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Anoctamin 1 - a Member of a Novel Family of Ion Channels with Extended Functions and Significance in Disease

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Anoctamin 1 - a Member of a Novel Family of Ion Channels with Extended Functions and Significance in Disease UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS Anoctamin 1 - A Member of a Novel Family of Ion Channels with Extended Functions and Significance in Disease Doutoramento em Biologia Especialidade: Biologia de Sistemas Joana Ramos Rapaz Lérias Tese orientada por: Prof. Dr. Karl Kunzelmann e Prof. Dr. Margarida D. Amaral Documento especialmente elaborado para a obtenção do grau de doutor 2018 UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS Anoctamin 1 - A Member of a Novel Family of Ion Channels with Extended Functions and Significance in Disease Doutoramento em Biologia Especialidade: Biologia de Sistemas Joana Ramos Rapaz Lérias Tese orientada por: Prof. Dr. Karl Kunzelmann e Prof. Dr. Margarida D. Amaral Júri: Presidente: ● Doutor Rui Manuel dos Santos Malhó, Professor Catedrático da Faculdade de Ciências da Universidade de Lisboa Vogais: ● Doutor Karl Kunzelmann, Kunzelmann, Professor, Faculty of Biology and Pre-Clinical Medicine da University of Regensburg, Alemanha (orientador); ● Doutor Ana Colette Pereira de Castro Osório Maurício, Professora Associada com Agregação, Instituto de Ciências Biomédicas Abel Salazar (ICBAS) da Universidade do Porto; ● Doutor Peter Jordan, Investigador Principal, Departamento de Genética Humana do Instituto Nacional de Saúde Doutor Ricardo Jorge; ● Doutora Maria da Graça Tavares Rebelo de Soveral Rodrigues, Professora Associada com Agregação, Faculdade de Farmácia da Universidade de Lisboa. Documento especialmente elaborado para a obtenção do grau de doutor Fundação para a Ciência e Tecnologia do Ministério da Educação e Ciência (FCT/ SFRH / BD / 52489 / 2014) 2018 De acordo com o disposto no artigo 24º do Regulamento de Estudos de Pós- Graduação da Universidade de Lisboa, Despacho nº 7024/2017, publicado no Diário da República – 2ª Série – nº 155 – 11 de Agosto de 2017, foram utilizados nesta dissertação resultados incluídos nos seguintes artigos: 1. Lérias JR*, Pinto MC*, Botelho HM, Awatade NT, Quaresma MC, Silva IAL, Wanitchakool P, Schreiber R, Pepperkok R, Kunzelmann K, Amaral MD (2018). A novel microscopy-based assay identifies extended synaptotagmin-1 (ESYT1) as a positive regulator of anoctamin 1 traffic. Biochim Biophys Acta 1865: 421- 431. [*Shared first authorship] 2. Lérias J*, Pinto M*, Benedetto R, Schreiber R, Amaral MD, Aureli M, Kunzelmann K (2018). Compartmentalized Crosstalk of CFTR and ANO1 (ANO1) Through EPAC and ADCY1. Cell Signal 44:10-19. [*Shared first authorship] No cumprimento do disposto da referida deliberação, a autora esclarece serem da sua responsabilidade, exceto quando referido o contrário, a execução das experiências que permitiram a elaboração dos resultados apresentados, assim como da interpretação e discussão dos mesmos. Os resultados obtidos por outros autores foram incluídos com a autorização dos mesmos para facilitar a compreensão dos trabalhos e estão assinalados nas respetivas figuras e metodologias. Index Acknowledgments .................................................................................................... iii Abbreviations ............................................................................................................. v Summary ................................................................................................................... ix Resumo ..................................................................................................................... xi I. Introduction ........................................................................................................... 1 1. Cystic Fibrosis .................................................................................................. 3 1.1. General introduction .................................................................................... 3 1.2. Pathophysiology .......................................................................................... 3 1.3. Molecular basis of disease and mutation-specific therapies ........................ 4 2. Alternative (non-CFTR) chloride channels as potential targets for CF therapy: the anoctamin family .............................................................................. 6 2.1. The Anoctamin family: an overview ............................................................. 6 2.2. ANO1 structure and function ....................................................................... 6 2.3. ANO1 function in different organs ............................................................... 8 2.4. ANO1 and CFTR ......................................................................................... 9 2.5. “Bypass” therapies for Cystic Fibrosis ....................................................... 10 II. Objectives of the present work .......................................................................... 13 III. Materials and Methods ........................................................................................ 17 1. Production of double-tagged 3HA-ANO1-eGFP ........................................... 19 1.1. Cells and Culture conditions ...................................................................... 20 2. Biochemical and Cell Biology Techniques ................................................... 21 2.1. cDNA and siRNAs transfection ................................................................. 21 2.2. Semiquantitative Reverse-transcriptase PCR ........................................... 21 2.3. Western-blot .............................................................................................. 22 2.4. cAMP immunoassay ................................................................................. 22 2.5. Immunocytochemistry ............................................................................... 23 2.6. Chemiluminescence .................................................................................. 26 2.7. ANO1 Traffic Assay ................................................................................... 27 2.8. CLCA1 enriched media production ........................................................... 30 3. Functional assays ........................................................................................... 30 3.1. Iodide effluxes ........................................................................................... 30 3.2. Ussing Chamber assay ............................................................................. 31 3.3. Whole-cell patch-clamp ............................................................................. 31 3.4. Ca2+ measurements .................................................................................. 33 IV. Results and Discussion .................................................................................... 35 i CHAPTER 1 ........................................................................................................... 37 1. Production of stable inducible 3HA-ANO1-eGFP expressing cell lines ..... 39 2. Characterization of 3HA-ANO1-eGFP stably expressing cells lines ........... 40 2.1. Double-tagged ANO1 in HEK293T cells.................................................... 40 2.2. Double-tagged ANO1 in three novel cell lines: protein expression, tags fluorescence and channel functionality ............................................................... 42 2.3. Pilot screen and identification of hits ......................................................... 49 2.4. Validation of ESYTs and COPβ1 as ANO1 traffic regulators .................... 51 2.5. Validation of additional top hits .................................................................. 57 3. Conclusions ..................................................................................................... 63 CHAPTER 2 ........................................................................................................... 65 1. Compartmentalized crosstalk of CFTR and ANO1 through EPAC1 and ADCY1 ................................................................................................................... 67 1.1. Relationship between GPCRs and ANO1 ................................................. 67 1.2. Relationship between GPCRs and WT-/F508del-CFTR............................ 74 1.3. Role of EPAC1 and ADCY1 in ANO1 and CFTR activation and their relation to GPCRs ............................................................................................... 77 1.4 ANO1 and CFTR Golgi bypass ................................................................. 83 2. Conclusions ..................................................................................................... 85 CHAPTER 3 ........................................................................................................... 89 1. ANO1 and IL4-induced mucus production .................................................... 91 2. ANO1 and CLCA1 regulation .......................................................................... 93 3. Conclusions ..................................................................................................... 96 CHAPTER 4 ........................................................................................................... 99 1. Effect of ANO1 and ANO6 on CFTR function and expression .................. 101 2. Conclusions ................................................................................................... 106 V. General Discussion and Future Perspectives ................................................ 107 VI. Appendix – Supplementary Figures .............................................................
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