Zurich, Zurich, Switzerland

Honors and Awards

2006 Stéphanie Michlig, Prix de Faculté

Page 14 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Renal calcium reabsorption

Sharing time between the Department of Pharmacology and Toxicology (60%) and the Division of Nephrology of the University Hospital (40%) since December 2007, we took advantage of the first few months to start the lab and build up a team. Our projects aim at deciphering regulation of calcium reabsorption in the kidney. More precisely, we are studying:

1. The role of the sodium/calcium exchanger (NCX1) We generated a mouse kidney-specific knockout model for the Olivier Bonny sodium/calcium exchanger while in postdoctoral fellowship in Prof. O.W. Moe’s laboratory in Dallas. The sodium/calcium exchanger is Olivier Bonny is a physician- expressed in the distal and connecting tubules of the nephron, where scientist who earned his MD calcium is reabsorbed transcellularly under the control of PTH and at the University of Lausanne, vitamin D. Its location at the basolateral side of the cell allows Switzerland and a PhD from calcium reabsorption by transporting calcium to the interstitial the MD-PhD program of the Swiss Academy of Medical compartment in exchange for sodium. We have now established a Sciences. His PhD thesis was colony of these mice in Lausanne and we are addressing new led by Prof. Bernard C. questions arising from our initial characterization (manuscript in Rossier and was aimed at understanding the functional preparation). relevance of mutants of the 2. Circadian rhythms of renal calcium reabsorption epithelial sodium channel (ENaC). He subsequently Even though circadian rhythms for calcemia, calciuria, PTH and completed a post-doctoral fellowship with Prof. Orson vitamin D have been described for long, not much has been W. Moe at the University of described at the molecular level. We are dedicated to filling the gap Texas, Southwestern Medical between descriptive physiological circadian rhythms and the Center in Dallas on renal molecular reality. calcium reabsorption. He joined the Department of Pharmacology and Toxicology in December 2007 where he has been leading his own Collaborations research group since. In parallel, Dr Bonny Prof. O.W. Moe, Medical Center at Dallas, University of Texas completed a clinical training as a nephrologist at the Southwestern, Texas, USA University Hospital Bern with Prof. Felix J. Frey and hold Dr D. Firsov, Department of Pharmacology and Toxicology, University the Swiss board certifications of Lausanne, Lausanne, Switzerland in Internal Medicine (FMH). He is attending at the Division of Nephrology of the University Hospital of Lausanne. Selected references

Bonny O, Burnier M. Treatment of secondary hyperparathyroidism in renal insufficiency: role of calcitriol, sevelamer and cinacalcet. Rev Med Suisse. (2008) 4(147):589-92, 594-5 Suzuki Y, Pasch A, Bonny O, Mohaupt MG, Hediger MA, Frey FJ. Gain- of-function haplotype in the epithelial calcium channel TRPV6 is a risk factor for renal calcium stone formation. Hum Mol Genet. (2008) 17(11):1613-8 Pasch A, Frey FJ, Eisenberger U, Mohaupt MG, Bonny O. PTH and 1.25 vitamin D response to a low-calcium diet is associated with bone mineral density in renal stone formers. Nephrol Dial Transplant. (2008) 23(8):2563-70

Page 15 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Bonny O, Rubin A, Huang CL, Frawley WH, Pak CY, Moe OW. Mechanism of urinary calcium regulation by urinary magnesium and pH. J Am Soc Nephrol. 2008 19(8): 1530-7

Membres du groupe (période 2006-2008) Lectures as invited speaker Doctorante: - Vlasta Zavadova December 5th 2007, Basics in Nephrology: Nephrolithiasis. Annual Civiliste: meeting of the Swiss Society of Nephrology, Lausanne - Benoît Pochon 20 mars 2008, Investigation d’une néphrolithiase à Dallas. 15ème réunion Etudiante: romande de néphrologie, Lausanne - Valérie Bonvin Nyon, 18 septembre 2008, Nouveautés dans la prévention et le diagnostic de la néphrolithiase. 1ère journée romande d’uro-néphrologie

2 octobre 2008, Calcium et néphrolithiase, comment gérer? Grand Round de médecine interne, CHUV, Lausanne Key words 14 novembre 2008, Regulation of calcium reabsorption by magnesium Calcium, kidney, circadian and role of the sodium/calcium exchanger. Institut des Cordeliers, Paris rhythm, sodium/calcium exchanger, distal and 17 novembre 2008, Regulation of calcium reabsorption by magnesium connecting tubule, PTH. and by circadian rhythm. Grundlage Forschung der Nephrologie, Zürich

Honors and awards

2007 Best oral presentation, annual meeting of the Swiss Society of Nephrology, Lausanne.

Page 16 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Molecular and functional characterization of the epithelial Na channel and its regulators in vivo

Research Summary The research of our laboratory focuses on the molecular pharmacology/ physiology of the highly amiloride-sensitive epithelial sodium channel ENaC and its regulators. This sodium channel is implicated in several pathological conditions, like hypertension, respiratory distress syndrome and in skin diseases. We are interested in understanding the heterogeneity of stimulating/repressing systems that control ENaC activity in these Edith Hummler organs. Genetically engineered mice allow analyzing complex regulatory systems in vivo, and, in parallel, primary and/ or stable cell cultures can Edith Hummler was trained as be established to study any alteration of ENaC activity on a cellular and biologist at the Universities of molecular level. These studies are complemented by comparative Tübingen and Göttingen, microarray analysis of genes involved in pathological versus Germany, where she received her PhD in 1989. She carried physiological status of a given organ. Besides ENaC as the “effector” in out postdoctoral work with several organs, the analysis of regulatory proteins like the channel Günther Schütz at the German activating proteases will help to dissect the regulation of ENaC in distinct Cancer Research Center in tissues. Our laboratory focuses on two main lines of studies : Heidelberg, Germany, establishing mice deficient for the transcription factor CREB 1. Implication of the amiloride-sensitive epithelial sodium channel (cAMP responsive element in the control of blood pressure. Using homologous binding protein) and for the recombination in the mouse, we want to understand the glucocorticoid receptor. In 1992, she joined the group of underlying molecular and cellular mechanisms that lead to Bernard Rossier at the hyper- or hypoactivity of ENaC in human patients. We have Department of Pharmacology generated several mouse models bearing mutations in all three and Toxicology in Lausanne, subunits of the ENaC (alpha, beta, and gamma ENaC) that result Switzerland. In 1996, she became Assistant Professor at in reduced or complete abolishment of channel activity. Reduced this Department and ENaC activity in ENaC mutant mice leads to clinical symptoms established her own research similar to the PHA-1 (Pseudohypoaldosteronism type 1) group at the same Department. phenotype, ranging from mild (e.g. mutation in the beta ENaC In 2003, she was additionally appointed head of the gene locus to severe forms (e.g. gamma ENaC knockout mice) of transgenic facility at the this disease. When a Liddle mutation was introduced into the Faculty of Medicine and beta ENaC gene locus, these mice exhibited a salt-sensitive Biology of the University of hypertension. Using Cre-loxP technology, we are now able to Lausanne. Her research group has generated and analyzed genetically dissect ENaC-mediated sodium reabsorption along several mouse models for the nephron that is under the tight control of aldosterone and to diseases caused by the reveal the role of the colon in whole body sodium homeostasis. epithelial sodium channel These findings will help to develop new targets for treatment of ENaC and its regulators. hypertension.

Model for the regulation of Na+ reabsorption through epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN): Insights from genetic mouse models

Copyright ©2005 American Society of Nephrology

Page 17 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

2. Role of ENaC and channel activating proteases (CAPs) in epidermal remodeling and differentiation. The primary function of the epidermis is to form a barrier between an organism and the Membres du groupe outside environment designed to advert the invasion of bacteria (période 2006-2008) and other foreign entities while simultaneously preventing the Technicienne de laboratoire: escape of water required for terrestrial life. In humans, defective - Anne-Marie Merillat epidermal barrier function is seen in a variety of skin disorders

Doctorants: resumed generally as ichthyosis. The molecular nature of the - Elena Gonzalez-Rodriguez epidermal permeability barrier is still under investigation and it - Ditte Andreasen has been proposed that during phylogenetic evolution, the - Roch-Philippe Charles mammalian skin has obtained at least two independent systems - Simona Frateschi - Philippe Suarez for forming a strong barrier, namely a protein-lipid layer (CE/lipid lamellae) and the tight junction complex in the stratum Postdoc: corneum. Our recent findings unveiled that disturbed expression - Romain Perrier of the membrane-bound serine protease CAP1/Prss8 and of its

Chargé de recherche: potential substrate, the amiloride-sensitive sodium channel - Mathieu Membrez ENaC results in severe dehydration phenotypes, caused by a defective epidermal permeability barrier. Disturbed protease – Laborantine: protease inhibitor balance causes faulty differentiation processes - Jessika Guiu in the epidermis, thereby affecting the epidermal barrier Etudiants: homeostasis. Regulated expression of channel-activating - Sylvain Jacques proteases (CAPs) is crucial in the prenatal development of the - Valério Nesca embryo, and most likely implicated in transdifferentiation - Aline Dousse processes of distinct organs. Stagiaires: - Audrey Rieder - Nathalie Ramirez Cobos - Sylvie Perroset Expression of TJ proteins and TJ permeability assay - Sandrine Egli - Alexi Lambert - Alexandra Iouranova - Christelle Pahud - Sarah Rieser - Sumedha Ram Malsure

Apprenti: - Arnaud Pichon

Key words epithelial sodium channel, channel-activating proteases, serine protease inhibitors, conditional gene targeting, Copyright ©2005 Rockefeller University Press transgenic mouse models, sodium and potassium homeostasis, epidermal barrier homeostasis, mouse development Collaborations

Prof. Bernard Rossier, Department of Pharmacology and Toxicology, Université de Lausanne, Lausanne, Suisse Dr Friedrich Beermann, ISREC/ EPFL, Epalinges, Suisse Prof. Bernard Thorens, CIG, Université de Lausanne, Lausanne, Suisse Dr Samuel Rothman, Department of Pathology, Université de Lausanne, Lausanne, Suisse

Page 18 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Prof. Michel Burnier, Division of Hypertension, CHUV, Lausanne, Suisse Dr Konrad Sandhoff, Kékulé-Insitut, Organische Chemie, Bonn, Germany Dr Rivkah Isserhoff, Professor of Dermatology, University of California, Davis, USA Prof. Daniel Hohl, Department of Dermatology, UNIL, Lausanne, Suisse Dr Ric Boucher, Cystic Fibrosis Center, University of North Carolina, Chapel Hill, USA

Selected references

Charles, R-P, Guitard M, Leyvraz C, Breiden B, Haftek M, Haftek- Terreau Z, Stehle J-C, Sandhoff K and E Hummler, Postnatal requirement of the epithelial sodium channel for maintenance of epidermal barrier function. J. Biol. Chem. (2008) 283: 2622-2630 Bertog M, Cuffe J E, Pradervand S, Hummler E, Hartner A, Porst M, Hilgers K F, Rossier B C and C Korbmacher, Aldosterone responsiveness of the epithelial sodium channel (ENaC) in colon is increased in a mouse model for Liddle’s syndrome. J. Physiol. (2008) 586: 459-475 Randrianarison N, Clerici C, Ferreira C, Fontayne A, Pradervand S, Fowler-Jaeger N, Hummler E, Rossier BC, Planès C. Low expression of the beta-ENaC subunit impairs lung fluid clearance in the mouse. Am J Physiol Lung Cell Mol Physiol. (2008) 294(3):L409-16 Randrianarison N, Escoubet B, Ferreira C, Fontayne A, Fowler-Jaeger N, Clerici C, Hummler E, Rossier BC, Planès C. beta-Liddle mutation of the epithelial sodium channel increases alveolar fluid clearance and reduces the severity of hydrostatic pulmonary oedema in mice. J Physiol. (2007) 582(Pt 2):777-88. Epub 2007 Apr 12 Kovacikova J, Winter C, Loffing-Cueni D, Loffing J, Finberg KE, Lifton RP, Hummler E, Rossier B, Wagner CA. The connecting tubule is the main site of the furosemide-induced urinary acidification by the vacuolar H+-ATPase. Kidney Int. (2006) 70(10):1706-16 Oskarsson T, Essers MA, Dubois N, Offner S, Dubey C, Roger C, Metzger D, Chambon P, Hummler E, Beard P, Trumpp A. Skin epidermis lacking the c-Myc gene is resistant to Ras-driven tumorigenesis but can reacquire sensitivity upon additional loss of the p21Cip1 gene. Genes Dev. (2006) 20(15):2024-9 Membrez M, Hummler E, Beermann F, Haefliger JA, Savioz R, Pedrazzini T, Thorens B. GLUT8 is dispensable for embryonic development but influences hippocampal neurogenesis and heart function. Mol Cell Biol. (2006) 26(11):4268-76 Porret, A, Mérillat, A.M, Guichard, S, Beermann, F and E Hummler, Tissue-specific transgenic and knockout mice. Methods Mol. Biol. (2006) 337: 185-205 Andreasen D, Vuagniaux G, Fowler-Jaeger N, Hummler E, Rossier BC. Activation of epithelial sodium channels by mouse channel activating proteases (mCAP) expressed in Xenopus oocytes requires catalytic activity of mCAP3 and mCAP2 but not mCAP1. J Am Soc Nephrol. (2006) 17(4):968-76

Page 19 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Rubera I, Hummler E, Beermann F. Transgenic mice and their impact on kidney reseach. Pflügers Arch. Europ. J. Physiol. 2008, in press

Mérillat AM, Charles R-P, Porret A, Maillard M, Rossier BC, Beermann F, Hummler E. Conditional gene targeting of the ENaC subunit genes Scnn1b and Scnn1g. Am. J. Physiol. Renal Physiol. (2008) in press

Lectures as invited speaker

September 6 2006, E2BRN (European Epidermal Barrier Research Network, Satellite Meeting ESDR), Paris, France. “ Molecular physiology of ENaC-mediated sodium transport in skin” November 29 2006, Seminar Dermatology, Lausanne. “Molecular physiology of ENaC-mediated sodium transport in skin” December 15 2007, ESAC meeting (European Section of the Aldosterone Council), Paris, France. “Genetic dissection of ENaC function along the Aldosterone Sensitive Distal Nephron (ASDN)” December 5 - 7, 2007, SGN-SSN (Annual Meeting of the Swiss Society of Nephrology), , Lausanne, Switzerland. “ Collecting duct-specific gene inactivation of alpha ENaC protects mice against lithium-induced nephrogenic diabetes insipidus” October 3 – 7, 2007, 6th International Meeting on Aldosterone and ENaC: Aldosterone and ENaC: from gene to disease, Zermatt, “ How does ENaC keep the skin tight” August 12-13, 2008, CARD (Congres Annuel de Recherche Dermatologique), Toulouse, France. “Rôle du canal epithelial à sodium ENaC sensible à l’amiloride dans la barrière épidermique” August 29, 2008, Seminar, Aahus, Denmark “ Lessons from ENaC mutant mice” August 27 – 29, 2008, PhD Course, , Odense, Denmark. “ Epithelal sodium channel, salt intake and hypertension”. “ ENaC and barrier function” 19 – 21 June, 2008, DaCRA (Danish Cardiovascular Research Academy), Sandbjerg Summer Meeting, Sandbjerg, Denmark. “ New insights in aldosterone effects on epithelial and cardiovascular target tissues with gene-targeted mice” Avril 8, 2008, Seminar, Sion, Switzerland. “ Knockout and transgenic animals to study epithelial sodium channel (ENaC) function” March 19 – 21, 2008, CoPIP 2008 (Colloque Francophone Thématique de Biologie Cutanée Humaine), Lyon, France. “ Rôle du canal epithelial à sodium ENaC sensible à l’amiloride dans la fonction de la barrière d’épiderme”

Page 20 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Honors and Awards

2006 Prix de Faculté de l’Université de Lausanne: recipient Dr Celine Leyvraz, group Edith Hummler, Department of Pharmacology and Toxicology, for her thesis work: Role of the epithelial sodium channel (ENaC) and its positive regulator, the channel-activating protease 1 (CAP1) in skin. 2008 Prix de Faculté de l’Université de Lausanne: recipient Dr Roch-Philippe Charles, group Edith Hummler, Department of Pharmacology and Toxicology, for her thesis work: Role of the epithelial sodium channel (ENaC) and its positive regulator, the channel-activating protease 1 (CAP1) in skin.

Page 21 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

FXYD proteins: New regulators of Na,K-ATPase

Na,K-ATPase is an ubiquitous ion transporter that is responsible for the maintenance of the characteristic Na+ and K+ gradients between the intra- and extracellular milieu of animal cells. These gradients are important to maintain the basic cellular homeostasis and also control specialized tissue Käthi Geering functions such as renal Na+ reabsorption, muscle contaraction and neuronal excitability. In view of its important physiological role, NaK- Käthi Geering a obtenu son ATPase expression and/or activity must be finely regulated. Recently, we PhD en 1971 à l’Université de Bâle. Elle a occupé une have identified auxiliary subunits of Na,K-ATPase, FXYD proteins, that position post-doctorale à modulate Na,K-ATPase transport properties in a tissue- and isozyme- l’Institut Tropicale à Bâle specific way. The functional effects of each FXYD protein on Na,K- jusqu’en 1975 avant de ATPase activity are distinct, most likely reflecting the differential needs joindre le Département de Pharmacologie et Toxicologie of Na,K-ATPase activity in different tissues. During the last 3 years, we de l’Université de Lausanne. have mainly concentrated on the elucidation of the physiological and Elle a effectué deux stages pathophysiological relevance of Na,K-ATPase regulation by several post-doctorales à l’European FXYD proteins. Molecular Biology Laboratories’ à Heidelberg en 1) FXYD6: We could define the last FXYD protein of unknown function 1883 et 1988. Käthi Geering a été nommée Professeure as a modulator of Na,K-ATPase, and its unique expression among FXYD ordinaire en 2004 et est proteins in the inner ear. Immunohistochemistry of the cochlea shows actuellement Professeure marked similarity in the developmental expression pattern of Na,K- honoraire (2008). Ses ATPase and FXYD6 suggesting functional cooperation betweeen the two recherches se sont focalisées sur la relation structure- proteins in the generation and maintenance of the endocochlear potential fonction et la régulation de la and and ion composition of the endolymph. Na,K-ATPase et en particulier sur le rôle de ses sous-unités 2) FXYD1 or phospholemman (PLM): PLM is the main plasma accessoires. membrane substrate for protein kinase A and C in the heart. We have studied the role of PLM phosphorylation by PKA and PKC in the modulation of different Na,K-ATPase isozymes present in the heart. Our results show that PKA phosphorylation of PLM had similar effects on α1/β and α2/β isozymes. PKA phosphorylation of PLM has no effect on the apparent K+ affinity or the turnover of both isozymes but increases their apparent Na+ affinity. On the other hand, PKC phosphorylation of PLM produces a differential effect on α1/β and α2/β isozymes. PKC phosphorylation of PLM produces an increase in the turnover number of α2/β but not of α1/β isozymes. This latter result may be of physiological relevance in cardiac myocytes. Activation of PKA by stimulation of β- adrenergic receptors may phosphorylate PLM associated with Na,K- ATPase α1/β and α2/β isozymes and increase their apparent Na affinity. Moreover, activation of PKC, by stimulation of α-adrenergic receptors, may phosphorylate, in addition, PLM associated with α2/β isozymes and increase their turnover number. Physiologically, the overall response to concomitant PKA and PKC activation would be an additive increase in Na extrusion that favours Ca extrusion through the Na/Ca exchanger. This may limit positive inotropy and Na and Ca overloads, diastolic dysfunction and arrythmias during sympathetic stimulation of the heart. 3) FXYD3 or Mat-8: In normal tissue, FXYD3 is mainly expressed in stomach and colon but it is also overexpressed in cancer cells suggesting a role in tumorogenesis. We could show that FXYD3 silencing has no effect on cell proliferation but promotes cell apoptosis and prevents cell differentiation and polarization of human colon adenocarcinoma cells (Caco-2), which is reflected by a reduction in alkaline phosphatase and villin expression, a change in several other differentiation markers, and by a decrease in transepithelial resistance.

Page 22 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Membres du groupe Inhibition of cell differentiation in FXYD3-deficient cells is accompanied (période 2006-2008) by an increase in the apparent Na+ and K+ affinities of Na,K-ATPase reflecting the absence of Na,K-pump regulation by FXYD3. In addition, Technicienne de laboratoire : we observe a decrease in the maximal Na,K-ATPase activity due to a - Danièle Schaer decrease in its turnover number, which correlates with a change in Na,K- Postdocs: ATPase isozyme expression that is characteristic of cancer cells. Overall, - Stéphanie Bibert our results suggest an important role of FXYD3 in cell differentiation of - Benjamin Delprat Caco-2 cells. We propose that FXYD3 silencing prevents proper + Collaboratrice de recherche: regulation of Na,K-ATPase, which leads to perturbation of cellular Na + - Stéphanie Bibert and K homeostasis and changes in the expression of Na,K-ATPase isozymes, whose functional properties are incompatible with Caco-2 cell Etudiants: differentiation. - David Aebischer - Juliane Aebischer - Florian Desgranges In several collaborative studies, we contributed to the characterization of - Manuel Jung 1) the 3rd Na+ binding site of Na,K-ATPase and its relation to pH- - Daniel Pouly activated currents, 2) the ouabain binding site in Na,K-ATPase, 3) the role of a fifth member of X,K-ATPase β subunits, 4) a novel β subunit- interacting protein, 5) Glut9, a member of the facilitative glucose transporter familiy and 6) glutathionylation of Na,K-ATPase β subunit as a novel regulatory mechanism of Na,K-ATPase.

Collaborations

Prof. Jean-Daniel Horisberger, Department of Pharmacology and Toxicology, Université de Lausanne, Lausanne, Suisse

Key words Dr Nikolai Modyanov, Department of Physiology and Pharmacology, University of Toledo College of Medicine, Toledo, USA Na,K-ATPase structure- function Svetlana Gorokhova, Laboratory of Molecular Biology, Howard Hughes relationship/regulation, FXYD Medical Institute, The Rockefeller University, New York, USA proteins structure-function relationship/physiological and Donata Rimoldi, LICR, Université de Lausanne, Lausanne, Switzerland pathophysiological relevance, protein phosphoryaltion, Prof. Helge Rasmussen, Department of Cardiology, Royal North Shore Xenopus oocytes, cell Hospital and Department of Medicine, University of Sydney, Australia differentiation Prof. Bernard Thorens, CIG, Université de Lausanne, Lausanne, Suisse

3 β isoforms

4 α isoforms β α FXYD proteins out

1 2 3 4 5 6 7 8 9 10 in N C N C

Morth et al. (2007) Nature 450, 1043-1049

Présentation schématique et la structure crystallographique de la Na,K-ATPase contenant la sous-unité α catalytique et ses deux sous-unités acessoires, la sous- unité β et les protéines FXYD

Page 23 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Selected references

Bibert S, Roy S, Schaer D, Felley-Bosco E and Geering K, Structural and functional properties of two human FXYD3 (MAT-8) isoforms. J. Biol. Chem. (2006) 281, 39142-39151 Delprat B, Schaer D, Roy S, Wang J, Puel J-L and Geering K, FXYD6 is a novel regulator of Na,K-ATPase expressed in the inner ear. J. Biol. Chem. (2007) 282, 7450-7456 Bibert S, Roy S, Schaer D, Horisberger J-D and Geering K, Phosphorylation of phospholemman (FXYD1) by protein kinases A and C modulates distinct Na,K-ATPase isozymes. J. Biol. Chem. (2008) 283, 476-486 Pestov N B, Ahmad N, Korneenko T V, Zhao H, Radkov R, Schaer D, Roy S, Bibert S, Geering K, Modyanov N N. Evolution of Na,K-ATPase βm-subunit into a coregulator of transcription in placental mammals. Proc. Natl. Acad. Sci. USA (2007) 104, 11215-11220

Lectures as invited speaker

June 16 -18, 2006, Molecular Mechanism and Regulation in Cation Transport ATPases an Related Genetic Diseases, Satellite meeting for 20th IUBMB International Congress of Biochemistry and Molecular Biology, Kyoto, Japan. ‘FXYD3 and FXYD6, new regulators of Na,K- ATPase’ January 26, 2007, Mini-Symposium ‘Physiology, Pathphysiology and Genetics of Renal Ion Transport’, Lausanne, Switzerland. ‘Regulation of Na,K-ATPase by FXYD proteins’ August 12-16, 2007, 5th International Research Conference on ‘Biomedical Transporters’, Bern, Switzerland. ‘Protein-protein interaction and transport regulation: FXYD proteins and Na,K-ATPase’ September 11-12, 2007, 6th Annual FXYD Symposium, London, UK. ‘Functional aspects of 2 FXYD proteins, FXYD1 (PLM) and FXYD3 (Mat-8)’ August 5-10, 2008, 12th International ATPase Conference, , University of Aarhus, Aarhus, Denmark, ‘Physiological and pathophysiological aspects of Na,K-ATPase regulation by FXYD proteins’ November 27-29, 2008, International Symposium on ‘Perspectives of Cell Signaling and Molecular Medicine’, Bose Institute, Kolkota, India. ‘A link between FXYD3-mediated regulation of Na,K-ATPase and differentiation of Caco-2 intestinal epithelial cells’

Page 24 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Structure-function relation of Na,K-ATPAse, regulation of the epithelial Na channel

Aim 1 Molecular mechanisms regulating the secretion and the reabsorption of K+ in the distal nephron segments.

Aim 2 Mutagenesis studies and molecular modeling of the binding of cardiac steroids to Na,K-ATPase

Aim 3 Regulation of ENaC by extracellular ligand, in particular by proteases and by the concentration of extracellular Na+ (self- Jean-Daniel inhibition) Horisberger

Jean-Daniel Horisberger a obtenu son diplôme de médecin à Lausanne en 1977, Collaborations puis son doctorat en médecine en 1982 à l'Institut de Prof. Olivier Michielin, SIB, Institut Ludwig, Lausanne, Suisse Pharmacologie de l'UNIL (prof. J. Diezi). Après une Prof. Martin Gijs, Institut de microtechnique, EPFL, Lausanne, Suisse formation complète en médecine interne clinique Prof. N. Modyanov, Dept. Physio Pharm Meta Cardio, University of dans divers hôpitaux et au Toledo, Ohio, USA CHUV, il est aller se former dans le domaine de la physiologie rénale, plus particulièrement avec des techniques Selected references électrophysiologiques à l'Université de Yale de 1984 à Original articles 1987 (prof. G. Giebisch). Il est revenu à l'Institut de Bibert S, Roy S, Schaer D, Horisberger J-D and Geering K, Pharmacologie de l'UNIL, Phosphorylation of Phospholemman (FXYD1) by Protein Kinases A and comme prof-assistant, puis prof. associé de 1992 à 2002, C Modulates Distinct Na,K-ATPase Isozymes. J Biol Chem.(2008) 283: et a été promu à la fonction de 476-486 professeur ordinaire en 2002. th Il s'est intéressé à la structure Capendeguy O, Iwaszkiewicz J, Michielin O and Horisberger J-D. The 4 et à la fonction de plusieurs extracellular loop of the a subunit of Na,K-ATPase: Functional evidence système de transport for close proximity with the 2nd extracellular loop. J Biol Chem.(2008) membranaire, en particulier la Na,K-ATPase et le canal 283: 27850-27858 épithélial à sodium et en a étudié divers modes de Bize V and Horisberger J-D. Sodium self-inhibition of human epithelial régulation, hormonale ou sodium channel: selectivity and affinity of the extracellular sodium autres. sensing site. American Journal of Physiology-Renal Physiology (2007) Il a pris la charge de vice- 293: F1137-F1146 doyen de la relève dès 2006. Dahan E, Bize V, Lehnert T, Horisberger J-D and Gijs MAM. Integrated microsystem for non-invasive electrophysiological measurements on Xenopus oocytes. Biosensors & Bioelectronics (2007) 22: 3196-3202

Guennoun-Lehmann S, Fonseca JE, Horisberger J-D and Rakowski RF. + + Palytoxin acts on Na ,K -ATPase but not nongastric H+,K+-ATPase. J Membrane Biol (2007) 216: 107-116 Radkov R, Kharoubi-Hess S, Schaer D, Modyanov NN, Geering K and Horisberger J-D. Role of homolgous ASP334 and GLU319 in human non-gastric H,K- and Na,K-ATPase in cardiac glycoside binding. Biochem Biophys Res Commun (2007) 356: 142-146

Page 25 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Capendeguy O, Chodanowski P, Michielin O and Horisberger J-D. Access of extracellular cations to their binding sites in Na,K ATPase: Membres du groupe (période 2006-2008) Role of the 2nd extracellular loop of the a subunit. J Gen Physiol (2006) 127: 341-352 Technicienne de laboratoire: - Solange Kharoubi Hess Reviews articles, Book chapters and conference proceedings

Doctorants: - Oihana Capendeguy Horisberger J-D and Geering K. Brain Na,K-ATPase. In: The New - Vincent Bize Encyclopedia of Neuroscience, edited by Squires L. Elsevier, 2008

Horisberger J-D and Doucet A. Renal Ion-Translocating ATPases: The P- Postdocs: type Family. In: Seldin and Giebisch's The Kidney, edited by Alpern RJ - Vincent Bize and Hebert SC. Amsterdam: Elsevier, 2007, p. 57-90 - Heidi Fodstad Horisberger J-D. Mécanisme du transport des cations Na+ et K+ par la pompe à sodium. Médecine/Sciences 22: 27-28, 2006

Lectures as invited speaker

Key words 14-22 June, 2006, "Extracellular cation access to their binding site in Na+ transepithelial transport, Na,K-ATPase". Cation transporting ATPases, Kyoto, Japan K+ transepithelial transport, September 18 to 20, 2006, "Evolution of Na,K-ATPases". 99th Annual Na,K-ATPase, H,K-ATPase, Meeting of the German Zoological Society, Münster, Germany ENaC, ouabain, WINK, Na+ self inhibition, proteases. October 3 – 7, 2007, "Regulation of ENaC Activity by Extracellular

Factors: Cations and Proteases". 6th International Symposium on “Aldosterone and ENaC: from gene to disease”, Zermatt, Switzerland

27. 08. – 30. 08. 2008, "K+ homeostasis, a hormonal control for urinary K+ excretion ?" TRANSPORTERS 2008 Centre Loewenberg, Murten, Switzerland

Page 26 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Activation of the epithelial Na channel by membrane-bound proteases

Aim 1 to examine key components of ENaC -mediated sodium transport in the kidney, the lung and the colon

Aim 2 to examine key components of ENaC regulation by serine proteases

Bernard C. Rossier Selected references

Bernard Rossier received his MD in 1966 from the Reviews University of Lausanne(UNIL). After his Harris M, Garcia-Caballero A, Stutts MJ, Firsov D, Rossier BC, physiology training at the Activation of the Epithelial Sodium Channel (ENaC) by Serine Proteases. Institute of Physiology at Rossier BC, Stutts MJ. Annu Rev Physiol. (2008). [Epub ahead of print] UNIL, followed by his medical training in the Epithelial sodium channel: mendelian versus essential hypertension. Departement of Medicine of Rossier BC, Schild L. Hypertension. (2008) 52(4):595-600 the University of Geneva and his post-doctoral training with Articles Isidore Edelman at the University of California San Harris M, Garcia-Caballero A, Stutts MJ, Firsov D, Rossier BC Francisco, he joined the Preferential assembly of epithelial sodium channel (ENaC) subunits in Department of Pharmacology Xenopus oocytes: role of furin-mediated endogenous proteolysis. J Biol of UNIL in 1974. He has been leading his own research team Chem. (2008) 283(12)7455-63 since then. He has been Professor of Phamacology and Zuber AM, Singer D, Penninger JM, Rossier BC, Firsov D. Increased Toxicology (1981-2006), renal responsiveness to vasopressin and enhanced V2 receptor signaling Chairman of the Department in RGS2-/- mice. J Am Soc Nephrol. (2007) 1672-8 of Pharmacology (1990 - 2006), Dean of the Faculty of Gonzalez-Rodriguez E, Gaeggeler HP, Rossier BC. IGF-1 vs insulin: Medicine (1996-200.). He his respective roles in modulating sodium transport via the PI-3 kinase/Sgk1 presently Emeritus Professor of Pharmacology and pathway in a cortical collecting duct cell line. Kidney Int. (2007) 71(2) Toxicology (2006 - ). He is 116-25 european co-ordinator of the Transatlantic Network of Harris M, Firsov D, Vuagniaux G, Stutts MJ, Rossier BC. A novel Excellence in Hypertension of neutrophil elastase inhibitor prevents elastase activation and surface the Leducq Foundation. He is cleavage of the epithelial sodium channel expressed in Xenopus laevis recipient of many international oocytes. J Biol Chem. (2007) (1):58-64 Prizes in the field of nephrology. He is a member Andreasen D, Vuagniaux G, Fowler-Jaeger N, Hummler E, Rossier BC. of EMBO, the Academia Europaea, the deutsche Activation of epithelial sodium channels by mouse channel activating Akademie der Naturforscher proteases (mCAP) expressed in Xenopus oocytes requires catalytic Leopoldina, and is a Foreign activity of mCAP3 and mCAP2 but not mCAP1. J Am Soc Nephrol. Honorary member of the (2006) (4):968-76 American Academy of Arts and Sciences. He is Dr. honoris causa of the University Pierre et Marie Curie in Paris. He is member Lectures as invited speaker of the Louis Jeantet Foundation in Geneva and the Cloetta Foundation in Zurich 19-21.2.2006, San Raffaele Scientific Retreat, Bardolino (VR) "Epithelial and the Leenards Foudation in sodium channels in health and disease" Lausanne. 19.4.2006, Université de Montréal, Groupe d'études des protéines membranaires. "Activation of epithelial sodium channels by serine proteases: physiological and pathophysiological implications"

Page 27 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

20.4.2006, Merck Frosst Center for Therapeutic Research, Dept of Biochemistry and Molecular Biology. Seminar. Montreal. "Channel

activating proteases (CAPs) and the regulation of the epithelial sodium Membres du groupe channel (ENaC)" (période 2006-2008) 21-23.4.2006, National Kidney Foundation (Chicago)."ENaCting Techniciens de laboratoire: function or dysfunction". Donald W. Seldin Award - Nicole Fowler Jaeger - Hans-Peter Gaeggeler 25.4.2006, Collège de France, Paris. Séminaire de la Chaire de Médecine - Anne-Marie Mérillat Expérimentale "Genetics of arterial hypertension: lessons from the Last Doctorante: Ten Years". "ENaC function and dysfunction: lessons from the Liddle - Irène Ryvicki syndrome"

Postdocs: 18-19.5.2006, Royal Swedish Academy of Sciences, Stockholm. 3rd Key - Ditte Andreasen Symposium on "Membrane transport in Health and Diseases": ENaC: - Michael Harris lessons from human diseases and animal models

Boursière: 26.9.2006, Cardiovascular Sciences Seminar. Edinburgh, "Epithelial - Birgitte Christensen sodium channel: lessons from human diseases and animal models" Stagiaires: - Nicolas Markadieu 28.9.2006 CHUV, Lausanne, Auditoire César Roux. "Le charme discret - Sylvie Perroset des transitions..." 3.10.2006, Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven. "Regulation of sodium transport and osmotic gradient generation: a novel cellular model" 4.10.2006, MGH, Nephrology Division, Boston. "Hormonal control of sodium and water transport in a cortical collecting duct cell line"

5.10.2006, MGH Medical Grand Round, 3rd Annual MGH Kidney Care Day. "ENaC function or dysfunction: revisiting diuretic therapy" Key words 1.12.2006, Croissance et signalisation. Faculté de Médecine René ENaC, hypertension, epithelial Descartes site Necker, Paris. "Aldosterone and ENaC: revisiting our sodium transport, amiloride sodium balance"

26.1.2007, Physiology, Pathophysiology and Genetics of Renal Ion Transport. Minisymposium Cardiomet. Lausanne. "Activation of ENaC by serine proteases" 2.2.2007, EurReGene Symposium on Renal Pathophysiology. Bruxelles.. "ENaC: New insights from human diseases and animal models" 19.2.2007, Renal Regulation of Water and Sodium Balance: Molecular, Physiological and Systems Biology. Arhus University. "Activation of ENaC by serine proteases" 18.9.2007, William E. Mitch Lecture. Emory University School of Medicine. "ENaC function and dysfunction: lessons from human diseases and animal models" 20.9.2007, Physiology Grand Round. Emory University School of Medicine. "ENaC activation by serine proteases".25.9.2007, University of North Carolina - CF Center. "ENaC activation by serine proteases" 7.10.2007, 6th International Meeting on aldosterone and ENaC: from gene to disease. Parkhotel Beau-Site, Zermatt, Switzerland. "To be cleaved or not to be..." 13.11.2007, Conférence Revillod. Hôpitaux Universitaires de Genève. "De la découverte du canal sodique au rôle du sodium dans l'hypertension artérielle: Reconsidérons le rôle des diurétiques"

Page 28 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

30.11.2007, ISN Nexus. Hypertension and the Kidney. Guyton's concept 2007. Vienna. "Blood Pressure Control - Special role of ENaC and body fluids" 14-15.12.2007, Symposium AMGEN. Trois décennies de recherche translationnelle du chercheur vers le néphrologue. Paris "Le canal à sodium épithélial: 14 ans après" 18.12.2007, Seminar - Physiologisches Institut der Universität Zürich. "Epithelial sodium channels in health and disease" March 8 - 12, 2008, ESF Research Conference Rare Diseases: channel and transporters Sant Feliu de Guixols, Costa Brava October 2 - 3, 2008 Epithelial sodium channels in health and disease, 14th Cardiovascular Biology & Clinical Implications Meeting Morat, Hypertension is a renal disease November 12, 2008 T. E. Andreoli Lecture, University of Alabama Birmingham, "Epithelial Sodium Channel, Salt Intake and Blood Pressure Control: The Story of our Internal Environment revisited"

Honors and Awards

2006 Donald W. SELDIN Award of the National Kidney Foundation (USA)

Page 29 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

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 they affect their function and if they are playing a physiological role. We have particularly been interested in the role of Nedd4/Nedd4-like ubiquitin-protein ligases, which we have shown to directly interact with channel proteins. Ubiquitin-protein ligases are at the end point of an enzymatic cascade, which covalently attaches Olivier Staub ubiquitin-moieties on target proteins. Proteins included in this cascade are E1 (ubiquitin-activating enzyme), E2s (ubiquitin-conjugating enzymes) Olivier Staub received his and E3s (ubiquitin-protein ligases). In recent year it has become apparent bachelor degree (chemistry) at that ubiquitylation is a reversible process, involving the activity of the University of Bern, deubiquitylating enzymes. We have indeed identified two Switzerland (1987), and his deubiquitylating enzymes that are able to regulate the expression of ENaC PhD degree at the University of Lausanne (1992). He was at the cell surface. then trained as a postdoc at + UCLA and at the Hospital for Regulation of the epithelial Na channel ENaC by ubiquitylation and Sick Children in Toronto, deubiquitylation before joining the Department of Pharmacology & ENaC represents the prototype for the regulation of ion channels by Toxicology as an independent Nedd4-like proteins. This protein complex, composed of three researcher in 1997. Currently homologous subunits (αβγ), which is localized to the aldosterone- he holds the position of an + Associate Professor. 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 of human hypertension. The disease is caused by

mutations, which delete/alter PY motifs in the C-termini of β or γENaC, resulting in increased Na+ channel activity due to elevated channel

number and open probability. We have shown that these PY-motifs act as binding sites for the ubiquitin-protein ligase Nedd4-2, that ENaC becomes ubiquitylated and that Nedd4-2 suppresses ENaC activity by controlling channel number at the cell surface, providing an explanation for the regulatory defect in Liddle's syndrome. We have found that the aldosterone induced Sgk1 kinase phosphorylates Nedd4-2 in Xenopus laevis oocytes, and that such phosphorylation reduces ENaC-Nedd4-2 interaction, resulting in decreased ENaC ubiquitylation and accumulation at the plasma membrane. The importance of Nedd4-2 and Sgk1 in Na+ homeostasis and blood pressure has been demonstrated with Nedd4-2 and Sgk1 deficient mice. ENaC is also regulated by deubiquitylation catalyzed by deubiquitylating enzymes (DUBs). We have identified 2 DUBs that stimulate ENaC activity: 1) Usp2-45 is an aldosterone-induced protein that binds to and deubiquitylates ENaC. Thereby it increases cell surface density of ENaC and stimulates extracellular cleavage of ENaC, leading to increased ENaC activity. On the other hand, vasopressin induced Usp10 promotes indirectly ENaC cell surface expression by interacting with and deubiquitylating sorting nexin 3 (SNX3), a protein involved in endosomal trafficking. We are currently investigating the physiological role of these proteins in the regulation of Na+ balance and blood pressure. Moreover, we are also interested in the assembly and quality control of ENaC (involving ER associated degradation), and in the regulation of Na+ balance in vivo in different mice models.

Page 30 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Membres du groupe Collaborations (période 2006-2008) Technicienne de laboratoire: -Pierrette Hausel Dr Frédéric Jaisser, INSERM U772, Collège de France, Paris, France Doctorants: - Anne Debonneville Dr Xavier Jeunemaître, INSERM U36, Collège de France, Paris, France - Dagmara Lagnaz - Daniel Pouly Dr Aniko Naray-Fejes-Toth, Dept. of Physiology, Dartmouth Medical - Fabien Terranova School, Lebanon, USA - Jean-Jacques Vitagliano Dr David Pearce, Cellular and Molecular Pharmacology, UCSF, San Postdocs: Francisco, USA - Sheerazed Boulkroun - Nourdine Faresse Dr Ermanno Rossi, Department of Internal Medicine, Reggio Emilia, - Caroline Ronzaud Italy

Etudiants: Dr Daniela Rotin, The Hospital for Sick Children, Toronto, Ontario, - Carole Bacca - Martino Bardelli Canada - Simon Crausaz Dr John B. Stokes, Internal Medicine, University of Iowa, USA - Marianna Di Chiara - Christine Greiner Dr Baoli Yang, Departments of Obstetrics and Gynecology, University of - Dagmara Lagnaz - Aris Maquelin Iowa, USA

Stagiaires universitaires: - Pedro San Cristobal Zepeda

Apprentis: - Sting Chabanel - Romain Groux

Stagiaires: - Nadia Rainoldi - Yoan Rappaz - Sylvia Verdon

Boursier: - Cécilia Lindholm

Key words Ubiquitin-protein ligase, Nedd4, PY-motif, protein- Regulatory pathways controlling Nedd-42 dependant ubiquitylation and protein interaction, Na+- internalization/degradation of ENaC. High sodium diet increases Nedd4-2 homeostasis, hypertension, expression in the cortical collecting duct. Aldosterone increases Sgk1 and Usp2- kinase, ENaC, 45 expression, and consequently interferes with ENaC deubiquitylation. deubiquitylation, Deubiquitylation also favors proteolytic cleavage and activation of ENaC. phosphorylation, Sgk1, Usp2- 45, Usp10

Page 31 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Selected References

Boulkroun S, Ruffieux-Daidié D, Vitagliano J-J, Poirot O, Lagnaz D, Charles R-P, Firsov D, Kellenberger S and Staub O, Vasopressin inducible ubiquitin specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3. Am.J.Physiol./Renal Physiol. (2008) 295:F889-900 Ruffieux-Daidié D, Poirot O, Boulkroun S, Verrey F, Kellenberger S and Staub O, Deubiquitylation regulates proteolytic cleavage and activation of the epithelial Na+ channel ENaC. J.Am.Soc.Nephrol. (2008) 19:2170-80 Flores S Y, Loffing-Cueni D, Kamynina E, Daidié D, Gerbex C, Chabanel C, Dudler J, Loffing J and Staub O, Aldosterone induced Serum- and Glucocorticoid induced kinase 1 expression is accompanied by Nedd4-2 phosphorylation and increased Na+ transport in cortical collecting duct cells. J.Am.Soc.Nephrol. (2005) 16:2279-87 Debonneville C, and Staub O, Participation of the ubiquitin-conjugating enzyme UBE2E3 in Nedd4-2 dependent regulation of the epithelial Na+ channel ENaC. Mol.Cell.Biol, (2004) 24:2397-2407 Debonneville C, Flores S Y, Kamynina E, Plant P J, Tauxe C, Thomas M A, Münster C, Chraibi A, Pratt J H, Horisberger J D, Pearce D, Loffing J, and Staub O, Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na+ channel cell surface expression. EMBO J. (2001) 20:7052-7059 Verrey F, Fakitsas P, Adam G and Staub O, Early transcriptional control of ENaC:(de)ubiquitylation by aldosterone. Kidney International (2008) 19:298-309,(IF07: 4.92) Staub O and Rotin D, The role of ubiquitylation in membrane transport. Physiol.Rev. (2006) 86:669-707, (IF06: 31.44) Abriel H and Staub O, Regulation of ion channels by ubiquitylation. Physiology (2006) 20:398-407, (IF06: 6.27) Loffing J, Flores S Y and Staub O. Epithelial Transport regulation by Sgk. Annual Review of Physiology (2006) 68:461-490, (IF06: 15.36)

Lectures as invited speaker

31/5/07 26th International Aldosterone Conference (31/5-1/6/2007), Toronto, Canada. The Aldosterone-induced Deubiquitylating Enzyme Usp2-45 Enhances Cell Surface Expression of the Epithelial Na+ Channel ENaC 12/8/07 5th International Conference on Membrane Transporters in Disease and Drug Development, (12-16/8/07), Bern, Switzerland The role of ubiquitylation and deubiquitylation in the control of transepithelial Na+ transport 31/8/07 4th Annual Cell Signalling Symposium: The Interplay Between Protein Phosphorylation and Ubiquitylation in Cell Signaling, (30/8- 2/9/07) Dundee, UK 21/9/07 EASD Islet Study Group, Symposium 2007, (21-23/9/07) Brussels, Belgium Key note lecture: Ubiquitylation of ion channels 4/10/07 6th International Meeting on Aldosterone and ENaC: from gene to disease, (3-7/10/2007), Zermatt Switzerland (Organizer). ENaC regulation by ubiquitylation and deubiquitylation

Page 32 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

1/11/07 Annual Meeting of the American Society of Nephrology, (31/10 – 5/11/07) San Francisco, USA. Regulation of ENaC trafficking by deubiquitylation 8/4/08 Dutch Nephrology Days (8-9/4/2008), Veldhoven, The Netherlands. Regulation of the epithelial Na+ channel ENaC by ubiquitylation 30/8/08 Transporters 2008 (27-30/8/2008), Murten Switzerland Concerted action of deubiquitylation and proteolysis in the regulation of the epithelial Na+ channel EnaC 27/11/08 8ème Réunion Commune : Société de Néphrologie et Société Francophone de Dialyse (26-29/11/2008), Marrakech, Morocco. Ubiquitylation de canaux ioniques

Page 33 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Structure and function of the epithelial sodium channel

Epithelia form barriers that separate the body fluids from the outside world, and serve to maintain their water and solute composition. To achieve this essential function, epithelial cells are capable of vectorial transport of solutes and water thanks to ion pumps, transporters or channels located in plasma cell membranes. The epithelial sodium channel ENaC allows the entry of Na+ ions into the cell and in serie with the Na+/K+-ATPase, mediates vectorial transcellular transport of sodium. In the kidney, ENaC is responsible for the fine tuning of urinary Na+ Laurent Schild excretion that allows the maintenance of a strict balance between the daily salt intake and Na+ elimination. In the lung ENaC participates in + Laurent Schild obtained a Na secretion in the airway epithelia, in order to maintain constant the diploma in medicine in 1979 airway surface liquid. Human genetic studies have demonstrated the role from the Medical School of of ENaC in the control of extracellular fluid volume and blood pressure. the University of Lausanne and a Doctorate in Medicine In mice, ENaC gain of function targeted in the lung leads to a cystic in 1984. From 1984 to 1990, fibrosis-like lung disease. he was postdoctoral fellow at Yale University Medical ENaC belongs to the family of ion channels, including the Acid- School, first in the laboratory Sensing Ion Channels (ASIC) for which a crystal structure has recently of Prof. Giebisch at the been obtained. The long term aim of our research is to understand at the Department of Cellular and + Molecular Physiology, then at atomic level, the molecular basis of ENaC function comprising Na ions the Department of flux through the protein, channel opening or closing, interactions with Pharmacology in the pharmacological ligands, regulation of channel activity by intracellular laboratory of Prof. associated proteins. Moczydlowski. He returned to Lausanne in 1990, at the Department of Pharmacology & Toxicology, and started an indepedendent group of research, working on the pharmacology of ion channels. He closely collaborated with Prof. B. C. Rossier for the understanding of the structure, the function and the regulation of the epithelial sodium channel (ENaC). Presently, he is full Professor of Pharmacology and Director of the Department of Pharmacology.

Figure 1. Homology model of ENaC based on crystal structure of ASIC1a channel. ENaC is a heterotrimer made of 3 homologous abg subunits. The transmembrane domain of the channel is made of parallel a helices (bottom); a large extracellular domain extends several tens of Å above the plane of the membrane and makes more than half of the mass of the protein.

Page 34 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

We have recently focused our work on the identification of ENaC Membres du groupe structures facing the cytosolic side of the membrane that are important for (période 2006-2008) the regulation of ENaC activity. We provided evidences showing that Technicien de laboratoire: modifications of intracellular cysteines on ENaC and ASIC1 channels can - Ivan Gautschi modulate channel activity. (Pfister et al.), indicating that the N-terminus of ENaC and of channel homologs also represents an important functional Doctorants: - Sylvian Bron domain of the channel protein that participates in the internal vestibule of - Aris Maquelin the channel pore that undergoes conformational changes during channel - Armelle-Natsuo Takeda gating. Finally Cys residues in the distal part of the second transmembrane domain of the g ENaC subunit can coordinate metal Postdocs: 2+ - Muriel Auberson cations such as Cd and block the channel acting as a strict pore blocker - Christophe Debonneville to inhibit ENaC.

Chargé de recherche: The results of our research have several implications for our - Miguel Van Bemmelen understanding of ENaC function. First they identify a novel molecular mechanism of ENaC regulation that is mediated by intracellular cysteine Etudiants: - Nina Ausderau residues. The physiological relevance of this regulation of channel - Benoît Genoud activity is not yet clear and opens new ways for investigations to elucidate - Delphine Huser novel regulatory ENaC regulatory mechanisms, such as the role of the - Martial Mbefo Kamdem intracellular redox potential to control Na+ transport in ENaC expressing

Apprentie: epithelia. Such regulatory mechanism might be important in the - Aurélie Calame pathogenesis of pulmonary edema, of renal salt loosing in acute renal ischemia. Second our results have implications for ENaC structure and Stagiaires: drug discovery. They demonstrate that specific ligands that target defined - Alexandre Ansorge - Nicolas Faller intracellular domains of the ENaC channel are able to modulate channel - Cora Liardon activity. This represents the proof of principle for futur development of - Julien Zaldivar-Jolissaint highly efficient intracellular ligands to block ENaC channel. So far only extracellular ENaC blockers such as amiloride or triamterene are used clinically as diuretics to promote renal Na+ excretion, with relatively poor efficiency.

Key words Ion channel, ENaC, ion Collaborations transport, epithelia, Na homeostasis, aldosterone, amiloride Collaborations dans le cadre du projet ‘Transatlantic Network on Hypertension : R.P. Lifton, M.J. Caplan, Yale, University, X. Jeunemaître , Collège de France, Paris Dr D. Rotin, Sick Children Hospital Toronto, Canada: ENaC regulation via protein-protein interactions Prof. Kimmo Kontula, Biomedical Centrum, Helsinki : genetic of hypertension Dr Xiao-Dan Li, Biomolecular Research Unit (Structural Biology of Membrane Proteins) Paul Scherrer Institut, Villigen: structure du canal ENaC Prof. Olivier Michielin, LICR, Université de Lausanne, Lausanne, Suisse

Page 35 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Selected references

Pfister Y, Gautschi I, Takeda A N, Van Bemmelen M, Kellenberger S and Schild L. A gating mutation in the internal pore of ASIC1a. J Biol Chem (2006) 281: 11787-11791 Takeda, A N, Gautschi I, Van Bemmelen M and Schild L, Cadmium trapping in an epithelial sodium channel pore mutant. J. Biol. Chem. (2007) 282:31928-31936 Riepe F G, Van Bemmelen M, Cachat F, Plendl H, Gautschi I, Krone N, Holterhus P M, Theintz G, and Schild L. Revealing a subclinical salt- loosing phenotype in heterozygous carriers of the novel S562P mutation in the alpha subunit of the epithelial sodium channel. Clin. Endocrinol. 2008 Jun 10

Lectures as invited speaker

October 3 – 7, 2007, 6th International Symposium on “Aldosterone and ENaC: from gene to disease”, Zermatt, Switzerland

November 29 to December 2, 2007, International Society of Nephrology: Hypertension and the Kidney, Vienna, Austria

November 6th to November 8th, 2008, American Society of Nephrology. Philadelphia, USA

Page 36 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Cardiovascular system and associated pathologies

The Role of the A-kinase anchoring proteins signaling complexes in G protein-coupled receptor-induced cardiac remodeling

The main interest of our laboratory is to define the role of scaffolding and anchoring proteins in the pathological remodeling process associated with cardiac hypertrophy and heart failure. In this context, we investigate how signals that control hypertrophy and fibrosis are integrated, coordinated and processed within cardiomyocytes and cardiac fibroblasts by these families of signal transduction proteins. During the last years we have mainly focused on a family of molecular scaffolds named A-kinase anchoring proteins (AKAPs), which target the cAMP-dependent kinase (PKA) and other enzymes at precise subcellular sites where they can be accessed by activators and, in turn, interact with particular substrates. In Dario Diviani particular, our recent studies identified a novel cardiac anchoring protein expressed both in cardiomyocytes and cardiac fibroblasts, called AKAP- Dario Diviani received his Lbc, that assembles a multienzyme transduction complex critically PhD in 1998 from the University of Lausanne for involved in the integration and processing of a variety of hypertrophic research on alpha-1 adrenergic signals. We are currently investigating the precise molecular architecture receptors performed with of the AKAP-Lbc signaling complex using a variety of biochemical and Susanna Cotecchia. Between proteomic approaches and we are defining AKAP-Lbc-activated 1998 and 2001 he performed his post doctoral trainig with pathways that control hypertrophy and fibrosis both in vitro and in vivo. John D. Scott at the Vollum These findings will serve as ground information to develop molecular Institute of the Oregon Health strategies to selectively disrupt and inhibit signaling complexes involved Sciences University, Portland, in the transduction of pro-hypertrophic or pro-fibrotic signals. where he worked on the molecular mechanims Aim 1 controlling signaling specificity in heart cells. In To determine the functional role of the AKAP-Lbc complex in 2001, he joined the cardiomyocyte hypertrophy. Department of Pharmacology of the University of Lausanne, Our recent findings indicate that AKAP-Lbc is a key mediator of the where he currently holds a hypertrophic responses initiated by G protein-coupled receptors including tenure track assistant professor a1-adrenergic-, angiotensin II- and endothelin I-receptors. position. However, the signaling pathways linking AKAP-Lbc to the activation of the transcription of hypertrophic genes are yet to be identified. Interestingly, our current results suggest that AKAP-Lbc can bind and activate mitogen-activated protein kinases (MAPKs), which are known to play a key role in the activation of transcription factors that regulate cardiomyocyte growth.

A) Schematical representation of the hypertrophic signaling pathway mediated by AKAP-Lbc in cardiomyocytes. AKAP-Lbc functions as a guanine nucleotide exchange factor that promotes hypertrophic signaling through the activation of the small GTP binding protein RhoA. The Rho-GEF activity of AKAP-Lbc is induced by G protein coupled receptors such as a1-adrenergic- and type I Angiotensin- receptors via a signaling pathway that involves the a subunit of the heterotrimeric G protein G12.

Page 37 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Based on these findings, we are currently characterizing the molecular organization of the complex formed by AKAP-Lbc and MAPKs, identifying the signal transduction molecules that link AKAP-Lbc to the Membres du groupe stimulation of MAPKs in neonatal ventricular cardiomyocytes and (période 2006-2008) addressing questions aimed at elucidating whether AKAP-Lbc-MAPK Technicienne de laboratoire: signaling complexes are required for regulation of transcriptional - Monique Nenniger Tosato responses involved in cardiomyocyte hypertrophy.

Doctorants: Aim 2 - Laurent Alain Baisamy - Sabrina Cavin To determine the functional relevance of AKAP-Lbc in cardiac fibrosis. - Luca Cariolato This is accomplished by characterizing the molecular mechanisms as well - Damiano Del Vescovo - Irene Pérez-Lopez as the signaling pathways underlying the activation of the AKAP-Lbc signaling complex by pro-fibrotic stimuli such angiotensin II and Etudiants: endothelin 1. In particular, we are determining whether and how AKAP- - Fabian Gerster Lbc promotes cellular responses associated with cardiac fibrosis both in - Julien Quebatte vitro, using primary cultures of cardiac fibroblast as a model system, and in vivo.

Keywords

Cardiac hypertrophy, cardiac fibrosis, A-kinase anchoring proteins, G protein coupled receptors.

B)This figure illustrates the inhibitory effect of silencing AKAP-Lbc expression in primary cultures of rat neonatal ventricular myocytes on the hypertrophic response induced by phenylephrine. The sarcomeric organization of cardiomyocytes is visualized using anti-a-actinin antibodies.

Collaborations

Dr Enno Klussmann, Department of Pharmacology, Freie Universität Berlin, Germany Prof. Thierry Pedrazzini, Department of Medicine, CHUV, Lausanne Suisse Prof. John D. Scott, Department of Pharmacology, Washington State University, Seattle, USA

Page 38 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Selected references

Stanasila L, Abuin L, Diviani D, Cotecchia S, Ezrin directly interacts with the a1b-adrenergic receptor and plays a role in receptor recycling. J. Biol. Chem. (2006) 281: 4354- 4363 Diviani D, Baisamy L, Appert-Collin A, AKAP-Lbc: a molecular scaffold for the integration of cAMP and Rho transduction pathways. (review) Eur. J. Cell Biol., (2006) 85: 603-10 Appert-Collin A, Baisamy L, Diviani D, Regulation of G protein coupled receptor signaling by A kinase anchoring proteins (review). J. Recept. Signal Transduct. Res. (2006) 26: 631-646 Appert-Collin A, Cotecchia S, Nenniger-Tosato M, Pedrazzini T, Diviani D, The AKAP-Lbc signaling complex mediates alpha-1 adrenergic receptor-induced cardiomyocyte hypertrophy. Proc. Natl. Acad. Sci. U.S.A. (2007) 104: 10140-10145 Diviani D, Regulation of cardiac function by A-kinase anchoring proteins (review). Curr. Opin. Pharmacol (2008) 8: 166-173 Carnegie G K, Soughayer S, Pedroja B S, Smith F D, Zhang F, Diviani D, Bristow M R, Kunkel M T, Newton A C, Langeberg L K, Scott J D, AKAP-Lbc mobilizes a hypertrophic signaling pathway in cardiomyocytes. Mol. Cell. (2008) 32: 169-179

Lectures as invited speaker

15th Protein kinase meeting and FEBS/EMBO Workshop. Spatial and temporal regulation of signaling. September 20-24, 2006. Oslo, Norway. Regulation of Rho signaling by the AKAP-Lbc transduction complex

Leibniz Institut für Molekulare Pharmakologie. Berlin, Germany. “AKAP-Lbc: a transduction complex that regulates cardiomyocytes hypertrophy”. June 19, 2007

Biotechnology Center, Oslo, Norway. “AKAP-Lbc: a transduction complex that regulates cardiomyocytes hypertrophy”. June 21, 2007

2nd International Meeting on Anchored cAMP Signaling Pathways, September 12-14, 2007. “Regulation of cardiomyocyte hypertrophy by the AKAP-Lbc transduction complex”

Page 39 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Molecular bases of cardiac arrhythmias

The main goal of our research group is to elucidate novel molecular and cellular mechanisms underlying cardiac arrhythmias causing sudden death. To this end, on the one hand, we are investigating ion channels mutations found in patients and families presenting with genetic forms of lethal arrhythmias such as the congenital long QT syndrome and Brugada syndrome. On the other hand, we are studying new forms of regulation of cardiac ion channels relevant to arrhythmogenic mechanisms.

Hugues Abriel

Hugues Abriel studied life sciences at the Swiss Federal Institute of Technology in Zurich (ETHZ, 1989). He continued his education to become a physician (MD, 1994) and received a PhD degree in Physiology from the University of Lausanne in Switzerland (1995). He has spent two years as a research Schematic presentation of the cardiac action potential and the three main ion scientist at Columbia channels that we are currently studying in the laboratory. The channel Nav1.5 University in New York, mediates the rapid depolarization of the membrane, whereas the hERG and USA. Hugues Abriel is a KCNQ1 channels are involved in the repolarization of the cardiac cell. group leader at the Department of Pharmacology and Toxicology at the University of Lausanne. He is also affiliated to the Division Collaborations of Cardiology of the University Hospital of Institut du thorax, Nantes, France Lausanne (CHUV). His research work focuses on the Stéphane Hatem, Inserm, Paris, France roles of ion channels in human diseases (channelopathies). Thomas Jespersen, Department of Biomedical Sciences, University of Currently, he is mainly Copenhagen, Denmark exploring the molecular and cellular bases of cardiac Jan Kucera, Institut für Physiologie, University of Bern, Switzerland arrhythmias. Jean-Marc Burgunder, DKF, Forschungsgruppe Neurologie, University of Bern, Switzerland

Selected references

Gavillet B, Rougier J-S, Domenighetti A, Behar R, Boixel C, Ruchat P, Lehr H-A, Pedrazzini T and Abriel H, Cardiac Sodium Channel Nav1.5 is Regulated by a Multiprotein Complex Composed of Syntrophins and Dystrophin. Circulation Research (2006) 99(4): 407-14

Eap C B ∗, Crettol S, Rougier J-S, Schlaefer J, Sintragrilo L, Deglon J-J, Besson J, Croquette-krokkar M, Carrupt P-A and Abriel A ∗, (∗both corresponding authors) Stereoselective block of hERG channel by (S)- methadone and QT interval prolongation in CYP2B6 slow metabolizers. Clinical Pharmacology and Therapeutics, (2007) 81(5): 719-28

Page 40 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Membres du groupe Jespersen T, Membrez M, Nicolas C S, Pitard B, Staub O, Olesen S-P, (période 2006-2008) Baro I and Abriel H, The KCNQ1 Potassium Channel is Down-Regulated by Ubiquitylating Enzymes of the Nedd4/Nedd4-like Family. Techniciennes de laboratoire: Cardiovascular Research, (2007) 74:64-74. Published with accompanying - Sophie Roy - Florine Apotheloz editorial - Stéphanie Sutter - Anne-Flore Zmoos Petitprez S, Tiab L Chen L, Kappeller L, Rosler K M, Schorderet D-F, Abriel H ∗ and Burgunder J-M ∗ (∗both corresponding authors). A novel Doctorants: - Maxime Albesa dominant mutation of the Nav1.4 a-subunit domain I leading to sodium - Cédric Laedermann channel myotonia. (2008) In press Neurology - Séverine Petitprez - Liliana Sintra Grilo

Postdocs: - Bruno Gavillet Lectures as invited speaker - Konstantin Gusev - Jean-Sébastien Rougier 27 January 2006, Abriel H., Molecular mechanisms of sudden cardiac death in heart failure patients, Gstaad, Swiss Cardiology Foundation Boursiers: - Athanasia Aggeli meeting - Thomas Jespersen 24 February 2006 Abriel H., Ubiquitylation of Voltage-Gated Sodium - Elena Zaklyaziminskaya Channels, USGEB meeting, Geneva Chargé de recherche: - Mathieu Membrez 10 May 2006, Abriel H. Régulation des canaux ioniques cardiaques par les ubiquitine-ligases appartenant à la famille Nedd4, GRRC-French Apprentie: Society of Cardiology Meeting, Toulouse, France - Sophie Mermoud 18 May 2006, Abriel H., Ubiquitylation of cardiac ion channels, Etudiants: Molecular function of ion channels Meeting, Copenhagen, Danemark - Romina Behar - Alessandro Zordan 23 June 2006, Abriel H., Cardiac sodium channel Nav1.5 is part of a multiprotein complex composed of syntrophins and dystrophin: Stagiaires: - Saïd ElHaou biochemical and functional studies, University of Berne, Department of - Katarzyna Krystanek Physiology

27 June 2006, Abriel H., Régulation du canal sodique cardiaque Nav1.5 par son ubiquitylation et le complexe de la dystrophine. Search for a position in Physiology, Faculty of Medicine, University of Sherbrooke,

Quebec 14 September 2006, Abriel H., Cardiac sodium channel Nav1.5 is part of Key words a multiprotein complex composed of syntrophins and dystrophin: sodium channel, potassium biochemical and functional studies, Academic Medical Center, channel, hERG channel, Amsterdam, Netherlands cardiac electrophysiology, congenital long QT syndrome, 5 October 2006, Abriel H., Drug-Induced Long QT Syndrome: an Brugada syndrome Introduction to the Problem, Fall meeting of the Swiss Society of Pharmacology, CHUV, Lausanne 23 November 2006, Abriel H., Molecular and biophysical characterization of mutation-induced ion channel dysfunction. Training Day in Cardiogenetics of the Swiss Cardiovascular Network of Research and Education, University of Basel 14 December 2006, Abriel H., Ubiquitylation of cardiac voltage-gated ion channels. University of Berne, Department of Biochemistry and molecular medicine 8 March 2007, ABRIEL H., Regulation of the cardiac sodium channel Nav1.5 by the dystrophin-multiprotein complex and its ubiquitylation. University of Geneva, Division of Cardiology

Page 41 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

13 March 2007, Abriel H., KCNQ1 Potassium channel is down-regulated by ubiquitylating enzymes of the Nedd4/Nedd4-like family, USGEB meeting, Basel 8 June 2007, Abriel H., Regulation of voltage-gated cardiac channels by Nedd4-like ubiquitin ligases, Hamburg, The ubiquitin-proteasome system in cardiovascular disease meeting 22 June 2007, Abriel H., Regulation of the cardiac sodium channel: recent insights from mouse models, Nantes, Denis Escande Symposium, Cardiovascular diseases 21 September 2007, Abriel H., Genetic and molecular bases of cardiac arrhythmias, Zagreb, Croatian Society of Cardiology 6 October 2007, Abriel H., Mineralocorticoid receptor is essential for aldosterone-induced up-regulation of Ca-current in cardiac myocytes, 6th international meeting on ENaC and Aldosterone, Zermatt 4 December 2007, Abriel H., Modulation of the cardiac sodium channel by associated proteins, 13th World Congress of Pacing and Electrophysiology, Rome 22 octobre 2008, Abriel H., Fonction et régulation du canal sodique dans les pathologies rythmiques. Atelier d’expertise rythmique, Paris

Honors and Awards

2008 Séverine Petitprez, Prix Novartis au 14th Cardiovascular Biology and Clinical Implication Meeting, octobre 2008

Page 42 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Neuronal ion channels and G-protein coupled Receptors

Adrenergic receptors: molecular mechanisms of receptor function and physiological implications

Projects Our research activity concerns the molecular mechanisms underlying the function and regulation of the adrenergic receptor subtypes in vitro and in vivo. The adrenergic receptors (ARs) mediate the effects of adrenaline and noradrenaline in several organs including heart, vessels, brain and liver. They belong to the family of the heptahelical G protein-coupled receptors which transduce a large number of signals across the cell membrane and are the targets for the majority of clinically used drugs. Susanna Cotecchia The AR family includes nine gene products divided in three groups, three alpha1 (alpha1a, alpha1b and alpha1d), three alpha2 (alpha2A, alpha2B Susanna Cotecchia graduated and alpha2C) and three beta (beta1, beta2 and beta3). Our investigation is from medical school in 1979 predominatly focussed on the alpha1-AR subtypes coupled to the and qualified in neurology at the University of Bari, Italy. Gq/phospholipase C signaling pathway. The main research projects After this training, she worked ongoing in our laboratory are the following: as researcher in neuropharmacology at the A) Molecular basis of receptor function and regulation "Mario Negri" Institute of Pharmacological Research in Combining site-directed mutagenesis and molecular modelling of Milano (1980-1984). In 1984, receptors and G proteins, we investigate the molecular basis of receptor she moved to the laboratory of activation and G protein coupling. We have generated a large number of Dr. R.J. Lefkowitz at Duke either inactive or constitutively active receptor mutants and studied their University, Durham, NC, USA, where she spent eight theoretical structures by molecular dynamics analysis (in collaboration years working, initially, as with Dr. F. Fanelli, Italy). Another area of investigation concerns the post-doctoral fellow and, molecular mechanisms involved in receptor endocytosis and recycling. afterwards, as assistant Using a proteomic approach we have identified two novel proteins professor. In September 1992, she became full professor at interacting with the alpha1-AR subtypes, AP50 and ezrin, which play a the Department of role in internalization and recycling of the alpha1b-AR, respectively. Pharmacology and Toxicology Beyind its role in receptor processing, ezrin might play a role in receptor- of the University of Lausanne. mediated signaling. She also acted as vice-dean of the Faculty of Medecine B) Molecular basis of drug action and selectivity. (2000-2003) and President of the Basic Sciences Section of We investigate the pharmacological behaviour, i.e. agonism, inverse the Faculty of Biology and agonism and neutral antagonism, of a large number of drugs at different Medecine (2003-2006). Her main scientific adrenergic receptor subtypes. Our goal is to elucidate the mechanisms of contribution was the cloning action of adrenergic drugs at a molecular level as well as to provide of the different alpha1- further insight into their action in vivo. adrenergic receptor subtypes and the investigation of the C) Study of the physiological role of the alpha1-AR subtypes using knock structure-function relationship out mice. We have created a knock out mouse model lacking the alpha1b- of G protein coupled receptors, constitutive activity AR. The knock out mice display changes in their blood pressure response, and drug action on these in glucose metabolism as well as in some behavioural parameters. A receptors. number of collaborations have been established with groups of the University of Lausanne as well as with other institutions to further explore the phenotype of the alpha1b-AR knock out mice and of those crossed with knock out mice lacking other AR subtypes.

Page 43 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Membres du groupe Collaborations (période 2006-2008) Dr Francesca Fanelli and Prof. P.G. DeBenedetti, Universitá di Modena, Technicienne de laboratoire : - Liliane Abuin Italy

Postdoc: Dr Tommaso Costa, Istituto Superiore di Sanitá, Rome, Italy - Aline Appert-Collin Bennasroune Prof. Martin Lohse, Department of Pharmacology, University of Wurzburg, Germany Chargée de recherche: - Stanasila Vollmer

Hôte sabbatique: Selected references - Gabriella Garruti

Stanasila L, Abuin L, Diviani D, Cotecchia S. Direct interaction of ezrin with the alpha1b-adrenergic receptor regulates recycling of the internalized receptors. J Biol Chem. (2006) 281, 4354-63

Cotecchia S, Constitutive activity and inverse agonism at the alpha1adrenoceptors. Biochem Pharmacol. (2007) 73, 1076-83

Faber JE, Szymeczek CL, Cotecchia S, Thomas SA, Tanoue A, Tsujimoto G, Zhang H. Alpha1-adrenoceptor-dependent vascular hypertrophy and remodeling in murine hypoxic pulmonary hypertension. Key words Am J Physiol, Heart Circ Physiol. (2007) 292, H2316-23 Receptors - G proteins - Desensitization - Molecular Hosoda C, Hiroyama M, Sanbe A, Birumachi J, Kitamura T, Cotecchia S, dynamics - Inverse agonism - Simpson PC, Tsujimoto G, Tanoue A. Blockade of both alpha1A- and Constitutive activity- knock alpha1B-adrenergic receptor subtype signaling is required to inhibit out mice neointimal formation in the mouse femoral artery. Am J Physiol, Heart Circ Physiol. (2007) H514-9 Appert-Collin A, Cotecchia S, Nenniger-Tosato M, Pedrazzini T, Diviani D. The A-kinase anchoring protein (AKAP)-Lbc-signaling complex mediates alpha1 adrenergic receptor-induced cardiomyocyte hypertrophy. Proc Natl Acad Sci U S A. (2007) 104, 10140-5 Sanbe A, Tanaka Y, Fujiwara Y, Tsumura H, Yamauchi J, Cotecchia S, Koike K, Tsujimoto G, Tanoue A. Alpha1-adrenoceptors are required for normal male sexual function. Br J Pharmacol. (2007) 152, 332-40 Garruti G, Giusti V, Nussberger J, Darimont C, Verdumo C, Amstutz C, Puglisi F, Giorgino F, Giorgino R, Cotecchia S. Expression and secretion of the atrial natriuretic peptide in human adipose tissue and preadipocytes. Obesity (2007) 15, 2181-9 Stanasila L, Abuin L, Dey J, Cotecchia S. Different internalization properties of the alpha1a and alpha1b-adrenergic receptor subytpes: the potential role of receptor interaction with beta-arrestins and AP50. Mol Pharmacol. (2008) 74, 562-573 Garruti G, Cotecchia S, Giampetruzzi F, Giorgino F, Giorgino R. Neuroendocrine deregulation of food intake, adipose tissue and the gastrointestinal system in obesity and metabolic syndrome. J. Gastrointestin Liver Dis. (2008) 17, 193-198

Page 44 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Lectures as invited speaker July 2006, Pharmacology of Adrenoceptors 3rd annual ASPET Symposium, of the IUPHAR, World Congress of Pharmacology, Beijing, China January 2007 (co-chair), Gordon Conference "Molecular Pharmacology", Ventura, CA, USA

Page 45 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Acid-sensing ion channels (ASICs) : function in sensory neurons and structure-function relationship

ASICs are novel members of the amiloride-sensitive Na channel/degenerin family of ion channels. They are activated by protons, but not by any known neurotransmitter or by voltage. ASICs are expressed in the central nervous system and in sensory neurons. All ASICs respond with a rapidly activating and subsequently desensitizing (inactivating) current to extracellular acidification. There is increasing Stephan Kellenberger evidence for roles of ASICs of the CNS in memory functions, fear conditioning and cell death during ischemic conditions. ASICs in the Stephan Kellenberger received peripheral nervous system have been shown to be involved in pain his PhD in 1994 from the sensation. Our laboratory investigates ASIC function on the cellular and University of Bern for research on GABAA receptor molecular level. function performed with Erwin Sigel. After his post- Aim 1 Regulation of ASIC function by proteases doctoral training with W.A. Catterall at the University of The ASICs themselves are targets of various regulatory mechanisms. We Washington, Seattle, he joined have identified as novel modulators serine proteases such as trypsin. the Department of Pharmacology of the UNIL in Extracellular trypsin cleaves ASIC1a in the extracellular loop and thereby 1997. He has been leading his shifts the pH dependence of channel activation and inactivation to more own research team since 2001. acidic pH. This regulation may adapt the ASIC1a pH dependence to situations in which the extracellular pH is constitutively lowered, as e.g. ischemia. In addition, the trypsin-modified ASIC1a channel shows a reduced permeability towards divalent cations. Regulation of ASIC1a by trypsin involves channel cleavage. Mutagenesis experiments showed that trypsin cleaves ASIC1a in the N-terminal part of the extracellular loop, between a highly conserved domain and a channel domain that is critical for ASIC1a inhibition by the venom of the spider Psalmopoeus cambridgei. This channel portion undergoes conformational changes during channel inactivation. Ongoing experiments aim at determining the physiological relevance of ASIC regulation by serine proteases.

Aim 2 Function of ASICs in nociceptive neurons The peripheral nerve endings of nociceptive neurons contain different pH-dependent ion channels, such as ASICs, P2X receptors and the capsaicin receptor TRPV1. pH changes occur during ischemia, inflammation and increased cellular metabolism. To determine whether in sensory neurons there is evidence for a role of ASICs in pH sensing, we have analyzed ASIC current properties in small-diameter DRG neurons. We detected ASIC currents in ~70% of DRG neurons of small diameter. ASIC currents in small-diameter DRG neurons were all preferentially expressed in putative nociceptors, as judged by co-expression of the capsaicin receptor TRPV1 and from the action potential shape. We distinguished three different types of ASIC currents with different pH dependencies. In small-diameter neurons, the current induced by acidification to pH 6 mediated by TRPV1 was in most neurons much smaller than the ASIC-mediated pH 6-induced current. Within small- diameter DRG neurons about half of the neurons contain neuropeptides such as CGRP and SP and depend on nerve growth factor for survival.

Page 46 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

We observed a much higher probability of finding ASIC currents in Membres du groupe peptidergic as compared to non-peptidergic neurons. Type 1 ASIC (période 2006-2008) currents were 7 times more frequent in peptidergic neurons, suggesting a Doctorants: possible involvement in peptidergic functions. In the second part of this - Benoîte Bargeton - Aurélien Boillat project we have tested in collaboration with Isabelle Décosterd - Maxime Blanchard (Département de Biologie Cellulaire et de Morphologie, Université de Lausanne) whether ASIC function is affected by a model of nerve injury, Postdocs: the spared nerve injury, SNI. The SNI model consists in a section and - Olivier Poirot ligation of two of the three sciatic nerve branches, the common peroneal Etudiants: and tibial nerves, leaving the sural nerve intact. Pain hypersensitivity - Thierry Bouduban (mechanical and thermal allodynia and hyperalgesia) develops in the - Delphine Jacot-Descombes territory of the non-injured nerve. We observed a complex regulation of - Luz Angelica Liechti ASIC subunit expression and function in DRG neurons of injured and Stagiaires: non-injured nerves leading to an overall decrease in ASIC currents in - Ariane Gloor injured and adjacent non-inured neurons, suggesting a reorganization of - Pauline Riond ASICs during the development of neuropathic pain, that may contribute - Aurélie Durret to the development or maintenance of neuropathic pain.

Aim 3 Functional changes of voltage-gated Na channels after

nerve injury

In a project with Isabelle Décosterd we have characterized the consequences of nerve injury (using the SNI model) on the function of voltage-gated Na channels (Navs) in DRG neurons. The changes of Nav expression in SNI had so far been well documented by biochemical methods, however, this was the first analysis of Nav current changes in

SNI. The functional analysis showed changes in the voltage-dependant properties of tetrodotoxin (TTX)-sensitive and –resistant Nav currents, Key words and a substantial decrease of TTX-resistant Nav current densities in the Acid-sensing ion channel, injured neurons after SNI. neuronal signaling, action potential, sensory neuron, channel gating, channel regulation, structure-function relationship, proteases Collaborations

PD Dr. Isabelle Decosterd, Dpt. de biologie cellulaire et morphologie, Université de Lausanne, Lausanne, Suisse Prof. H. Vogel and Dr. Ruud Hovius, Laboratory of physical chemistry of polymers and membranes, EPFL, Lausanne, Suisse Prof. Edward Farmer, Département de biologie moléculaire végétale, Universtié de Lausanne, Lausanne, Suisse Prof. Olivier Staub, Département de Pharmacologie et de Toxicologie, Université de Lausanne, Lausanne, Suisse Prof. L. Schild, Département de Pharmacologie et de Toxicologie, Université de Lausanne, Lausanne, Suisse

Selected references

Ruffieux-Daidie D, Poirot O, Boulkroun S, Verrey F, Kellenberger S, Staub O., Deubiquitylation regulates activation and proteolytic cleavage of ENaC. J Am Soc Nephrol (2008) 19(11): 2170-2180

Page 47 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Boulkroun S, Ruffieux-Daidie D, Vitagliano J J, Poirot O, Charles R P, Lagnaz D, Firsov D, Kellenberger S, Staub O. Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3. Am J Physiol-Renal Physiol (2008) 295(4):F889-F900

Cardozo A K, Buchillier V, Mathieu M, Chen J, Ortis F, Ladriere L, Allaman-Pillet N, Poirot O, Kellenberger S, Beckmann JS, Eizirik DL, Bonny C, Maurer F. Cell-permeable peptides induce dose- and length- dependent cytotoxic effects. Biochim Biophys Acta (2007) 1768(9): 2222-2234

Berta T, Poirot O, Pertin M, Ji R R, Kellenberger S, Decosterd I Transcriptional and functional profiles of voltage-gated Na+ channels in injured and non-injured DRG neurons in the SNI model of neuropathic pain. Mol Cell Neurosci (2008) 37(2):196-208

Poirot O, Berta T, Decosterd I, Kellenberger S. Distinct ASIC currents are expressed in rat putative nociceptors and are modulated by nerve injury. J Physiol (2006) 576:215-234

Pfister Y, Gautschi I, Takeda A-N, van Bemmelen M, Kellenberger S, Schild L A gating mutation in the internal pore of ASIC1a. J Biol Chem (2006) 281.11787-11791

Vukicevic M, Weder G, Boillat A, Boesch A, Kellenberger S Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating. J Biol Chem (2006) 281: 714-722

Book chapter Kellenberger S (2008) Epithelial sodium and acid-sensing ion channels. In: Sensing with ion channels, Editor Boris Martinac, Springer Verlag, Berlin Heidelberg

Lectures as invited speaker

16.3.06, Institute of Biochemistry and Molecular Medicine, University of Bern: "Acid-sensing ion channels: modulators in the nervous system?" 20.4.06, Medical Faculty, University of Bern: "Molecular analysis of ion conduction and gating of epithelial sodium and acid-sensing ion channels" 30.6.06, Department of Pharmacology and Toxicology, University of Lausanne (“Leçon d’épreuve” for a position of MER) "Acid-sensing ion channels: from molecular mechanisms to cellular functions" 7. 3.07, Novartis, Basel, "Regulation of acid-sensing ion channels (ASICs) by serine proteases” 5. 6. 07, Physiologisches Institut, Würzburg, Germany, “Acid-sensing ion channels: Modulators in the nervous system?"

Page 48 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Chemosensory systems: Ion channels and receptors involved in odor and pheromone transductions

Aim 1 "Cyclic Nucleotide-Gated Channels: Multiple isoforms, multiple roles" Cyclic nucleotide-gated (CNG) channels are non selective cation channels first identified in retinal photoreceptors and olfactory neurons. They are opened by the direct binding of the cyclic nucleotides cAMP and cGMP. The function of CNG channels has been established in retinal photoreceptors (rods and cones) and in main olfactory neurons where six different genes encoding four A subunits (A1 to A4) and two B subunits Marie-Christine (B1 and B3) give rise to three different ion channels. Through the direct Broillet gating of nitric oxide, we have been able to show that CNGA4 subunits can form functional homomeric channels in olfactory neurons. CNG Marie-Christine Broillet channels gated by NO or by cyclic nucleotides are highly permeable to received her PhD in 1993 from the University of calcium. They are good candidates for transducing chemosignals received Lausanne for research on renal by olfactory sensory neurons at different stages in their development. K channels performed with Prof. Jean-Daniel Horisberger. In 1998, After a post-doctoral Our laboratory investigates the roles of CNG channels in the development training with Prof. Stuart and the chemosensory properties of olfactory neurons focusing on the Firestein at Columbia vomeronasal organ, an olfactory subsystem which is responsible for University (New York) where pheromone recognition. She studied olfactory cyclic nucleotide-gated channels, She joined the Department of Aim 2 “The mouse Grueneberg ganglion” Pharmacology and Toxicology of the UNIL as a START Studies using gene-targeted mice have revealed the presence of multiple fellow to create and lead her olfactory subsystems. Indeed, in mammals, the reception of olfactory cues own research group working seems to be primarily mediated by chemosensory neurons localized in on ion channels and distinct nasal compartments: the main olfactory epithelium, the membrane receptors in olfactory neurons. vomeronasal organ and the septal organ. Based on its expression in mature sensory cells of these compartments, the olfactory marker protein (OMP) is generally considered as indicative of mature olfactory sensory neurons. In mice, OMP-positive neurons have also been identified recently in the so-called Grueneberg ganglion, a ganglion present at the

tip of the nasal cavity, found in different mammalian species including humans. Recently, we have morphologically characterized this ganglion using electron microscopy and showed that GG neurons bear primary cilia and have their cell bodies ensheathed by glial cells. We also demonstrated that alarm pheromones evoked calcium responses in GG neurons in vitro and induced freezing behavior in vivo, which completely disappeared when the GG was deleted by axotomy. We concluded that mice detect alarm pheromones that give intraspecies information about the presence of a danger through the activation of olfactory GG neurons. Our laboratory tries to identify the molecular pathway(s) of mouse alarm pheromone detection focusing on the electrophysiological characterization of the Grueneberg ganglion cells. We also try to determine the chemical nature of mouse alarm pheromones.

Page 49 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Collaborations

Membres du groupe Dr. I. Rodriguez, Dep. of Zoology and Animal Biology, University of (période 2006-2008) Geneva, Switzerland Technicien de laboratoire: - Olivier Randin Dr Rolf Gruetter, Centre d'Imagerie BioMedicale, University of Lausanne, Switzerland Doctorants: - Julien Brechbühl Dr Christian Giroud, University of Lausanne, Switzerland - Sarah Vollery Dr Frank Sporkert, University of Lausanne, Switzerland Chargé de recherche: - Vincent Bize

Etudiants: - Magali Klaey - Tesa Menendez - Aline Pichon - Gaëlle Tschopp - Julien Brechbühl - Nathalie Droz (UNIGE)

Stagiaire: - Aurélie Comand Expression of the cyclic nucleotide-gated channel (CNGA2) (red) in the cilia of mouse olfactory neurons (green)

Key words

Olfaction, pheromone Selected references communication, ion channels, GPCR, imaging, electrophysiology, behavioral Roppolo D, Vollery S, Kan C D, Lüscher C, Broillet M-C, Rodriguez I. analysis, microscopy. Gene cluster lock after pheromone receptor choice. EMBO J. (2007) 26(14) :3423-30 Brechbühl J, Klaey M, Broillet M-C. Grueneberg ganglion cells mediate alarm pheromone detection in mice. Science (2008) 321(5892):1092-5

Book chapter Broillet, M-C. (2008). Olfactory Cyclic Nucleotide-Gated Ion Channels. In : The Senses: A Comprehensive Reference - Volume 4 - Olfaction & Taste. Elsevier Inc. 4(29) 511-526

Mouse olfactory cilia stained with

Acetylated-tubulin (purple)

Page 50 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Lectures as invited speaker

December 1st 2008, “The skin and the nose” Beiersdorf AG, Hamburg, Germany December 8th2008, “The mouse Grueneberg ganglion, a danger detector”, Séminaires de Neurosciences Fondamentales, Dept. des Neurosciences, University of Geneva, Geneva December 19th 2008, “Le danger par le bout du nez”. Cérémonie de remises des Bachelors en Biologie 2008, Lausanne Chairperson: July 1-4th2008, 12th Weurman Falvour Research Symposium. Interlaken, Switzerland, (Symposium 1: Biology)

Abstracts

Brechbühl J., Klaey M., Broillet M-C., Deletion of the CNGA4 protein affects the turnover of mouse vomeronasal neurons. Swiss Society for Neuroscience, Poster A16, 03 (2007). Brechbühl J., Klaey M., Monin A., Broillet M-C., Neuronal renewal in the mouse vomeronasal organ: a role for CNGA4. Changins NEUROSCIENCES, Poster NEURO - 55, 11 (2007).

Honors and Awards

2006 Prix de Faculté de l’Université de Lausanne : recipient Julien Brechbühl, group Marie-Christine Broillet, for his master thesis work. 2008 Prix de Faculté de l’Université de Lausanne : recipient Magali Klaey, group Marie-Christine Broillet, for her master thesis work.

Calcium imaging experiments performed on HEK cells expressing a pheromone receptor

Page 51 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Publications

Publications

Andreasen D, Vuagniaux G, Fowler-Jaeger N, Hummler E, Rossier BC. Activation of epithelial sodium channels by mouse channel activating proteases (mCAP) expressed in Xenopus oocytes requires catalytic activity of mCAP3 and mCAP2 but not mCAP1. J Am Soc Nephrol. 2006 Apr;17(4):968-76. Epub 2006 Mar 8.

Appert-Collin A, Cotecchia S, Nenniger-Tosato M, Pedrazzini T, Diviani D., 2007. The A-kinase anchoring protein (AKAP)-Lbc-signaling complex mediates alpha1 adrenergic receptor-induced cardiomyocyte hypertrophy. Proc Natl Acad Sci U S A. 104, 10140-5. APPERT-COLLIN A., BAISAMY L., DIVIANI D. (2006) Regulation of G protein coupled receptor signaling by A kinase anchoring proteins (review). J. Recept. Signal Transduct. Res. 26: 631-646. Berta T, Poirot O, Pertin M, Ji RR, Kellenberger S, Decosterd I (2008) Transcriptional and functional profiles of voltage-gated Na+ channels in injured and non-injured DRG neurons in the SNI model of neuropathic pain. Mol Cell Neurosci 37(2):196-208. Bertog, M., Cuffe, J.E., Pradervand, S., Hummler, E., Hartner, A., Porst, M., Hilgers, K.F., Rossier, B.C. and C. Korbmacher. Aldosterone responsiveness of the epithelial sodium channel (ENaC) in colon is increased in a mouse model for Liddle’s syndrome. J. Physiol. 2008, 586: 459-475. Bibert S, Roy S, Schaer D, Horisberger J-D and Geering K. Phosphorylation of Phospholemman (FXYD1) by Protein Kinases A and C Modulates Distinct Na,K-ATPase Isozymes. J Biol Chem 283: 476-486, 2008. Bibert, S., Aebischer, D., Desgranges, F., Roy, S., Schaer, D., Kharoubi-Hess, S., Horisberger, J.-D. and Geering, K. (2008) A link between FXYD3 (Mat-8)-mediated Na,K-ATPase regulation and differentiation of Caco-2 intestinal epithelial cells. Mol. Biol. Cell, in press. Bibert, S., Roy, S., Schaer, D., Felley-Bosco, E. and Geering, K. (2006). Structural and functional properties of two human FXYD3 (MAT-8) isoforms. J. Biol. Chem. 281, 39142-39151. Bize V and Horisberger J-D. Sodium self-inhibition of human epithelial sodium channel: selectivity and affinity of the extracellular sodium sensing site. American Journal of Physiology-Renal Physiology 293: F1137-F1146, 2007. Bochud M, Eap CB, Elston RC, Bovet P, Maillard M, Schild L, Shamlaye C and Burnier M. Association of CYP3A5 genotypes with blood pressure and renal function in African families. J Hypertens 24: 923-929, 2006.

BOIXEL C.*, GAVILLET B.*, ROUGIER J.-S. and ABRIEL H. (2006) Aldosterone Increases the Voltage- Gated Sodium Current in Ventricular Cardiomyocytes. Am J Physiol Heart; Jun;290(6):H2257-66. Published with accompanying editorial. Bonny O, Burnier M. Treatment of secondary hyperparathyroidism in renal insufficiency: role of calcitriol, sevelamer and cinacalcet. Rev Med Suisse. 2008 Mar 5;4(147):589-92, 594-5. Bonny, O. and M. Burnier. 2008. Place de la vitamine D, du sevelamer et du cinacalcet dans la prise en charge des patients en insuffisance rénale. Revue Médicale Suisse. 147: 589-595. Bonny, O., Rubin, A., Huang, C.L., Frawley, W.H., Pak, C.Y.C. and O.W. Moe. 2008. Regulation of urinary calcium excretion by urinary magnesium and pH. J Am Soc Nephrol. 19(8):1530-1537. Boulkroun., D. Ruffieux-Daidié, J.-J. Vitagliano, O. Poirot, D. Lagnaz, R.P. Charles, D. Firsov, S. Kellenberger, and O. Staub. Vasopressin inducible ubiquitin specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3. Am.J.Physiol./Renal Physiol.. 295:F889-900, 2008.

Brechbühl J., Klaey M., Broillet M.-C. (2008). Grueneberg ganglion cells mediate alarm pheromone detection in mice. Science 321(5892):1092-5. Bruce, M.C., V. Kanelis, F. Fouladkou, A. Debonneville, O. Staub and D. Rotin. Regulation of Nedd4-2 self- ubiquitylation and stability by a PY motif located within its HECT-domain. Biochemical J. 415:155-63, 2008.

CAMACHO, J. A., HENSELLEK, S., ROUGIER, J.-S., BLECHSCHMIDT, S., ABRIEL, H., BENNDORF, K., and ZIMMER, T. (2006). Modulation of Nav1.5 channel function by an alternative spliced sequence in the DII/DIII linker region. The Journal of Biological Chemistry, Apr 7;281(14):9498-506. Capendeguy O, Chodanowski P, Michielin O and Horisberger J-D. Access of extracellular cations to their binding sites in Na,K ATPase: Role of the 2nd extracellular loop of the a subunit. J Gen Physiol 127: 341-352, 2006.

Page 52 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Capendeguy O, Iwaszkiewicz J, Michielin O and Horisberger J-D. The 4th extracellular loop of the a subunit of Na,K-ATPase: Functional evidence for close proximity with the 2nd extracellular loop. J Biol Chem 283: 27850- 27858, 2008. Cardozo AK, Buchillier V, Mathieu M, Chen J, Ortis F, Ladriere L, Allaman-Pillet N, Poirot O, Kellenberger S, Beckmann JS, Eizirik DL, Bonny C, Maurer F (2007) Cell-permeable peptides induce dose- and length- dependent cytotoxic effects. Biochim Biophys Acta 1768(9):2222-2234. Carnally,S.M., H.S.Dev, A.P.Stewart, N.P.Barrera, M.X.van Bemmelen, L.Schild, R.M.Henderson, and J.M.Edwardson. 2008. Direct visualization of the trimeric structure of the ASIC1a channel, using AFM imaging. Biochem. Biophys. Res. Commun. 372:752-755 CARNEGIE G.K., SOUGHAYER S., PEDROJA B.S., SMITH F.D., ZHANG F., DIVIANI D., BRISTOW M.R., KUNKEL M.T., NEWTON A.C., LANGEBERG L.K., SCOTT J.D. (2008) AKAP-Lbc mobilizes a hypertrophic signaling pathway in cardiomyocytes. Mol. Cell. 32: 169-179. Charles, R.-P., Guitard, M., Leyvraz, C., Breiden, B., Haftek, M., Haftek-Terreau, Z., Stehle, J.-C., Sandhoff, K. and E. Hummler, Postnatal requirement of the epithelial sodium channel for maintenance of epidermal barrier function. J. Biol. Chem. 2008, 283: 2622-2630. Cotecchia , 2007. Constitutive activity and inverse agonism at the alpha1adrenoceptors. S. Biochem Pharmacol. 73, 1076-83. Dahan E, Bize V, Lehnert T, Horisberger J-D and Gijs MAM. Integrated microsystem for non-invasive electrophysiological measurements on Xenopus oocytes. Biosensors & Bioelectronics 22: 3196-3202, 2007. Delprat, B., Puel, J.-L., Geering, K. (2007). Dynamic expression in the inner ear suggests a role of the protein in endolymph homeostasis and neuronal activity. Dev. Dyn. 236, 2534-2540. Delprat, B., Schaer, D., Roy, S., Wang, J., Puel, J.-L. and Geering, K. (2007). FXYD6 is a novel regulator of Na,K-ATPase expressed in the inner ear. J. Biol. Chem. 282, 7450-7456. DIVIANI D. (2008) Regulation of cardiac function by A-kinase anchoring proteins (review). Curr. Opin. Pharmacol 8: 166-173. DIVIANI D., BAISAMY L., APPERT-COLLIN A. (2006) AKAP-Lbc: a molecular scaffold for the integration of cAMP and Rho transduction pathways. (review) Eur. J. Cell Biol., 85: 603-10. DOMENIGHETTI A.A., BOIXEL C., CEFAI D., ABRIEL H., and PEDRAZZINI T. (2007) Cardiac angiotensin

II overproduction leads to an acquired long QT syndrome associated with IK1 downregulation and action potential prolongation in hypertrophic cardiomyocytes. Journal of molecular and cellular cardiology, Jan;42(1):63-70.

EAP, C. B.#, CRETTOL, S., ROUGIER, J.-S., SCHLAEPFER, J., SINTRA GRILO, L., DEGLON, J.-J., BESSON, J., CROQUETTE-KROKKAR, M., CARRUPT, P.-A., and ABRIEL, H.# (#both corresponding authors) (2007). Stereoselective block of hERG channel by (S)-methadone and QT interval prolongation in CYP2B6 slow metabolizers. Clinical Pharmacology and Therapeutics, May;81(5):719-28. Faber JE, Szymeczek CL, Cotecchia S, Thomas SA, Tanoue A, Tsujimoto G, Zhang H., 2007. Alpha1- adrenoceptor-dependent vascular hypertrophy and remodeling in murine hypoxic pulmonary hypertension. Am J Physiol, Heart Circ Physiol. 292, H2316-23. Fakitsas,P., G. Adam, D. Daidié, D, M.X., van Bemmelen, F. Fouladkou, A. Patrignani, U. Wagner, R. Warth, O. Staub, and F. Verrey. Early aldosterone-regulated gene product controls epithelial Na+ channel by deubiquitylation. J.Am.Soc.Nephrol. 18:1084-1092, 2007 (IF07: 7.11). FG Riepe MX Bemmelen, F. Cachat, H.Plendl, I.Gautschi, N.Krone, P.M.Holterhus, G.Theintz, and L.Schild. 2008. Revealing a subclinical salt-loosing phenotype in heterozygous carriers of the novel S562P mutation in the alpha subunit of the epithelial sodium channel. Clin Endocrinol (Oxf). 2009 Feb;70(2):252-8 FODSTAD H., BENDAHHOU S., ROUGIER J.-S., LAITINEN P., BARHANIN J., ABRIEL H., SCHILD L., KONTULA K., and SWAN H. (2006) Genetics of long QT syndrome in Finland: molecular characterization of two founder mutations and identification of compound heterozygous patients. Annals of Medicine, 38:4, 294- 304. Garruti G, Cotecchia S, Giampetruzzi F, Giorgino F, Giorgino R. , 2008. Neuroendocrine deregulation of food intake, adipose tissue and the gastrointestinal system in obesity and metabolic syndrome. J Gastrointestin Liver Dis. 17, 193-198. Garruti G, Giusti V, Nussberger J, Darimont C, Verdumo C, Amstutz C, Puglisi F, Giorgino F, Giorgino R, Cotecchia S, 2007. Expression and secretion of the atrial natriuretic peptide in human adipose tissue and preadipocytes. Obesity, 15, 2181-9.

GAVILLET, B., ROUGIER, J.-S., DOMENIGHETTI, A. A., BEHAR, R., BOIXEL, C., RUCHAT, P., LEHR, H.-A., PEDRAZZINI, T., and ABRIEL, H. (2006). Cardiac Sodium Channel Nav1.5 is Regulated by a Multiprotein Complex Composed of Syntrophins and Dystrophin. Circulation Research Aug 18; 99(4):407-14.

Gorokhova, S., Bibert, S., Geering, K. and Heintz, N. (2007) A novel family of transmembrane proteins interacting with b subunits of the Na,K-ATPase. Hum. Mol. Genet. 16, 3394-3410.

Page 53 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Guennoun-Lehmann S, Fonseca JE, Horisberger J-D and Rakowski RF. Palytoxin acts on Na+ ,K+ -ATPase but not nongastric H+,K+-ATPase. J Membrane Biol 216: 107-116, 2007. Hagens, O., Ballabio, A., Kalscheuer, V., Kraehenbuhl, J.P., Schiaffino, M.V., Smith, P., Staub O., Hildebrand, J., and Wallingford, J.B. A new standard nomenclature for proteins related to Apx and Shroom. BMC Cell Biol 7:18, 2006 (IF06: 2.74). Harris M, Firsov D, Vuagniaux G, Stutts MJ, Rossier BC. A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes. . J Biol Chem. 2007 Jan 5;282(1):58-64. Harris M, Garcia-Caballero A, Stutts MJ, Firsov D, Rossier BC. Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus oocytes: role of furin-mediated endogenous proteolysis. J Biol Chem. 2008 Mar 21;283(12):7455-63 Hosoda C, Hiroyama M, Sanbe A, Birumachi J, Kitamura T, Cotecchia S, Simpson PC, Tsujimoto G, Tanoue A., 2007. Blockade of both alpha1A- and alpha1B-adrenergic receptor subtype signaling is required to inhibit neointimal formation in the mouse femoral artery. Am J Physiol, Heart Circ Physiol H514-9. Jespersen T, Gavillet B, van Bemmelen M.X, Cordonier S, Thomas M.A, Staub O, and Abriel H. Cardiac sodium channel Na(v)1.5 interacts with and is regulated by the protein tyrosine phosphatase PTPH1. Biochem Biophys Res Commun 348: 1455-1462, 2006 (IF06: 2.86). Jespersen,T., Membrez,M., Nicolas,C., Pitard,B., Staub,O., Olesen,S.P., and Abriel, H., The KCNQ1 channel is downregulated by ubiquitylating enzymes of the Nedd4/Nedd4-like family. Cardiovas.Res. 74: 64-74, 2007 (IF07: 6.13). Kovacikova J, Winter C, Loffing-Cueni D, Loffing J, Finberg KE, Lifton RP, Hummler E, Rossier B, Wagner CA. The connecting tubule is the main site of the furosemide-induced urinary acidification by the vacuolar H+- ATPase. Kidney Int. 2006 Nov;70(10):1706-16. Epub 2006 Sep 20. KRANJCEC, D., BERGOVEC, M., ROUGIER, J.-S., RAGUZ, M., PAVLOVIC, S., JESPERSEN, T., CASTELLA, V., KELLER, D.I., and ABRIEL, H. (2007). Brugada syndrome unmasked by accidental inhalation of gasoline vapors. PACE, 30:1294-1298. Li, C., Geering, K. and Horisberger, J.-D. (2006). The third sodium binding site of Na,K-ATPase is functionally linked to acidic pH-activated inward current. J. Membr. Biol. 213, 1-9. Loffing-Cueni,D., S.Y. Flores, D. Sauter, D. Daidié, N.Siegrist, P. Meneton, O. Staub, and J. Loffing. Dietary Na+ intake regulates the expression of the ubiquitin-protein ligase Nedd4-2 in the renal collecting system. J.Am.Soc.Nephrol. 17:1264-1274, 2006 (IF06: 7.37). Membrez M, Hummler E, Beermann F, Haefliger JA, Savioz R, Pedrazzini T, Thorens B. GLUT8 is dispensable for embryonic development but influences hippocampal neurogenesis and heart function. Mol Cell Biol. 2006 Jun;26(11):4268-76. Mérillat AM, Charles R-P, Porret A, Maillard M, Rossier BC, Beermann F, Hummler E. Conditional gene targeting of the ENaC subunit genes Scnn1b and Scnn1g. Am. J. Physiol. Renal Physiol. 2008, in press. Muller O, Firsov D, Seydoux C. Rev Med Suisse. 2007 May 30 [Aldosterone receptor antagonists] ;3(113):1401- 4. Review. French. Muller O, Pradervand S, Berger S, Centeno G, Milet A, Nicod P, Pedrazzini T, Tronche F, Schütz G, Chien K, Rossier BC, Firsov D. Identification of corticosteroid-regulated genes in cardiomyocytes by serial analysis of gene expression. Genomics. 2007 Mar;89(3):370-7. O. Bonny. 2008. The Epithelial Sodium Channel, Chapter 2 in Sodium in Health and Diseases, edited by M. Burnier, published by Informa Healthcare USA Inc., New York. pp 27-65. Oskarsson T, Essers MA, Dubois N, Offner S, Dubey C, Roger C, Metzger D, Chambon P, Hummler E, Beard P, Trumpp A. Skin epidermis lacking the c-Myc gene is resistant to Ras-driven tumorigenesis but can reacquire sensitivity upon additional loss of the p21Cip1 gene. Genes Dev. 2006 Aug 1;20(15):2024-9. Pasch A., Frey, F.J., Eisenberger, U., Mohaupt, M.G. and O. Bonny. 2008. PTH and 1,25-vitamin D response to a low calcium diet is associated with bone mineral density in renal stone formers. Nephro Dial Transplant. 23(8):2563-2570. Pestov, N. B., Ahmad, N., Korneenko, T. V., Zhao, H., Radkov, R., Schaer, D., Roy, S., Bibert, S., Geering, K., Modyanov. N. N. (2007). Evolution of Na,K-ATPase bm-subunit into a coregulator of transcription in placental mammals. Proc. Natl. Acad. Sci. USA 104, 11215-11220. PETITPREZ, S.*, JESPERSEN, T.*, PRUVOT, E., KELLER, D.I., CORBAZ, C., SCHLAEPFER, J., ABRIEL, H.#, and KUCERA, J.P.# (#both corresponding authors) (2008). Analyses of a Novel SCN5A Mutation (C1850S): Conduction vs. Repolarization Disorder Hypotheses in the Brugada Syndrome. Cardiovascular Research, 78 :494-504. PETITPREZ, S.*, TIAB, L.*, CHEN, L., KAPPELLER, L., ROSLER, K.M., SCHORDERET, D.F., ABRIEL, H.#, and BURGUNDER, J.-M.# (#both corresponding authors) (2008). A novel dominant mutation of the Nav1.4 alpha-subunit domain I leading to sodium channel myotonia. In press Neurology.

Page 54 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Pfister Y, Gautschi I, Takeda A-N, van Bemmelen M, Kellenberger S, Schild L (2006) A gating mutation in the internal pore of ASIC1a. J Biol Chem 281.11787-11791. Poirot O, Berta T, Decosterd I, Kellenberger S (2006) Distinct ASIC currents are expressed in rat putative nociceptors and are modulated by nerve injury. J Physiol 576:215-234. Porret, A., Mérillat, A.M., Guichard, S., Beermann, F. and E. Hummler, Tissue-specific transgenic and knockout mice. Methods Mol. Biol. 2006, 337: 185-205. Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus oocytes: role of furin-mediated endogenous proteolysis. Harris M, Garcia-Caballero A, Stutts MJ, Firsov D, Rossier BC. J Biol Chem. 2008 Mar 21;283(12):7455-63. Radkov R, Kharoubi-Hess S, Schaer D, Modyanov NN, Geering K and Horisberger J-D. Role of homolgous ASP334 and GLU319 in human non-gastric H,K- and Na,K-ATPase in cardiac glycoside binding. Biochem Biophys Res Commun 356: 142-146, 2007. Randrianarison N, Clerici C, Ferreira C, Fontayne A, Pradervand S, Fowler-Jaeger N, Hummler E, Rossier BC, Planès C. Low expression of the beta-ENaC subunit impairs lung fluid clearance in the mouse. Am J Physiol Lung Cell Mol Physiol. 2008 Mar;294(3):L409-16. Epub 2007 Nov 16. Randrianarison N, Escoubet B, Ferreira C, Fontayne A, Fowler-Jaeger N, Clerici C, Hummler E, Rossier BC, Planès C. beta-Liddle mutation of the epithelial sodium channel increases alveolar fluid clearance and reduces the severity of hydrostatic pulmonary oedema in mice. J Physiol. 2007 Jul 15;582(Pt 2):777-88. Epub 2007 Apr 12. Roppolo D., Vollery S., Kan C. D., Lüscher C., Broillet, M.-C., Rodriguez, I. (2007). Gene cluster lock after pheromone receptor choice. EMBO J. 26(14) :3423-30 Rossi,E., E. Farnetti, A. Debonneville, D. Nicoli, C. Grasselli, G. Regolisti, A. Negro, F. Perazzoli, F. Mantero, O. Staub. Liddle's syndrome caused by a novel missense mutation (P617L) of the epithelial sodium channel b subunit. J.Hypertension 26:921-927, 2008 Rotin,D. and L.Schild. 2008. ENaC and its regulatory proteins as drug targets for blood pressure control. Curr. Drug Targets. 9:709-716. Rougier JS, Muller O, Berger S, Centeno G, Schütz G, Firsov D, Abriel H. Mineralocorticoid receptor is essential for corticosteroid-induced up-regulation of L-type calcium currents in cultured neonatal cardiomyocytes. Pflugers Arch. 2008 May;456(2):407-12. Rubera I, Hummler E, Beermann F. Transgenic mice and their impact on kidney reseach. Pflügers Arch. Europ. J. Physiol. 2008, in press. Ruffieux-Daidié, D., O. Poirot, S. Boulkroun, F. Verrey, S. Kellenberger, and O. Staub. Deubiquitylation regulates proteolytic cleavage and activation of the epithelial Na+ channel ENaC. J.Am.Soc.Nephrol. 19(11):2170-80, 2008 Sanbe A, Tanaka Y, Fujiwara Y, Tsumura H, Yamauchi J, Cotecchia S, Koike K, Tsujimoto G, Tanoue A, 2007. Alpha1-adrenoceptors are required for normal male sexual function. Br J Pharmacol. 152, 332-40 Shi,P.P., X.R. Cao, E. Sweezer, T.S. Kinney, N. Williams, R.F. Husted, R. Nair, R.A. Williamson, C.D. Sigmund, P.M. Snyder, O. Staub, J.B. Stokes, B. Yang. Salt-Sensitive Hypertension and Cardiac Hypertrophy in Mice Deficient in the Ubiquitin Ligase Nedd4-2. Am.J.Physiol./Renal Physiol. 295:F462-70, 2008 Stanasila L, Abuin L, Dey J, Cotecchia S., 2008. Different internalization properties of the alpha1a and alpha1b- adrenergic receptor subytpes: the potential role of receptor interaction with beta-arrestins and AP50. Mol Pharmacol. 74, 562-573. Stanasila L, Abuin L, Diviani D, Cotecchia , 2006. Direct interaction of ezrin with the alpha1b-adrenergic receptor regulates recycling of the internalized receptors. S. J Biol Chem. 281, 4354-63. Suzuki, Y., Pasch, A., Bonny, O., Mohaupt, M.G., Hediger, M.A. and F.J. Frey. 2008. Gain of function haplotype in the epithelial calcium channel TRPV6 is a risk factor for renal calcium stone formation. Human Molecular Genetics. 17(11):1613-1618. Takeda,A.N., I.Gautschi, M.X.van Bemmelen, and L.Schild. 2007. Cadmium trapping in an epithelial sodium channel pore mutant. J. Biol. Chem. 282:31928-31936. TFELT-HANSEN,J., JESPERSEN, T., HOFMAN-BANG, J., RASMUSSEN, H. B., CEDERGREEN, P., SKOVBY, F., ABRIEL, H., SVENDSEN, J. H., OLESEN, S. P., CHRISTIANSEN, M., and HAUNSO, S. (2008). Ventricular tachycardia in a Brugada syndrome patient caused by a novel deletion in SCN5A. Canadian Journal of Cardiology. Vukicevic M, Weder G, Boillat A, Boesch A, Kellenberger S (2006) Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating. J Biol Chem 281 :714-722. Zuber AM, Singer D, Penninger JM, Rossier BC, Firsov D. Increased renal responsiveness to vasopressin and enhanced V2 receptor signaling in RGS2-/- mice. J Am Soc Nephrol. 2007 Jun;18(6):1672-8. Epub 2007 May 2.

Page 55 of 56 Rapport d’activités 2006-2008 Département de Pharmacologie et de Toxicologie

Books, book chapters, reviews

ABRIEL H. (2007) Review - Cardiac Sodium Channel Nav1.5 and its Associated Proteins. Archives des maladies du coeur et des vaisseaux, 100(9) :787-793.

ABRIEL H. (2007) Roles and regulation of the cardiac sodium channel Nav1.5: Recent insights from experimental studies. Cardiovascular Research, 76:381-389.

ABRIEL H. (2008) Voltage-Gated Sodium Channel Nav1.5. AfCS-Nature Molecule Pages, www.signaling- gateway.org, doi:10.1038/mp.a001614.01 Abriel H. and Staub O. Regulation of ion channels by ubiquitylation. Physiology 20:398-407, 2006 (IF06: 6.27)

ABRIEL H., and DIZON J. (2006) Brugada syndrome. eMedicine pages, www.emedicine.com.

Andreasen D, Vuagniaux G, Fowler-Jaeger N, Hummler E, Rossier BC. .Activation of epithelial sodium channels by mouse channel activating proteases (mCAP) expressed in Xenopus oocytes requires catalytic activity of mCAP3 and mCAP2 but not mCAP1. J Am Soc Nephrol. 2006 Apr;17(4):968-76. Epub 2006 Mar 8. Broillet, M-C. (2008). Olfactory Cyclic Nucleotide-Gated Ion Channels. In : The Senses: A Comprehensive Reference - Volume 4 - Olfaction & Taste. Elsevier Inc. 4(29) 511-526 CRETTOL S., SCHLAEPFER J., ABRIEL H., and EAP C.P. (2008) Response to “Methadone induced QTc prolongation: Is it due to stereoselective block of hERG or to inappropriate QT interval correction”, Clinical Pharmacology and Therapeutics, 83(5):672. Gonzalez-Rodriguez E, Gaeggeler HP, Rossier BC. IGF-1 vs insulin: respective roles in modulating sodium transport via the PI-3 kinase/Sgk1 pathway in a cortical collecting duct cell line. Kidney Int. 2007 Jan;71(2):116- 25. Epub 2006 Dec 13. Harris M, Firsov D, Vuagniaux G, Stutts MJ, Rossier BC.Harris M, Firsov D, Vuagniaux G, Stutts MJ, Rossier BC. A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes. J Biol Chem. 2007 Jan 5;282(1):58-64. Epub 2006 Nov 7. Harris M, Garcia-Caballero A, Stutts MJ, Firsov D, Rossier BC. Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus oocytes: role of furin-mediated endogenous proteolysis. J Biol Chem. 2008 Mar 21;283(12):7455-63. Epub 2008 Jan 14. Horisberger J-D and Doucet A. Renal Ion-Translocating ATPases: The P-type Family. In: Seldin and Giebisch's The Kidney, edited by Alpern RJ and Hebert SC. Amsterdam: Elsevier, 2007, p. 57-90. Horisberger J-D and Geering K. Brain Na,K-ATPase. In: The New Encyclopedia of Neuroscience, edited by Squires L. Elsevier, 2008. Horisberger J-D. Mécanisme du transport des cations Na+ et K+ par la pompe à sodium. Médecine/Sciences 22: 27-28, 2006. Kellenberger S (2008) Epithelial sodium and acid-sensing ion channels. In: Sensing with ion channels, Editor Boris Martinac, Springer Verlag, Berlin Heidelberg Loffing, J., S.Y. Flores and O. Staub. Epithelial Transport regulation by Sgk. Annual Review of Physiology 68:461-490, 2006 (IF06: 15.36) PETITPREZ S., and ABRIEL H. (2008) Editorial – Crosstalk between cardiac ion channel subunits: An encrypted language to be deciphered, Journal of molecular and cellular cardiology, 45(3):333-5. PETITPREZ S., and ABRIEL H. (2008) Letter to the Editor - Effects of heart failure on brain-type Na+ channels in rabbit ventricular myocytes, Europace, 10(2):257. Rossier BC, Schild L. Epithelial sodium channel: mendelian versus essential hypertension. Hypertension. 2008 Oct;52(4):595-600. Rossier BC, Stutts MJ. Activation of the Epithelial Sodium Channel (ENaC) by Serine Proteases. Annu Rev Physiol. 2008 Oct 17. [Epub ahead of print] Scheffner,M. and O. Staub, HECT E3 and human disease, BMC Biochemistry 8 Suppl 1:S6, 2007. Scheffner,M. and O. Staub. Hect ubiquitin-protein ligases in human disease. In: The ubiquitin-proteasome system and disease, Protein degradation 4 (Chapter 4). Mayer R.J., Ciechanover A., and Rechsteiner,M. (eds.), Wiley-VCH Verlag GmbH & Co., Weinheim. pp. 77-105, 2008. SINTRA GRILO, L., and ABRIEL H. (2008) Perspectives – Male and female equality: still far from goal, Journal of Physiology, 586(12):2825. Staub,O., and Rotin D. The role of ubiquitylation in membrane transport. Physiol.Rev. 86:669-707, 2006 (IF06: 31.44). Verrey,F., P. Fakitsas, G. Adam, and O.Staub, Early transcriptional control of ENaC:(de)ubiquitylation by aldosterone. Kidney International 19:298-309, 2008 (IF07: 4.92).

Page 56 of 56