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Role of BK Channels in Macrophage Activation

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Role of BK Channels in Macrophage Activation Role of BK channels in macrophage activation A thesis submitted by Minae Yoshida to University College London for the Degree of Doctor of Philosophy March 2017 Department of Neuroscience, Physiology and Pharmacology University College London, Gower Street, London WC1E 6BT 1 Declaration I, Minae Yoshida, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract The cells of the immune system expresses an array of different ion channels, yet their roles in these cells are not fully understood. The large-conductance voltage and calcium-activated potassium channel (BK channel) is a potassium ion channel, which is found in a wide variety of cells and tissues. Their biophysical properties and expression in excitable cells are extensively studied. However, their role in immune cells is unclear. In this thesis I have investigated the role of BK channels in macrophages. The expression of BK channels in the RAW264.7 mouse macrophages cell line was characterised by Western blot, immunofluorescence imaging and electrophysiological recordings. BK channels were predominantly associated with intracellular compartments in resting macrophages. Activation of RAW264.7 with ultrapure lipopolysaccharide (LPS) resulted in the upregulation of BK channel protein on the plasma membrane and the channel activity. To investigate the function of BK channels in these cells, both genetic and pharmacological approaches were used. These studies suggested that the role of the BK channel in macrophages is dependent on the sub-cellular location of the channel. Importantly this thesis suggests that plasma membrane located BK channels regulate the release of tumour necrosis factor- (TNF- and interleukin-6 receptor (IL-6R from activated macrophages. IL-6 release was not affected. These results together suggested that a disintegrin and metalloprotease domain 17 (ADAM17) enzyme is negatively regulated by the plasma membrane BK channel. This thesis proposes a novel interaction between ion channels and the activity of a membrane metalloprotease. This project also identified the dynamic movement of BK channels during activation of macrophages. ADAM17 regulates the release of a diverse range of proteins including cytokines, growth factors, receptors and adhesion molecules, which are implicated in many fields including immunology, tissue regeneration, neurology and tumour growth. It is anticipated that this finding could stimulate further research. 3 Acknowledgement Firstly I owe my gratitude to my supervisor, Dr Dean Willis, for his support and guidance during my PhD study. I am grateful for Professor Alasdair Gibb for his advice. I would like to thank Dr Steve Marsh for his guidance. I am also indebted to my colleagues and friends at UCL and in Japan, especially Dr Kheng Peh, Mr Stuart Martin, Miss Emily Langron, Mrs Setsuko Ichikawa and Mr Jack Barton, whose friendship and help have been a great encouragement and made my time here most enjoyable. It is my honour to thank those who have financially supported my study, Miss Miyuki Miyazaki, my grandmother and parents. Finally I would like to express my heartfelt thanks to my family, my grandmother, mother, sister, dog, Luna, and cats, Moo and Lu, for their support and care. 4 Contents Title 1 Declaration 2 Abstract 3 Acknowledgements 4 Contents 5 List of figures 10 List of tables 13 List of equation 13 List of abbreviations 14 Chapter 1 Introduction ........................................................................................18 1.1 Inflammation .............................................................................................18 1.1.1 General concepts of inflammation .....................................................18 1.1.2 Pattern recognition receptors .............................................................20 1.2 Toll-like receptor pathways .......................................................................21 1.2.1 TLR structure .....................................................................................21 1.2.2 TLR4 .................................................................................................24 1.2.3 NF-B ................................................................................................28 1.3 Cells in innate immune system .................................................................29 1.4 Macrophages ............................................................................................30 1.4.1 Macrophages production of cytokines ................................................32 1.4.2 Control of TNF- mRNA ....................................................................34 1.4.3 Translational control of TNF- ...........................................................36 1.4.4 Regulation of TNF- release and actions ..........................................37 1.5 Ion channels in immune systems ..............................................................39 5 1.5.1 Evolution of ion channels and function in immune cells .....................39 1.5.2 Macrophages and ion channels .........................................................40 1.6 BK channels .............................................................................................42 1.6.1 Physiological roles for BK channels ...................................................42 1.6.2 Channel structure and gating mechanisms of BK channels ...............43 1.6.2.1 Crystal structure and function .....................................................43 1.6.2.2 The gating mechanism of BK channels .......................................44 1.6.3 BK channel auxiliary subunits ............................................................46 1.6.4 BK channel pharmacology .................................................................48 1.6.4.1 BK channel blockers ...................................................................48 1.6.4.2 BK channel openers ...................................................................49 1.6.4.3 BK channel modulation by endogenous signalling molecule .......50 1.6.5 BK channel expression in different cellular locations .........................51 1.7 Aims .........................................................................................................52 Chapter 2 Material and method ..........................................................................55 2.1 Materials ...................................................................................................55 2.1.1 Drugs .................................................................................................55 2.1.2 Chemicals and reagents ....................................................................57 2.1.3 Bioassay kits .....................................................................................57 2.1.4 Buffers and solutions .........................................................................57 2.1.5 Antibodies ..........................................................................................61 2.2 Methods....................................................................................................62 2.2.1 Cell culture ........................................................................................62 2.2.2 Mycoplasma detection .......................................................................62 2.2.3 Nitrite assay .......................................................................................63 2.2.4 MTT assay .........................................................................................66 2.2.5 Bradford assay ..................................................................................66 2.2.6 Whole cell protein preparation for Western blot analysis ....................66 2.2.7 Plasma membrane protein isolation. ..................................................68 2.2.8 Nuclei isolation ..................................................................................68 6 2.2.9 Rat brain homogenise preparation .....................................................69 2.2.10 Western blot analysis.........................................................................70 2.2.10.1 Calibration of the molecular weight .............................................72 2.2.10.2 Densitometry analysis ................................................................74 2.2.11 Electrophysiology ..............................................................................76 2.2.12 Cytokine and cytokine receptor assays ..............................................78 2.2.13 RNA extraction and cDNA synthesis ..................................................80 2.2.14 Real time-quantitative PCR................................................................80 2.2.15 Gene silencing ...................................................................................81 2.2.16 ADAM17 activity assay ......................................................................82 2.2.17 Immune fluorescence imaging ...........................................................84 2.2.18 Statistical analysis .............................................................................85 Chapter 3 How macrophages change TNF-, IL-6 and IL-6R release during TLR4 activation? .....................................................................................................86 3.1 Introduction ...............................................................................................86 3.2 Results .....................................................................................................86
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