Osmoregulation of chloride channels in epithelial cells Osmoregulatie van chloride kanalen in epitheekeUen Chien-hua (Christina) lim Cover: with the permission of the photographer ian Yang (Surform) Design: Legatron Electronic Publishing, Rotterdam Printed by PrintPartners lpskamp B.V., Enschede (www.ppi.nl) ISBN: 978·90·9023577·6 All rights reserved. No part of this thesis may be reproduced in any form by print, photoprint, microfilm or any other means without written permission of the rightful claimant(s). This restriction concerns the entire publication or any part of it. Osmoregulation of chloride channels in epithelial cells Osmoregulatie van chloride kanalen in epitheelcellen Proefschrift ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof.dr. S.W.J. Lamberts en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op vrijdag 12 december 2008 om 09:00 uur door Chien-hua (Christina) lim geboren te Taipei, Taiwan Promotiecommissie Promotor: Prof.dr. C. P. Verrijzer Overige led en: Prof.dr. J.A. Grootegoed Prof.dr. A.H.J. Danser Prof.dr. J.C. de Jongste Copromotoren: Dr. H.R. de Jonge Dr. B.C. Tilly Arnoldus Christiaan Breedveld (1950-2008) This thesis is dedicated to My parents and my brothers, {or always being there {or me &: Arnoldus Christiaan Breedveld (1950-2008), for believing in me. Contents Abbreviations 9 Chapter 1 Introduction 15 Chapter 2 Osmosignalling and Volume Regulation in Intestinal 35 Epithelial Cells Methods in Enzymology, 2007, 428: 325-342 Chapter 3 Regulation of the Cell Swelling-Activated Chloride 57 Conductance by Cholesterol-Rich Membrane Domains Acta Physiologica (Oxf), 2006, 187: 295-303 Chapter 4 Cholesterol Depletion and Genistein as Tools to 75 Promote F508delCFTR Retention at the Plasma Membrane Cellular Physiology and Biochemistry, 2007, 20: 473-482 Chapter 5 Osmotic Cell Swelling-Induced Recruitment of 93 Volume-Regulated Anion Channels to the Plasma Membrane Manuscript in preparation Chapter 6 Expression of Putative Chloride Channel Proteins 111 in Intestine 407 Cells Chapter 7 Aquaporins are Required for the Hypotonic Activation of 121 Osmolyte Release Pathways in Intestine 407 Cells Manuscript under revision Chapter 8 General Discussion 141 Summary 151 Samenvatting 155 Dankwoord 159 In memoriam 163 Acknowledgement and curriculum vitae (Chinese) 165 Publications 167 Appendix for color figures 169 Abbreviations Abbreviations AA arachidonic acids ABA hormonal abscisic acid ABCA1 ATP-binding cassette protein A1 ABP actin-binding protein AP-2 associated adaptin AQP aquaporins ATP adenosine 5' -triphosphate BAPTA-AM 1, 2 -bis(2 -aminophenoxy)ethane-N, N, N', N'-tetraacetic acid-acetoxymethyl ester cAMP cyclic adenosine 3':5'-monophosphoric acid CD 2 -hyd roxypropyl-j) -cyclodextri n eDNA complementary DNA CF cystic fibrosis CFTR cystic fibrosis transmembrane conductance regulator CPAE cells calf pulmonary artery endothelial cells DAG 1,2 diacylglycerol DIDS 4,4' -di-isothiocyanatostilbene-2, 2' -disulfonic acid DMEM Dulbecco's modified Eagle's medium DMSO dimethylsulfonyloxide DN dominant negative DPC diphenylamine-2-carboxylate ECL enhanced chemiluminescence ECM extracellular matrix EGF epidermal growth factor ENaC Epithelial Na• channel ER endoplasmic reticulum ERK extracellular signal-regulated protein kinase F-actin filamentous actin FAK focal adhesion kinase F508delCFTR deletion of phenylalanine at amino acid position 508 of CFTR FCS fetal calf serum FITC fluorescein isothiocyanate GAP GTPase activating protein GDI GDP dissociation inhibitor 10 Abbreviations GEF guanine nucleotide exchange factor GFP green fluorescent protein G protein guanine nucleotide-binding regulatory protein GTP guanosine 5' -triphosphate JH hydrogen isotope 3/ tritium HDL high-density lipoproteins he pes N- 2 -hydroxy-ethylpi perazine-N'-2 -ethane-sulfonic acid Hog1p high osmolarity glycerol response protein HRP horseradish peroxidase 1251 iodium isotope 125 IP inositol 1 ,4,5-triphosphate 3 K+ swell stretch-activated K+ channels and swelling activated K+ channels Kv voltage-gated K+ channels LDL low density lipoproteins MAP kinase mitogen-activated protein kinase Maxi-K+ Ca2+-dependent intermediate K+ channel MDCK cells Madin-Darby canine kidney cells MEK mitogen-activated I ERK-activating kinase MLCK myosin light chain kinase MMTS methylmethanethio-sulphonate Mscl mechanosensitive channel NEM N-ethylmaleimide NPC Niemann Pick C1 protein NPPB 5-nitro- 2-( 3 -phenyl-propylami no)-benzoate NSF N-ethylmaleimide-sensitive factor PAGE polyacrylamide gel electrophorese PBS phosphate-buffered saline PC-LUVs phosphatidylcholine large unilaminar vesicles PI-PLCp phosphatidylinositol-specific phospholipase cp PKA cAMP-dependent protein kinase PKC protein kinase C PLA phospholipase A Ptdlns-3K phosphatidyl inositol 3 kinase 86Rb rubidium isotope 86 RT-PCR reverse transcriptase polymerase chain reaction 11 Abbreviations RVD regulatory volume decrease RVI regulatory volume increase SD standard deviation SDS sodium dodecyl sulphate SEM standard error of mean SFK SRC family kinases SFK SiRNA small interfering RNA SITS 4-acetamido-4' -isothiocyanostilbene SNAP soluble NSF attachment protein SNARE soluble N-ethylmaleimide-sensitive factor attachment protein receptor TMAO trimethylamine N-oxide TKR tyrosine kinase receptor TRPC transient receptor potential channels t-SNARE target membrane SNARE TIBS Tris-Tween-buffered saline VAMP2 vesicle-associated protein on the v-SNARE VRAC volume regulated anion channels v-SNARE vesicle SNARE wt wild-type 12 Abbreviations List of chemical compounds and their action 2 ABA Increases the [Ca +] 1and activates the K+ channels Apyrase Nonspecific adenosine tri/biphosphatase (ATPase) that converts ATP into AMP and Pi BAPTA-AM A cell permeable calcium chelator Brefeldin A An inhibitor of vesicle transport between the ER and the Golgi. It disturbes intracellular membrane flow and Golgi function C. botulinium Zinc endopeptidase causing specific cleavage of vesicle neurotoxin F associated SNARE protein VAMP/synaptobrevin C. botulinium Enzyme that inactivates Rho A, B and C (not Rho D) through exoenzyme C3 ADP-ribosylation cytochalasin B Disrupts F-actin microfilaments DIDS o--channel blocker DPC o--channel blocker Herbimycin A Tyrosine kinases inhibitor KT5926 A potent and selective inhibitor of MLCK MMTS A sulfhydryl reagent methylmethanethio-sulphonate that inhibit AQP NEM Binds to the SNARE protein SNAP-25 and prevents vesicle fusion NPPB o--channel blocker SITS o--channel blocker Tamoxifen P-glycoprotein inhibitor and anion channel (VRAC) blocker TMAO Counteracts the protein denaturing effect of urea Vanadate Phosphotyrosine phosphatase inhibitor Wortmannin A specific inhibitor of phosphatidylinositol-3-kinase 13 Chapter 1 Introduction Water movement across the plasma membrane The plasma membrane of mammalian cells is formed by two layers of lipids (lipid bilayer), primarily phospholipids, glycolipids and cholesterol, in which many different proteins are embedded. Phospholipid consists of a glycerol backbone esterified to fatty acids (the "lipid tail") and, via a phosphate group, to either choline, serine, inositol or ethanolamine (the "head group"). Whereas the head group is hydrophilic and oriented towards the outer surface of the membrane, the lipid tail is hydrophobic and pointed towards the inner part. The plasma membrane is impermeable to large molecules like carbohydrates and proteins but readily permeable to small uncharged molecules like oxygen, water, and carbon dioxide. Molecules can move through the membrane either by direct diffusion or through specialized channels or transport proteins (facilitated diffusion). In an isolated system, as stated by the Second Law of Thermodynamics, all events move spontaneously from a higher energy state to a lower energy state and are driven by the tendency to increase the entropy (degree for randomness/ disorder) [1]. When molecules are evenly distributed throughout the available space, the entropy is at its maximum. Therefore, free moving molecules and atoms (i.e. not part of a crystal structure and not restrained by additional forces) tend to distribute themselves over an as large as possible area. As a consequence of the Second Law of Thermodynamics, differences in the concentrations of non-permeable solutes between the cell and its surrounding medium will result in a redistribution of the solvent (e.g. water) to maintain the lowest energy possible, a phenomenon known as osmosis. Therefore, in response to an osmotic imbalance, water will move across a semi-permeable membrane until the water molecules are equally distributed, resulting in swelling or shrinkage [2]. The Gibbs-Don nan effect refers to the observation that under certain conditions charged molecules or ions fail to distribute evenly across a semi-permeable membrane. At the Gibbs-Donnan equilibrium, the total number of positive charges balanced the total number of negative charges (bulk electroneutrality). Due to the presence of charged membrane-impermeable macromolecules and the electrogenic Na+ /K+ pump, however, the Gibbs Donnan equilibrium will never be obtained in an intact cell, leading to an asymmetric distribution of permeable ions and the generation of the membrane potential [3]. 16 Introduction A consequence of the presence of negatively charged membrane-impermeable proteins is the constant tendency of cells to accumulate water. To counteract a potential increase in volume,