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Molecular Identification and Electrophysiological Characterization of the Volume-Regulated Anion Channel VRAC

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Molecular Identification and Electrophysiological Characterization of the Volume-Regulated Anion Channel VRAC Molecular identification and electrophysiological characterization of the volume-regulated anion channel VRAC Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Florian Ullrich from Jena 2016 This work was prepared from July 1, 2012 to September 16, 2016 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. 1st Reviewer: Prof. Dr. Dr. Thomas J. Jentsch Department Physiology and Pathology of Ion Transport Max-Delbrück-Centrum für Molekulare Medizin (MDC) and Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin 2nd Reviewer: Prof. Dr. Stephan J. Sigrist Institute of Biology Department of Biology, Chemistry and Pharmacy Freie Universität Berlin Date of defense: January 6, 2017 PREFACE Part of this work has been published in the following peer-reviewed journal articles: ULLRICH, F., REINCKE, S.M., VOSS, F.K., STAUBER, T., AND JENTSCH, T.J. (2016). Inactivation and Anion Selectivity of Volume-regulated Anion Channels (VRACs) Depend on C-terminal Residues of the First Extracellular Loop. J. Biol. Chem. 291, 17040–17048. DOI: http://dx.doi.org/10.1074/jbc.M116.739342 VOSS, F.K., ULLRICH, F., MÜNCH, J., LAZAROW, K., LUTTER, D., MAH, N., ANDRADE- NAVARRO, M.A., VON KRIES, J.P., STAUBER, T., AND JENTSCH, T.J. (2014). Identification of LRRC8 heteromers as an essential component of the volume- regulated anion channel VRAC. Science 344, 634–638. DOI: http://dx.doi.org/10.1126/science.1252826 PLANELLS-CASES, R., LUTTER, D., GUYADER, C., GERHARDS, N.M., ULLRICH, F., ELGER, D.A., KUCUKOSMANOGLU, A., XU, G., VOSS, F.K., REINCKE, S.M., STAUBER, T., BLOMEN, V.A., VIS, D.J., WESSELS, L.F., BRUMMELKAMP, T.R., BORST, P., ROTTENBERG, S., AND JENTSCH, T.J. (2015). Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs. EMBO J. 34, 2993–3008. DOI: http://dx.doi.org/10.15252/embj.201592409 JENTSCH, T.J., LUTTER, D., PLANELLS-CASES, R., ULLRICH, F., AND VOSS, F.K. (2016). VRAC: Molecular identification as LRRC8 heteromers with differential functions (Review). Pflügers Arch. 468, 385–393. DOI: http://dx.doi.org/10.1007/s00424-015- 1766-5 Part of the experiments shown in this work was performed in collaboration with or exclusively by Felizia K. Voss, Tobias Stauber, S. Momsen Reincke and Jonas Münch. Detailed contributions: F.K.V. and T.S. established and conducted the siRNA screen (Figure 7), cloned LRRC8 genes (Table 8), purified and validated LRRC8 antibodies (Table 10), and performed immunostainings and microscopy (Figure 9). Knockout cell lines were generated and validated in collaboration with F.K.V. and T.S. (Figure 10, Tables 4–5 and 7). F.K.V. conducted qRT-PCR experiments (Figure 14, Table 11). F.K.V., T.S. and S.M.R. generated some of the LRRC8 mutants and chimeras. J.M. and S.M.R. contributed to patch clamp experiments. The parts of section 5.2 (Methods) that describe procedures performed exclusively by F.K.V. and T.S. (sections 5.2.1.2, 5.2.1.3, and 5.2.3) are based on Voss et al. (2014) and Voss (2015). I TABLE OF CONTENTS LIST OF FIGURES .......................................................................................................... III LIST OF TABLES ............................................................................................................IV LIST OF ABBREVIATIONS ..............................................................................................V ABSTRACT ....................................................................................................................VII ZUSAMMENFASSUNG .................................................................................................VIII 1. INTRODUCTION ........................................................................................................... 1 1.1. Mechanisms of cellular volume regulation ......................................................... 1 1.2. The volume-regulated anion channel VRAC ....................................................... 3 1.2.1. Biophysical properties ....................................................................................... 3 1.2.1.1. Permeation properties ................................................................................ 5 1.2.1.2. Voltage-dependent properties .................................................................... 6 1.2.1.3. Single channel properties ........................................................................ 10 1.2.2. Pharmacological properties ............................................................................. 12 1.2.3. Mechanisms of activation and regulation ........................................................ 14 1.2.4. Physiological and pathophysiological roles ..................................................... 19 1.2.5. Attempts at the molecular identification of VRAC ............................................ 24 1.3. The LRRC8 protein family ................................................................................... 27 2. AIM OF THE WORK ................................................................................................... 31 3. RESULTS ................................................................................................................... 32 3.1. Identification of LRRC8 proteins as essential VRAC components ................. 32 3.1.1. A genome-wide screen identifies LRRC8A as likely constituent of VRAC .............................................................................................................. 32 3.1.2. siRNA knockdown and overexpression of LRRC8A reduce ICl,vol .................... 33 3.1.3. Localization and trafficking of LRRC8 proteins ................................................ 34 3.1.4. LRRC8A and at least one other LRRC8 isoform is required for ICl,vol .............. 35 3.2. Molecular determinants of differential VRAC inactivation ............................... 43 3.2.1. Kinetic differences between ICl,vol mediated by LRRC8A/C and LRRC8A/E ....................................................................................................... 43 3.2.2. ICl,vol inactivation in LRRC8 triple knockout cells .............................................. 45 3.2.3. Molecular determinants of differential ICl,vol inactivation kinetics ...................... 47 3.2.4. LRRC8A also contributes to ICl,vol inactivation kinetics ..................................... 50 3.2.5. Concurrent mutation of key residues in LRRC8s drastically alters ICl,vol .................................................................................................................. 50 II 4. DISCUSSION .............................................................................................................. 54 4.1. LRRC8A as an essential component of VRAC .................................................. 54 4.2. VRAC is formed by LRRC8 heteromers ............................................................. 57 4.3. Molecular mechanisms of VRAC inactivation and permeation ....................... 58 4.4. Selected new findings since the molecular identification of VRAC ................ 61 4.4.1 The Lrrc8a knockout mouse ............................................................................ 61 4.4.2 Excitatory amino acid release from astrocytes requires LRRC8A ................... 62 4.4.3 LRRC8D is essential for cellular blasticidin S uptake ...................................... 63 4.4.4 Specialized VRACs mediate organic osmolyte and drug transport ................. 64 4.4.5 Functional reconstitution of purified LRRC8 complexes .................................. 69 4.5. Outlook ................................................................................................................. 72 5. MATERIAL AND METHODS ...................................................................................... 77 5.1. Material ................................................................................................................. 77 5.1.1. Chemicals........................................................................................................ 77 5.1.2. Bacteria strains ................................................................................................ 77 5.1.3. Cell lines .......................................................................................................... 77 5.1.4. Sequencing and genotyping primers ............................................................... 79 5.1.5. Plasmids .......................................................................................................... 80 5.1.6. Solutions for patch clamp experiments ........................................................... 81 5.1.7. Antibodies........................................................................................................ 82 5.2. Methods ................................................................................................................ 83 5.2.1. Molecular biology techniques .......................................................................... 83 5.2.1.1. Molecular cloning ..................................................................................... 83 5.2.1.2. Genome editing with CRISPR/Cas and zinc finger nucleases ................ 84 5.2.1.3. Quantitative real-time PCR .....................................................................
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