Molecular identification of the volume-regulated anion channel VRAC by a genome-wide siRNA screen Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) Submitted to the Department of Biology, Chemistry and Pharmacy of the Freie Universität Berlin by Felizia Katharina Voß from Heidelberg Berlin 2014 This work was prepared under the supervision of Prof. Dr. Dr. Thomas J. Jentsch at the Max-Delbrück-Centrum für Molekulare Medizin (MDC) and the Leibniz- Institut für molekulare Pharmakologie (FMP) in Berlin. 15.10.2010 – 21.10.2014 1. Gutachter: Prof. Dr. Dr. Thomas Jentsch 2. Gutachter: Prof. Dr. Volker Haucke Tag der Disputation: 25.2.2015 Preface Part of this work has been published in: Voss F.K., Ullrich F., Münch J., Lazarow K., Lutter D., Mah N., Andrade-Navarro M.A., von Kries J.P., Stauber T., Jentsch T.J. (2014). Identification of LRRC8 Heteromers as an Essential Component of the Volume- Regulated Anion Channel VRAC. Science Vol. 344 no. 6184 pp. 634-638. http://dx.doi.org/10.1126/science.1252826 Part of the experimental data shown in this work were produced by Dr. Tobias Stauber, Florian Ullrich and Jonas Münch. Dr. Tobias Stauber performed the immunostainings and microscopy for the topology determination. Florian Ullrich and Jonas Münch performed and evaluated the whole-cell patch-clamp measurements. Dr. Katina Lazarow of the FMP Screening Unit assisted in the upscaling of the YFP-assay and siRNA transfection to the high-throughput format. Dr. Nancy Mah performed the bioinformatics analysis of the primary screening data. Table of Contents List of Figures ............................................................................................................................... III List of Tables ................................................................................................................................ III List of Abbreviations .................................................................................................................... IV Abstract ........................................................................................................................................... 1 Zusammenfassung .......................................................................................................................... 2 1. Introduction ................................................................................................................................ 3 1.1 Cell volume regulation ........................................................................................................... 3 1.2 Biophysical and pharmacological properties of VRAC/ I Cl(swell) ............................................ 6 1.3 (Postulated) regulatory mechanisms of VRAC activity ......................................................... 7 1.4 Proposed (patho-) physiological importance of VRAC ....................................................... 11 1.5 Former candidates for the molecular identity of VRAC ...................................................... 15 1.6 The LRRC8 protein family .................................................................................................. 18 2. Aim of the work ........................................................................................................................ 21 3. Materials and Methods ............................................................................................................ 23 3.1 Materials ............................................................................................................................... 23 3.1.1 Chemicals ...................................................................................................................... 23 3.1.2 Cell lines........................................................................................................................ 23 3.1.3 Cell culture reagents ...................................................................................................... 23 3.1.4 Genome-wide siRNA library ........................................................................................ 23 3.1.5 Expression constructs .................................................................................................... 24 3.1.6 Commercial antibodies .................................................................................................. 24 3.1.7 Lab-generated antibodies .............................................................................................. 24 3.2 Methods ................................................................................................................................ 25 3.2.1 Generation of the HEK293-YFP Cell Line ................................................................... 25 3.2.2 Plate layout and control siRNAs ................................................................................... 26 3.2.3 High-throughput siRNA transfection ............................................................................ 26 3.2.4 YFP-based screening-assay ........................................................................................... 27 3.2.5 Bioinformatics analysis and hit assessment .................................................................. 28 3.2.6 Cloning and mutagenesis of candidate constructs ......................................................... 30 3.2.7 Electrophysiology ......................................................................................................... 31 3.2.8 Deglycosylation experiments ........................................................................................ 33 3.2.9 Co-immunoprecipitation, SDS-Page and Western blots ............................................... 33 3.2.10 Immunocytochemistry ................................................................................................. 35 3.2.11 Quantitative RT-PCR .................................................................................................. 35 I 3.2.12 Generation of monoclonal knockout cell lines using the CRISPR/Cas and zinc finger nuclease Technologies ........................................................................................................... 36 3.2.13 RVD Measurements .................................................................................................... 37 4. Results ....................................................................................................................................... 38 4.1 Assay set-up and up-scaling to high-throughput format ...................................................... 38 4.2. Prescreen ............................................................................................................................. 42 4.3 Genome-wide siRNA screen ................................................................................................ 44 4.4 Secondary screen .................................................................................................................. 47 4.5 Electrophysiological measurements of LRRC8A knockdown and overexpression ............. 49 4.6 Subcellular localization and trafficking of LRRC8 proteins ................................................ 50 4.7 Verification of the transmembrane topology of LRRC8A ................................................... 51 4.8 Heteromerization of LRRC8A with the other members of the LRRC8 protein family ....... 53 4.9 Generation of LRRC8 knockout cell lines ........................................................................... 55 4.10 Electrophysiological measurements of LRRC8 knockout cell lines .................................. 59 4.11 Inactivation kinetics in the reconstituted LRRC8 quintuple knockout .............................. 61 4.12 Differences in inactivation kinetics coincide with endogenous subunit expression .......... 62 4.13 LRRC8A as an essential component of regulatory volume decrease (RVD) .................... 63 5. Discussion .................................................................................................................................. 64 5.1 The genome-wide siRNA screen as superior approach for the identification of VRAC ..... 64 5.2 LRRC8A is essential for VRAC activity ............................................................................. 68 5.3 Heteromerization of LRRC8 proteins .................................................................................. 69 5.4 Evidence for a heteromeric LRRC8 channel complex ......................................................... 71 5.5 The molecular identity of VRAC from an evolutionary perspective ................................... 72 5.6 Transport of other osmolytes and substances through VRAC ............................................. 74 5.7 Possible regulatory sites in LRRC8 proteins ........................................................................ 78 5.8 Are we missing something? - The possibility of a limiting auxiliary subunit ...................... 79 5.9 Possible physiological significance of LRRC8 proteins ...................................................... 81 5.10 Expression pattern of LRRC8 proteins – a key to functional diversity? ............................ 85 6. References ................................................................................................................................. 87 7. Acknowledgements ................................................................................................................... 99 II List