ROLE OF Ca2+-PERMEABLE CATION CHANNELS IN Ca2+ SIGNALLING AND NECROTIC CELL DEATH by BRIAN J. WISNOSKEY Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY August 2004 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. 1 Table of Contents Table of Contents………………………………………………………………………….1 List of Tables……………………………………………………………………………...3 List of Figures……………………………………………………………………………..4 Statistical Analysis………………………………………………………………………...6 Acknowledgements………………………………………………………………………..7 List of Abbreviations……………………………………………………………………...8 Abstract…………………………………………………………………………………..10 Chapter 1: Introduction…………………………………………………………………. 12 Figures……………………………………………………………………………………31 Chapter 2: Activation of vanilloid receptor type I (TRPV1 channel) in the endoplasmic reticulum fails to activate store-operated Ca2+ entry…………………..………………...35 Introduction………………………………………………………………………………36 Materials and Methods…………………………………………………………………...38 Results..…………………………………………………………………………………..44 Discussion………………………………………………………………………………..52 Figures……………………………………………………………………………………56 2 Chapter 3: Maitotoxin-induced changes in plasmalemmal permeability in bovine aortic endothelial cells: Divalent cation specificity and selectivity…………………………….76 Introduction……………………………………………………………………………....77 Materials and Methods…………………………………………………………………...79 Results..……………………………………………………………………………….….84 Discussion…………………………………………………………………………….….93 Tables…………………………………………………………………………………….98 Figures……………………………………………………………………………………99 Chapter 4: Summary and future directions……………………………………………..124 Summary (Chapter 2)…………………………………………………………………...125 Future directions (Chapter 2)…………………………………………………………...127 Summary (Chapter 3)…………………………………………………………………...132 Future directions (Chapter 3)…………………………………………………………...134 Figures…………………………………………………………………………………..142 Bibliography……………………………………………………………………………146 3 List of Tables Table 1. Divalent cation selectivity of known Ca2+ binding domains…………………..98 4 List of Figures Chapter 1 Fig. 1.1. Possible effect of TRPV1 on store operated Ca2+ entry……………………..…32 Fig. 1.2. The maitotoxin-induced cell death cascade in bovine aortic endothelial cells...34 Chapter 2 Fig. 2.1. Time course of TRPV1 expression in Sf9 insect cells.……………..…………57 Fig. 2.2. Concentration-response relationship for TRPV1 agonists………………….....59 Fig. 2.3. Agonist-induced release of Ca2+ from internal stores in TRPV1-expressing Sf9 cells…………………………………………………………………………………..61 Fig. 2.4. Localization of TRPV1 to the Endoplasmic Reticulum…………………….…63 Fig. 2.5. Effect of thapsigargin and RTX on Ba2+ influx in control and TRPV1- expressing Sf9 cells………………………………………………………………………65 Fig. 2.6. Effect of 2-APB on thapsigargin- and RTX-induced Ba2+ influx in TRPV1-expressing Sf9 cells……………………………………………………………..67 Fig. 2.7. Overlap of RTX- and thapsigargin-sensitive Ca2+ stores………………..…….69 Fig. 2.8. Effect of 2-APB on thapsigargin-induced Ba2+ influx in absence or presence of RTX-induced store release in TRPV1-expressing Sf9 cells………......…71 Fig. 2.9. Concentration-response relationship for TRPV1 agonists in HEK cells stably expressing TRPV1……………………………………………………….….73 Fig. 2.10. Effect of 2-APB on thapsigargin- and RTX-induced Ca2+ influx in TRPV1-expressing HEK cells…………………………………………………...……75 Chapter 3 Fig. 3.1. Effect of MTX on plasmalemmal permeability………………………...…….100 5 2+ Fig. 3.2. Effect of ionomycin on the change in [Ca ]i and EB uptake in BAECs……..102 Fig. 3.3. Effect of divalent substitution on COP activation and cell lysis…………..….104 Fig. 3.4. Comparison of Ca2+ with Sr2+……………………………………………...…106 Fig. 3.5. Effect of Ca2+ replacement by Ba2+ on the MTX concentration-response curve…………………………………………………………………………….………108 Fig. 3.6. Effect of Ca2+ replacement by Ba2+ on the MTX concentration-response curve - dose response…………………………………………………………….…..…110 2+ 2+ Fig. 3.7. Effect of BAPTA-loading on MTX-induced change in [Ca ]i and [Ba ]i and the change in the associated EB uptake…………………………………………....112 Fig. 3.8. Effect of Ca2+ readmission on COP activation and cell lysis………………....114 Fig. 3.9. Simultaneous measurement of MTX-induced GFP loss and EB uptake in single BAECs………………………………………………………………….……..116 Fig. 3.10. Effect of Ba2+ on MTX-induced cell death cascade in single BAECs. ……..119 Fig. 3.11. Composite single cell fluorescence data……………………………….……121 Fig. 3.12. Average single cell responses..………………………………………….…..123 Chapter 4 2+ 2+ Fig. 4.1. Effect of Mg on MTX-induced rise in [Ca ]i and EB uptake……….……..143 Fig. 4.2. Effect of calpeptin on loss of cell associated GFP and EB uptake …………..145 6 Statistical analysis All experiments were performed at least 3 times. Unless otherwise indicated, the lines shown are mean values from 3 independent experiments. Symbols represent mean ± SE values at selected time points. Where indicated, values were compared using the paired Student t-test with p < 0.05 considered significant. 7 Acknowledgements I would like to express my sincere gratitude to my thesis advisor Dr. William Schilling, as well all of the members of the Schilling lab: Dr. Mark Estacion, Dr. Monu Goel, Dr. William Sinkins, Milana Belich, and Jabe Best. I would also like to thank the members of the Metrohealth Rammelkamep Center research staff including Dr. Eckhard Ficker, Dr. Diana Kunze, Dr. Angelina Ramirez-Navarro, Dr. David Kline, Pat Glazebrook, Maria Buniel, Kristie Takacs, Dr. Frank Sah, and Dr. Glenn Kirsch. I appreciate the knowledge and support each of you offered me as well as your friendship throughout my education. I would also like to express my thanks to the members of my dissertation committee including Dr. Tom Egelhoff, Dr. Stephen Jones, Dr. Robert Harvey, Dr. David Friel, Dr. Maureen McEnery, Dr. Michael Romero, Dr. Eric Glende and Dr. Richard Eckert for their time and guidance during the course of my education. Finally, I would like to thank the Department of Physiology and Biophysics for the educational opportunities that they have provided me. I would like to especially thank Dr. George Dubyak (Cell Physiology program), Dr. Ulrich Hopfer (Biophysics program), as well as the department chairman, Dr. Antonio Scarpa. 8 List of Abbreviations 2-ABP, 2-aminoethoxydiphenyl borate cADPR, cyclic adenosine diphosphate ribose CaNSC, Ca2+ permeable nonselective cation channel CaMKII, Calmodulin kinase II 2+ 2+ [Ca ]i, cytosolic free Ca concentration DAG, diacylglycerol DRG, dorsal root ganglion ER, endoplasmic reticulum EB, Ethidium bromide GPCR, G-protein coupled receptor 12-HETE, 12-hydroxyeicosatetraenoic acid 12-HPETE, 12-hydroperoxy-eicosatetraenoic acid HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HBS, Hepes buffered saline 2+ ICRAC, Ca release activated current InsP3, inositol-1,4,5-trisphosphate IP3R, inositol-1,4,5-trisphosphate receptor MES, 2-[N-morpholino]ethanesulfonic acid MTX, Maitotoxin NAD, Nicotanamide adenine dinucleotide NO, nitric oxide PI3K, phosphatidylinositol-3 kinase 9 PIP2, phosphatidylinositol-4,5-bisphosphate PLC, phospholipase C PrI, Propidium iodide RTK, receptor tyrosine kinase RTX, resiniferatoxin ROS, reactive oxygen species SOC, store-operated channel SOCE, store-operated Ca2+ entry TRP, transient receptor potential TRPV1, vanilloid receptor type 1, VR1 10 ROLE OF Ca2+-PERMEABLE CATION CHANNELS IN Ca2+ SIGNALLING AND NECROTIC CELL DEATH Abstract By Brian J. Wisnoskey To evaluate the interaction of the vanilloid receptor (TRPV1) with endogenous Ca2+ signalling mechanisms, TRPV1 was heterologously expressed in insect Sf9 and HEK cells. In the absence of extracellular Ca2+, stimulation of TRPV1 with agonists capsaicin and resiniferatoxin (RTX) caused a release of Ca2+ from internal stores. This release was not blocked by U73122 suggesting phospholipase C was not involved. Substantial overlap occurred between the TRPV1- and thapsigargin-sensitive Ca2+ pools, and TRPV1 immunofluorescence colocalized with the endoplasmic reticulum targeting motif "…KDEL…". To determine if TRPV1-induced release of Ca2+ from internal stores activated endogenous store-operated Ca2+ entry, the effect of 2-APB on Ba2+ was evaluated. 2-ABP blocked thapsigargin-induced Ba2+ influx, but not RTX-induced Ba2+ influx. In the combined presence of thapisgargin and RTX, the 2-APB sensitive component was essentially identical to the thapsigargin-induced component. These results indicate that TRPV1 forms agonist-sensitive channels in the endoplasmic reticulum, which when activated, release Ca2+ from internal stores but fail to activate store-operated Ca2+ entry. 11 Maitotoxin (MTX), a potent marine toxin, is a tool for the study of a Ca2+- overload induced necrotic/oncotic cell death. Upon stimulation with MTX, bovine aortic endothelial cells (BAEC) undergo sequential changes in plasmalemmal permeability. 2+ Initially, MTX activates CaNSC leading to an increase in [Ca ]i. Second is