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Investigations of the Functional Expression of SLC6A14 in Non-CF and CF Airway Epithelial Cells

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Investigations of the Functional Expression of SLC6A14 in Non-CF and CF Airway Epithelial Cells Investigations of the Functional Expression of SLC6A14 in Non-CF and CF Airway Epithelial Cells by Andrew Lloyd-Kuzik A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Physiology University of Toronto © Copyright Andrew Lloyd-Kuzik 2014 Investigations of the Functional Expression of SLC6A14 in Non-CF and CF Airway Epithelial Cells Andrew Lloyd-Kuzik Masters of Science Department of Physiology University of Toronto 2014 Abstract Recent studies have found single nucleotide polymorphisms significantly associated with more severe cystic fibrosis (CF) in the promoter region of the SLC6A14 gene1,2, encoding a NaCl- dependent neutral and cationic amino acid transporter highly expressed in the lung3,4. In a CF human bronchial epithelial cell line (CFBE41o-) transfected with wild-type- (CFBEwt) or dF505-CFTR (CFBEdf), 3H-arginine uptake followed Michaelis-Menten kinetics (CFBEwt Kt=95.01±14.05, CFBEdf Kt=72.8±16.14). Competitive inhibition experiments demonstrated function attributable to SLC6A14 and cationic amino acid transporter system y+ in both cells. Intracellular and transepithelial transport of 3H-arginine in non-CF and CF primary human airway cells was significantly reduced (~50%) in 0 Na+ or presence of a SLC6A14 blocker, α- methyl-DL-tryptophan (α-MT) apically, indicative of SLC6A14. There was no difference in SLC6A14 function between CF and non-CF cells, or CFBEwt and CFBEdf cells. These ii protocols allow isolation of SLC6A14 function in airway cells by competitive inhibition, Na+ dependence, and α -MT sensitivity. iii Acknowledgments I would like to thank my supervisor, Dr. Tanja Gonska, for providing me with direction and helping me through the many sticking points. Your patience, attention to detail and scientific rigor were instrumental in getting me where I am today. My co-supervisor Dr. Christine Bear, for offering her experienced opinions on my projects and always providing support whenever it is needed. Wan Ip, for helping with anything I could ever need, from permanent markers to protocol design; I couldn‟t imagine a kinder person to share a lab space with. The other members of the Gonska Lab Satti, Julie, and Katie; you might not have understood all of my talks, but thanks for helping where you could and providing support. To Dr. Anne-Marie Lam-Hon-Wah for valuable discussions that would critically influence my whole project. Dr. Kai Du for the early cell culture help, and Dr.‟s Welsh and Keshavjee for the primary human cell cultures. Saumel Ahmadi for the engaging discussion, PCR and cell culture help, lecture attendance partnership, and for the help dreaming up experiments. Of course everyone else along the way from the Bear lab as well: especially Stan and Steve, Ranj, and Ling-Jun. You guys were always ready to help and you always made me feel more than welcome on the 3rd floor. I also thoroughly enjoyed passing on my knowledge to the newest members of the Gonska lab (Ming Li) and Bear lab (Michelle Di Paola); I have learned a lot from our discussions. I would also like to acknowledge the valued input and guidance of my committee members, Dr. Patricia Brubaker and Dr. Johanna Rommens. Thank you to my parents Laurie and Brian, and my brother Nicholas. You have always offered me your unwavering support, love, and guidance in everything that I take on. iv Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents .............................................................................................................................v List of Tables ................................................................................................................................ vii List of Figures .............................................................................................................................. viii List of Abbreviations ..................................................................................................................... ix Introduction ......................................................................................................................................1 1 General Airway Physiology ........................................................................................................1 2 Cystic Fibrosis .............................................................................................................................5 2.1 Heterogeneity in CF .............................................................................................................8 2.2 The Search for Modifying Factors of CF .............................................................................9 3 Mammalian Amino Acid Transport Systems ............................................................................12 0,+ 3.1 System B (SLC6A14) .....................................................................................................14 3.1.1 The Expression of SLC6A14 in Human and Animal Tissues and Cell Lines .......17 0,+ 3.1.2 The Regulation of System B ..............................................................................19 3.1.3 Protein structure of SLC6 Transporters .................................................................21 0,+ 3.1.4 Association of B with Disease ............................................................................23 3.2 System y+L ........................................................................................................................24 0,+ 3.3 System b .........................................................................................................................25 3.4 System y+ ..........................................................................................................................25 Rationale ........................................................................................................................................27 Hypothesis and Aims .....................................................................................................................28 Methods..........................................................................................................................................29 1 Cell Line Culture .......................................................................................................................29 v 2 Primary Culture .........................................................................................................................29 3 Arginine Uptake Studies ...........................................................................................................30 3.1 Calculating Arginine Uptake .............................................................................................31 3.2 Dose Response Experiments ..............................................................................................33 3.3 Competitive Inhibition Experiments ..................................................................................33 3.4 Transepithelial Flux Experiments. .....................................................................................34 4 Cell Protein Assay .....................................................................................................................35 5 Solutions ....................................................................................................................................35 6 Statistics ....................................................................................................................................36 Results ............................................................................................................................................37 1 Kinetics of L-arginine uptake ...................................................................................................37 2 Competitive Inhibition experiments ..........................................................................................40 0,+ 2.1 B .....................................................................................................................................40 0,+ 2.2 b ......................................................................................................................................40 2.3 y+L .....................................................................................................................................43 2.4 y+ .......................................................................................................................................43 3 Primary Human Bronchial Culture Experiments ......................................................................44 3 3.1 Intracellular Uptake of L-[2,3- H]-arginine .......................................................................44 3 3.2 Transepithelial Transport of L-[2,3- H]-arginine ..............................................................46 Discussion ......................................................................................................................................48 References ......................................................................................................................................56 Appendix ........................................................................................................................................72 vi List of Tables Table 1 Descriptions of mammalian plasma membrane transporters
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