UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA Calcium-activated Chloride Channels in Cystic Fibrosis JOANA RAQUEL DELGADO MARTINS DOUTORAMENTO EM BIOQUÍMICA (Especialidade: Genética Molecular) 2011 UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA Calcium-activated Chloride Channels in Cystic Fibrosis JOANA RAQUEL DELGADO MARTINS Tese co-orientada pela Prof. Doutora Margarida D. Amaral e pelo Prof. Doutor Karl Kunzelmann DOUTORAMENTO EM BIOQUÍMICA (Especialidade: Genética Molecular) 2011 Joana Raquel Delgado Martins foi bolseira de doutoramento da Fundação para a Ciência e Tecnologia do Ministério da Ciência, Tecnologia e Ensino Superior SFRH / BD / 28663 / 2006 De acordo com o disposto no artigo 40° do Regulamento de Estudos Pós-Graduados da Universidade de Lisboa, Deliberação n°961/2003, publicada no Diário da República – IIa Série, n° 153 de 5 de Julho de 2003, foram incluídos nesta tese resultados dos artigos abaixo indicados: Ousingsawat, J., Martins, J. R., Schreiber, R., Rock, J. R., Harfe, B. D., Kunzelmann, K. Loss of TMEM16A causes a defect in epithelial Ca2+- dependent chloride transport. The Journal of Biological Chemistry 284, 28698- 703 (2009). Martins, J. R., Kongsuphol, P., Almaça, J., AlDehni, F., Clarke, L., Schreiber, R., Amaral, M.D., Kunzelmann, K. F508del-CFTR increases the intracellular Ca2+ signalling that causes enhanced calcium-dependent Cl- conductance in cystic fibrosis (2010) (Submitted to AJRCMB) No cumprimento do disposto na referida deliberação, a autora esclarece serem da sua inteira responsabilidade, excepto quando referido em contrário, a execução das experiências que permitiram a elaboração dos resultados apresentados assim como a interpretação e discussão dos mesmos. Os resultados obtidos por outros autores foram incluídos com autorização dos mesmos para facilitar a compreensão dos trabalhos e estão assinalados nas respectivas figuras. Outros artigos publicados em revistas internacionais contendo resultados obtidos durante o doutoramento: Spitzner, M., Martins, J. R., Barro Soria, R., Ousingsawat, J., Scheidt, K., Schreiber, R., and Kunzelmann, K. Eag1 and Bestrophin 1 are up-regulated in fast-growing colonic cancer cells. The Journal of Biological Chemistry 283, 7421-8 (2008). Aldehni, F., Spitzner, M., Martins, J. R., Schreiber, R. and Kunzelmann, K. Bestrophin 1 promotes epithelial-to-mesenchymal transition of renal collecting duct cells. Journal of the American Society of Nephrology : JASN 20, 1556-64 (2009). Schreiber, R., Uliyakina, I., Kongsuphol, P., Warth, R., Mirza, M., Martins, J. R. and Kunzelmann K. Expression and function of epithelial anoctamins. The Journal of Biological Chemistry 285, 7838-45 (2010). Kunzelmann, K.; Kongsuphol, P.; Chootip, K.; Toledo, C.; Martins, J. R.; Almaca, J.; Tian, Y.; Witzgall, R.; Ousingsawat, J.; Schreiber, R. Role of the Ca2+-activated Cl- channels bestrophin and anoctamin in epithelial cells. Biological chemistry 392, 125-34 (2011). Preface PREFACE Cystic Fibrosis (CF) is a life-threatening genetic disorder that primarily affects Caucasians. The disease is dominated by chronic bacterial infections resulting from the excessive build-up of a thick mucus in the respiratory system. Progressive loss of lung function and its ultimate failure is the main cause of mortality. Other symptoms include pancreatic insufficiency, male infertility and high sweat electrolytes. Life expectancy of patients suffering from CF is about 37 years of age. In the late 1980s the joint efforts of three independent laboratories unveiled that the gene coding for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel was the cause of CF when dysfunctional. CFTR activity as a Cl- channel is crucial for proper function and ionic transport of all epithelia, including those of the airways, intestinal tract (including pancreatic and bile ducts) as well as sweat glands and the male reproductive system. CFTR protein is a cAMP-dependent and phosphorylation-regulated chloride (Cl-) channel which, in healthy tissues, is present in the apical membrane of epithelial cells. In the majority of CF patients CFTR bears a mutation (F508del) that causes its intracellular retention at the endoplasmic reticulum (ER), thus preventing it from reaching the plasma membrane of epithelial cells. As there is up to now no cure for the disease, most current therapies aim to alleviate and minimize CF symptoms. However, several drug-discovery efforts have been attempted to find pharmacological agents capable of correcting F508del-CFTR intracellular mislocation and potentiating the function of rescued mutant channels. Although CFTR main function is undoubtedly to transport Cl- ions, this channel is also a key player in the physiology of epithelial tissues. Indeed, the complex regulation of ionic transport in tissues affected by CF compromises other players of which CFTR has been shown to be a key regulator. Nevertheless, alternatively to the cAMP second messenger pathway i Preface that leads to CFTR activation, Ca2+ is also able to trigger a generally transient Cl- conductance in a variety of tissues. It is believed that by stimulating alternative pathways that enable Cl- secretion, and are thus capable of replacing at least this aspect of CFTR function in epithelia, the basic CF defect might be overcome. In this regard, Ca2+-activated Cl- channels (CaCCs) appear as obvious targets to be stimulated. Moreover, it has been repeatedly reported that in tissues where CFTR is absent or defective, an enhanced Ca2+- dependent Cl- secretion is observed, prompting the hypothesis that the regulation of the two pathways might not be independent. Regardless that the Ca2+-dependent Cl- conductance has been well-known for more than twenty years, the molecular identity of CaCC has been considerably controversial. Multiple proteins have been suggested to account for the Ca2+-dependent Cl- 2+ currents (ICaCC) triggered upon rise in the intracellular Ca concentration 2+ ([Ca ]i) but a consistent candidate has just recently been found. The objective of this work was to investigate the role and contribution of two proteins described as CaCCs that could be potentially activated in a CF scenario. When this doctoral work started, significant experimental data pointed to bestrophin 1 as the major CaCC candidate, but during its course ANO1 was definitely identified as the major CaCC. The focus of the present work was thus both bestrophin 1 (Best 1) and anoctamin 1 (Ano 1 or TMEM16A). The impact of these proteins in the homeostasis and function of epithelial tissues known to be affected by CF was assessed here through biochemical and electrophysiological techniques. The observations found in this study contribute to a better understanding of how CaCC activity modulates (and is also modulated) in CF. It also gives new perspectives in using alternative approaches to overcome the CF basic defect (by the so-called "bypass therapies ") to the ultimate benefit of CF patients. ii Acknowledgments/Agradecimentos ACKNOWLEDGMENTS/AGRADECIMENTOS À Professora Margarida Amaral, por me ter dado a excelente oportunidade de trabalhar no seu laboratório. Agradeço a supervisão e o entusiasmo sempre presentes, que foram essenciais na realização deste trabalho. I would like to thank Karl Kunzelmann for accepting me in his group in Regensburg and for introducing me to the fascinating field of physiology when I first arrived in 2006. I am grateful for his support and guidance, as well as for his inspiring passion for science. Ao ministério da Ciência e Ensino Superior e à Fundação para a Ciência e a Tecnologia, por terem possibilitado a realização deste trabalho, através da concessão da bolsa de doutoramento de que fui recipiente. A number of scientists have contributed to this work in so many ways. To Eva Sammels and Jason Rock for essential work. Thank you for making this thesis more meaningful. A special thanks to Barbara Reich, for her kindness and patience while helping me with experiments. A PhD is a challenging test of determination and persistence, and I have been privileged enough to be surrounded by exceptional people, both in Lisbon and Regensburg. I thank everyone in the CF lab in Lisbon: à Marta, por toda a tua alegria e pela confiança em mim. Marisa, sempre uma fonte de energia e boa disposição. Filipa, Anabela e Ana Carina por todo o apoio e pela referência que foram principalmente no início do meu doutoramento. Toby for introducing me to protein purification, Luka and Shehrazade for your contributions to this work and for your friendship. Joana, Inna, Luisa Alessio e Mário pelas pessoas extraordinárias que são. André, (Prof.) Carlos, Carla, Simão e Francisco um muito obrigada! iii Acknowledgments/Agradecimentos To everyone I met in the in Regensburg: Brigitte, Julia, Agnes, Tini, Tina, Rene, Nesty, Melanie and Myriam, thank you for your priceless help and friendship. To Ji and Fadi, for helping me with a number of experiments and for making science even more fun! Aim and Diana, with whom I shared so many moments of my life, thank you for your friendship and for being there for me (also for the invaluable contributions to this work). I am grateful to Prof. Rainer Schreiber, for sharing his vast knowledge and for all his help and patience. I am very grateful to everyone who accepted reading and who helped me revising this thesis. A David, simplemente por todo. Gracias por el apoyo, sugestiones y el tiempo dedicado a esta tesis. A Miriam, por la ayuda