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(EGF) in the Regulation of Ion Channels in the Calu-3 Submucosal Cell Line

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(EGF) in the Regulation of Ion Channels in the Calu-3 Submucosal Cell Line The Novel Role of Epidermal Growth Factor (EGF) in the regulation of ion channels in the Calu-3 submucosal cell line Craig S. Clements BSc MSc A Thesis Presented to Faculty of Medicine and Health Sciences, Norwich Medical School University of East Anglia In Fulfilment of the Requirements of the University of East Anglia for the Degree of Doctor of Philosophy September 2012 © This copy of thesis has been supplied on the condition that anyone who consults it is understood to recognise that its copyright rests with the author and that no quotation from the thesis, nor any information derived from it, may be published without the author’s prior written consent. Declaration I hereby declare that the work in this thesis is my own work and effort and that it has not been submitted anywhere for any award. Where other sources of information have been used, they have been acknowledged. Signature: Date: 6th September, 2012 2 Abstract Cystic fibrosis transmembrane conductance regulator (CFTR) is a cell membrane bound chloride ion channel regulated by cyclic AMP-dependent phosphorylation and levels of intracellular ATP. Mutations in this channel, such as the common deletion of phenylalanine at residue 508 (CFTRΔF508), leads to a decrease in chloride transport seen in the disease condition cystic fibrosis (CF). The mutant CFTR is not processed in the normal way and consequently not delivered to the cell membrane. Currently, the effect of growth factors such as epidermal growth factor (EGF) on ion transport in the airway has not been previously researched and is consequently unknown. Therefore the aim of this thesis is to determine (i) if EGF has an effect on ion transport in the submucosal cell line Calu-3, (ii) what the mechanisms are behind this, and (iii) if the effect of EGF was due to induction of gelatinase activity or a transactivation process. Functional investigations looking at ion transport were carried out by using short circuit current. This technique was complemented by traditional molecular biology techniques such as RT-PCR, Western blotting, flow cytometry and gelatin zymography. The level of EGF, a potent inducer of gelatinases, is known to be elevated in the lungs during tissue repair in CF. Calu-3 cells preincubated with EGF on the basolateral membrane increased initial current at one hour via a EGFR-PI3K- PKC-δ-KCNN4/KCNQ1 signalling pathway. Similarly, preincubation with EGF also decreased forskolin induced short circuit current compared to untreated monolayers at 1 to 3 hours, with a recovery at 24 hours. The decreases were found to be dependent on the activation of KCNQ1 since chromanol 293B, a specific inhibitor for KCNQ1, restored the short circuit current back to untreated levels. Stimulation of the β2 adrenergic receptors with salbutamol were not reduced using metalloproteinase inhibitor, GM-6001 and EGFR inhibitor, AG1478. This suggested that stimulation of β2 adrenergic receptors does not lead to transactivation of EGFR via activation of sheddases and the release of EGF ligand. β3 adrenergic receptors are present in Calu-3, but produce negligible currents when stimulated. It was concluded that EGF induced potassium channel activation led to a change in chloride driving force. This activation of potassium channels has previously been linked to wound repair in the airway during disease. The implications of this study suggest that manipulation of the 3 EGF signalling pathway and / or potassium channel activity in the lungs may be beneficial in disease conditions such as CF for increasing chloride transport. 4 Papers and Conference Abstracts The following is a list of published works resulting from the work in this thesis. Papers: Clements CS and Winpenny JP (2012) EGF activates basolateral potassium channels and increases chloride driving force in the Calu-3 cell line. J Physiol (In prep) Clements CS and Winpenny JP (2012) EGF inhibits stimulated CFTR short circuit current in the Calu-3 cell line. J Physiol (In prep) Conference Abstracts: Clements CS, Gavrilovic J, Winpenny JP (2010) Epidermal growth factor (EGF) increases matrix metalloproteinase (MMP) activity and cystic fibrosis transmembrane conductance regulator (CFTR) chloride current in the Calu-3 cell line. Proc Physiol Soc 19, PC53. Abstract accepted and presented as a poster to The Physiological Society at the Physoc Manchester 2010 conference. Shortlisted by The Physiological Society for best poster prize. Clements CS and Winpenny JP (2012) EGF activates basolateral potassium channels in the Calu-3 cell line. Proc Physiol Soc 27, PC98. Abstract accepted and presented as a poster to The Physiological Society at the Physoc Edinburgh 2012 conference. 5 Table of Contents ABSTRACT ................................................................................................................. 3 PAPERS AND CONFERENCE ABSTRACTS ............................................................ 5 TABLE OF CONTENTS .............................................................................................. 6 LIST OF FIGURES .................................................................................................... 12 LIST OF TABLES ...................................................................................................... 14 LIST OF ABBREVIATIONS....................................................................................... 15 ACKNOWLEDGMENTS ............................................................................................ 17 CHAPTER 1 LITERATURE REVIEW .................................................................... 18 1.1 Types of Chloride Channels ........................................................................................................ 18 1.1.1 Ligand-gated chloride channels ............................................................................................. 18 1.1.1.1 GABAA receptor (GABAAR) ........................................................................................... 18 1.1.1.2 GABAA-rho receptor (GABAA-ρ) .................................................................................... 18 1.1.1.3 Glycine receptor (GlyR) ................................................................................................ 19 1.1.2 Voltage gated chloride channels (VGCLC) ............................................................................ 19 1.1.3 Volume-sensitive chloride channels (VSCC) ......................................................................... 20 1.1.4 Stretch-activated chloride channels ....................................................................................... 21 1.1.5 Calcium activated chloride channel (CaCC) candidates ....................................................... 21 1.1.5.1 Chloride channel, Calcium-activated (CLCA) ............................................................... 21 1.1.5.2 Bestrophins (BESTs)..................................................................................................... 22 1.1.5.3 Anoctamins (ANOs) ...................................................................................................... 24 1.1.6 Cystic fibrosis transmembrane conductance regulator (CFTR) ............................................ 26 1.1.6.1 Domain Structure of CFTR ........................................................................................... 26 1.1.6.2 Gating of CFTR is controlled by phosphorylation by protein kinases ........................... 28 1.1.6.3 Regulation via β2 adrenergic receptor........................................................................... 29 1.1.6.4 Regulation via β1 and β3 adrenergic receptors ............................................................. 30 1.1.6.5 Regulation via G Protein Coupled Receptors (GPCRs) ............................................... 30 1.2 Physiology of the Lung ................................................................................................................ 32 1.2.1 Lung structure ........................................................................................................................ 32 1.2.2 Lung function ......................................................................................................................... 34 1.2.3 Underlying tissue of the lung ................................................................................................. 34 1.2.4 Calu-3 cells as a model for lung ion transport ....................................................................... 36 1.2.4.1 Channels present in Calu-3 .......................................................................................... 36 1.3 Cystic Fibrosis (CF)...................................................................................................................... 39 1.3.1 Cystic Fibrosis in the lung ...................................................................................................... 39 6 1.3.1.1 High-salt hypothesis ...................................................................................................... 40 1.3.1.2 Low-volume hypothesis ................................................................................................ 40 1.3.2 Cystic fibrosis in the pancreas ............................................................................................... 41 1.3.3 Cystic fibrosis and the reproductive system .......................................................................... 42 1.3.3.1 Congenital absence of the vas deferens (CAVD) ........................................................
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