FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN Volume Regulated Channels - ICl, swell Regulation of Cell Physiology - Structure/Function Relation of TRPV4 and Chemical Agonists PhD thesis Thomas Kjær Klausen Section of Cell- and Developmental Biology Department of Biology University of Copenhagen Universitetsparken 13 2100 Copenhagen The Graduate School of Science Faculty of Science University of Copenhagen Academic advisors: Professor Else Kay Hoffmann Lektor Stine Falsig Pedersen Section of Cell- and Developmental Biology Department of Biology University of Copenhagen Universitetsparken 13 2100 Copenhagen Submitted: 02/10/09 PREFACE: I was just thinking what song to quote – from my own generation of course. Nothing from the old Beatles generation. Something that would reflect my thoughts about finishing my PhD thesis, something of intelligence, something of importance. However, nothing came up. Not that my generation haven’t generated wonderful music! We - whom are young in the 90’s and 00’s - have been blessed with some of most exciting music ever heard. I just couldn’t find anything to quote to reflect my feelings. Maybe, because I’m feeling a bit ambivalent about the project. It didn’t quite turn out to be, what I expected it to be, when I made the proposal. It didn’t generate the conclusions I was expecting and I didn’t perform as I expected myself to perform. During the last years we have left the August Krogh Institute and Hans Ussing’s Biokemisk Afdeling A for history but I’m not sure my work have completely left the influence of Krogh and Ussing. For this at least I’m very happy. I came ashore though – at least I hope so. Mostly because of my wonderful supervisors: Stine and Else. Truly the most intelligent and empathic person’s one could ever imagine. You have guided me whenever I had scientific or intellectual problems and more. You have arranged for me to visit fantastic laboratories in both Chile and Belgium at professors Andrés Stutzin and Bernd Nilius. Both of German origin, but with very different personalities. Both with a fantastic taste for science and very inspirational originality. Both in science and as persons. Definitely not everyday persons. Stine, you sometimes seems all excuses about your perfectionism. Don’t. As annoying as it may be for the rest of us to acknowledge our own imperfections it truly is necessary. I hope one day to acquire your interest for detail and your taste for true insight but am afraid it will never work out that way. Else, you have a very special talent of being both a very flamboyant person as well as being exceptionally practical. You can make things happen and this is what makes you a born leader. However it is the flamboyance that makes the everyday fun. You can always talk to everybody and always have interesting things to say. It is the latter that makes you inspiring. Actually, the last three years have not been bad at all. I started alone and now have a family with wife and kid. Hence, I sit as a happy man writing. Maybe this is the reason I cannot find the music in my collection of inexcusable noise? TAK Table of Contents Preface: ..................................................................................................................................... 2 List of Included Papers: ............................................................................................................... 5 List of Abbreviations: .................................................................................................................. 6 Thesis Objectives ........................................................................................................................ 7 Summary: .................................................................................................................................. 8 Resume ...................................................................................................................................... 9 CHAPTER I: Introduction ........................................................................................................... 10 CHAPTER II: Volume Regulated Cl- Channels: ............................................................................. 12 A Short Overview of Volume Regulated Cl- Channels ......................................................................... 12 The Volume Regulated Anion Current: .................................................................................................................... 12 Chloride Channel 3 (ClC-3): ...................................................................................................................................... 12 Chloride Channel 2 (ClC-2) ....................................................................................................................................... 13 Bestrophin 1 (Best1) ................................................................................................................................................ 13 Chloride Intracellular Channel 1 (CLIC1) .................................................................................................................. 14 TMEM16A ................................................................................................................................................................ 14 Cl- Channels in Cell Proliferation:....................................................................................................... 14 - Role of Cl in Volume Changes During Cell Cycle Progression .................................................................................. 15 - The Potential Role of Cl as a Messenger: ................................................................................................................ 16 - Cl as a pH Regulator: ............................................................................................................................................... 16 - Cl Channels as Regulators of Em: ............................................................................................................................. 17 What Channels are Important for Proliferation? ..................................................................................................... 17 Conclussion: ............................................................................................................................................................. 18 ICl, swell in Cell Migration: .................................................................................................................... 18 Signaling Mechanisms Involved in Activation of ICl, swell ....................................................................... 20 Role of ROS in Regulation of ICl,swell in Adherent Versus Suspension Cells ............................................................... 21 CHAPTER III: Structure/Function Relationship of TRPV4 and its Agonists ................................... 22 A Brief Introduction to TRPV4 ........................................................................................................... 22 The Role of Calcium in Volume Regulation: ............................................................................................................. 22 A Short Introduction to the TRP Channel Family: .................................................................................................... 22 The TRPV4 Channel and its Role in Volume Regulation: .......................................................................................... 23 TRPV4 in Physiology and Pathophysiology: ............................................................................................................. 24 Structure/Function Relationship between TRPV4 and its Chemical Agonist. ....................................... 25 A Few Notes about TRPV4 Antagonists: .................................................................................................................. 25 TRPV4 agonists and their interaction with the channel ........................................................................................... 25 Functional perspectives for TRPV4 gating ............................................................................................................... 27 Conclusion ................................................................................................................................................................ 28 References: .............................................................................................................................. 29 CHAPTER IV: Cell cycle-dependent activity of the volume- and Ca2+-activated anion currents in Ehrlich Lettre Ascites Cells ........................................................................................................ 38 CHAPTER V: Monovalent Ions Control Proliferation of Ehrlich Lettre Ascites cells ....................... 52 CHAPTER VI: H-ras transformation sensitizes volume-activated anion channels and increases migratory activity of NIH3T3 fibroblasts ................................................................................... 78 CHAPTER VII: Reactive oxygen species activate K,Cl co-transport in non-adherent Ehrlich ascites tumor cells but K+ and Cl- channels in adherent Ehrlich Lettré cells and NIH3T3 cells .................. 88 CHAPTER VIII: Modulation of the transient Receptor Potential Vanilloid 4 Channel by 4α-phorbol esters. A structural study .......................................................................................................