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University of Cincinnati UNIVERSITY OF CINCINNATI Date:__10/23/2007__ ______ I, __Stella A. Nicolaou________________________________________, hereby submit this work as part of the requirements for the degree of: Ph.D in: Pathobiology and Molecular Medicine It is entitled: K+ channel trafficking in the immunological synapse of human T cells in health and autoimmunity This work and its defense approved by: Chair: Dr. Laura Conforti Dr. Sean Davidson Dr. Alexandra Filipovich Dr. Robert Franco Dr. Judith Heiny K+ channel trafficking in the immunological synapse of human T cells in health and autoimmunity A dissertation submitted to the Graduate School of the University of Cincinnati In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY In the Department of Pathobiology and Molecular Medicine of the College of Medicine By STELLA A. NICOLAOU B.Sc., Medical Technology University of Indianapolis, 2002 Committee Chair: Dr. Laura Conforti THESIS ABSTRACT T cell receptor (TCR) engagement by an antigen presenting cell (APC) results in reorganization of intracellular and membrane molecules at the T/APC interface, forming a “signalosome”, the immunological synapse (IS). An early event associated with T/APC interaction is Ca2+ influx. K+ channels, Kv1.3 and KCa3.1, modulate Ca2+ signaling in human T cells. Resting and activated human T cells express both channels, albeit to different degrees. Kv1.3 channels modulate Ca2+ in resting, while KCa3.1 channels do so in activated, T cells. Although these channels play such an important role in Ca2+ homeostasis, very little is known about their localization in the IS. Furthermore, aberrant T cell responses in IS formation and Ca2+ influx have been documented in T cells from patients with systemic lupus erythematosus (SLE). The potential involvement of K+ channels in the etiology and progression of SLE remains unknown. Herein we determined K+ channel membrane distribution in resting and activated human T cells following TCR engagement and performed comparative studies with SLE T cells to decipher the role of K+ channels in the Ca2+ response anomaly. Our data show that in SLE T cells, Kv1.3 channels constitute the dominant channel and are functionally identical to their normal counterparts. We also found that resting SLE T cells show faster Kv1.3 kinetics out of the IS as compared to healthy T cells and comparable to healthy pre-activated T cells. However, normal pre-activated T cells recruit and maintain KCa3.1 channels in the IS after Kv1.3 channels leave, while SLE T cells do not express the appropriate KCa3.1 channel number to support this activated phenotype. This Kv1.3 mobility defect appears to be specific to SLE and not other autoimmune diseases, as it was not observed in rheumatoid arthritis (RA) patients. Further, transcription factor activation and gene expression relies on the shape of the Ca2+ response. Although SLE T cells demonstrate abnormal transcription factor regulation and gene expression, the potential contribution of the Ca2+ responses to these abnormalities remains unknown. We performed single T cell Ca2+ response analysis to better define the Ca2+ shape in SLE T cells. Our data suggest that there is an increase in cells with more sustained Ca2+ response in SLE as compared to normal and RA T cells, while a transient, short duration, Ca2+ response is more pronounced in normal T cells. We also found that during and upon termination of the Ca2+ response, Kv1.3 channels are retained in the IS in healthy T cells, implying a role for Kv1.3 in the termination of the Ca2+ response. We conclude that altered localization of Kv1.3 channel in the IS of SLE T cells is at least in part responsible for more sustained Ca2+ response in SLE T cells. This phenotype, in turn, supports T cell hyperactivity in SLE T cells. Therefore we propose that Kv1.3 channels may offer novel therapeutic targets for SLE. ACKNOWLEDGEMENTS First and foremost I would like to thank my advisor Dr. Laura Conforti whom I had the pleasure and honor of working with for the past four years. She has been a great and inspiring mentor and her guidance has been of tremendous value. Her ethical approach to science has taught me how to be a successful scientist myself. She pushed me to achieve more than I could have ever imagined and for that I thank her. In addition, I would like to thank my thesis committee members, Drs. Sean Davidson, Alexandra Filipovich, Robert Franco and Judith Heiny for all their input, guidance and assistance in completing this degree. Next, I would like to thank all the past and current members of the Conforti lab. Personal thanks to Lisa Neumeier, the senior research assistant in the lab, for her help and support. It has been a real pleasure working with her. I next wish to thank all of our collaborators whom without this work would not have been possible: Susan Molleran Lee, Dr. Heather Duncan, Dr. Shashi Kant, Dr. Anne Barbara Mongey, Dr. Daniel Devor (University of Pittsburg) and Dr. Koichi Takimoto (University of Pittsburg). Special thanks to my sister, Soulla, who was always there after a tough day, and my parents, Eleni and Antoni Nicolaou, and my brother, Nicos, for their encouragement and support. This work was supported by National Institute of Health Grant CA95286 to L. Conforti and a pre-doctoral fellowship from the American Heart Association, Southern and Ohio Valley Affiliate 0615213B to S. A. Nicolaou. TABLE OF CONTENTS Page Committee Approval i Title Page ii Thesis Abstract iii Acknowledgements v List of Tables 4 List of Figures 5 Abbreviations 8 CHAPTER I: Background and Thesis Scope 10 1.1 T cell activation, the Immunological Synapse and Calcium Signaling 11 1.1.1 Ion Channels in T cell Activation 14 1.1.2 T cell proliferation and K+ channel expression 16 1.2 Properties, structure and pharmacological characteristics of Ca2+ and K+ 18 channels 1.2.1 The CRAC channel. 19 1.2.2 The Kv1.3 channel 20 1.2.3 The KCa3.1 channel 25 1.3 K+ channels and disease 29 1.3.1 The Kv1.3 channel 29 1.3.2 The KCa3.1 channel 33 1.4 Systemic Lupus Erythematosus 35 1.4.1 The role of T cells in the etiopathogenesis of SLE. 37 1.4.2 T cell activation and Immunological Synapses in SLE T cells 38 1.4.3 Contribution of K+ channels in the pathophysiology of SLE 40 1.5 Scope of Thesis 40 1.5.1 Hypothesis 41 1.5.2 Organization of thesis 42 1.6 References 43 CHAPTER II: Altered dynamics of Kv1.3 channel 62 compartmentalization in the immunological synapse in systemic lupus erythematosus - 1 - 2.1 Abstract 63 2.2 Introduction 63 2.3 Materials and Methods 66 2. 4 Results 72 2.4.1 Determination of T cell phenotype 72 2.4.2 Biophysical and Pharmacological characteristics of Kv1.3 channels 74 in SLE T cells 2.4.3 Native Kv1.3 channels are recruited in the immunological synapse 77 upon activation of healthy and SLE T cells 2.4.4 Kv1.3 channel compartmentalization in the immunological synapse 79 is altered in SLE T cells. 2.4.5 The kinetics of Kv1.3 redistribution in the immunological synapse 86 of SLE T cells resemble those of pre-activated normal T cells 2.4.6 T lymphocytes from patients with SLE display a K channel 90 phenotype similar to healthy resting T cells. 2.5 Discussion 92 2.6 References 97 CHAPTER III: The Ca2+-activated K+ channel KCa3.1 103 compartmentalizes in the immunological synapse of human T lymphocytes 3.1 Abstract 104 3.2 Introduction 105 3.3 Materials and Methods 106 3.4 Results 112 3.4.1 Electrophysiological and pharmacological profile of the cloned 112 YFP-KCa3.1 channel in HEK 293 cells matches the native KCa3.1 channel in human T cells 3.4.2 Overexpression of functional YFP-tagged KCa3.1 channels in 114 human primary T lymphocytes 3.4.3 KCa3.1 channels and F-actin redistribute to the T cell and anti- 117 CD3/CD28 antibody coated bead contact site 3.4.4 Calcium influx during antigen presentation and its regulation by 119 KCa3.1 channels 3.4.5 Redistribution of KCa3.1 channels at the immunological synapse 122 3.5 Discussion 127 3.6 References 132 CHAPTER IV: Differential calcium signaling in T lymphocytes 135 from patients with systemic lupus erythematosus 4.1 Abstract 136 4.2 Introduction 137 - 2 - 4.3 Materials and Methods 140 4.4 Results 149 4.4.1 Pattern of [Ca2+]i signaling in SLE T cells 150 4.4.2 Magnitude of [Ca2+]i in SLE T cells 153 4.4.3 Biophysical and pharmacological profile of pEGFP-Kv1.3 channels 157 in HEK 293 cells 4.4.4 Overexpression of functional pEGFP-Kv1.3 channels in human 160 primary T cells 4.4.5 Redistribution of Kv1.3 channels in the IS in Jurkat T cells 162 4.4.6 Kv1.3 trafficking to the IS and [Ca2+]i in human T cells 163 4.6 Discussion 166 4.7 References 171 CHAPTER V: General Discussion 176 5.1 K+ channel trafficking in the immunological synapse in healthy T cells 177 5.1.1 Membrane distribution of Kv1.3 channels in healthy resting T cells 177 5.1.2 Membrane distribution of Kv1.3 and KCa3.1 channels in healthy 179 activated T cells 5.2 K+ channel trafficking in the immunological synapse and Ca2+ mobilization 181 in SLE T cells 5.2.1 Membrane distribution of Kv1.3 channels in SLE resting T cells 181 5.2.2 Ca2+ signaling in SLE T cells 182 5.3 Future Directions 185 5.4 Clinical Relevance and Therapeutic Indications 186 5.5 References 187 - 3 - LIST OF TABLES Page CHAPTER I: Background and Thesis Scope Table 1.1: Summary of T cell signaling defects in SLE 39 CHAPTER II: Altered dynamics of Kv1.3 channel compartmentalization in the immunological synapse in systemic lupus erythematosus Table 2.1: K+ channel expression in SLE and normal T lymphocytes 76 Table 2.2:
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