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

Andrew Lloyd-Kuzik

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Physiology University of Toronto

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

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.

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

3.1 System B0,+ (SLC6A14) ...... 14

3.1.1 The Expression of SLC6A14 in Human and Animal Tissues and Cell Lines ...... 17

3.1.2 The Regulation of System B0,+ ...... 19

3.1.3 Protein structure of SLC6 Transporters ...... 21

3.1.4 Association of B0,+ with Disease ...... 23

3.2 System y+L ...... 24

3.3 System b0,+ ...... 25

3.4 System y+ ...... 25

Rationale ...... 27

Hypothesis and Aims ...... 28

Methods...... 29

1 Cell Line Culture ...... 29

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

2.1 B0,+ ...... 40

2.2 b0,+ ...... 40

2.3 y+L ...... 43

2.4 y+ ...... 43

3 Primary Human Bronchial Culture Experiments ...... 44

3.1 Intracellular Uptake of L-[2,3-3H]-arginine ...... 44

3.2 Transepithelial Transport of L-[2,3-3H]-arginine ...... 46

Discussion ...... 48

References ...... 56

Appendix ...... 72

List of Tables

Table 1 Descriptions of mammalian plasma membrane transporters of cationic amino acids...... 13

Table 2 Protocol utilized for examining competing cationic amino acid transporters. .... 34

Table 3.3H-arginine uptake data from CFBEwt and CFBEdf cells...... 72

Table 4 Primary culture intracellular 3H-arginine uptake data ...... 72

vii

List of Figures

Figure 1 Schematic of major fluid regulating ion transporters of the polarized airway epithelium...... 4

Figure 2 Comparison of Non-CF and CF airway ion and fluid scenarios...... 7

Figure 3 Schematic of selected transporters on a polarized airway epithelium...... 16

Figure 4 Time course of 100 μM arginine uptake in CFBE41o- cells...... 38

Figure 5 Michaelis-Menten transport kinetics in CFBEwt and CFBEdf cells...... 39

Figure 6 Na+-dependent and α -MT-sensitive arginine uptake in CFBEdf and CFBEwt cells...... 41

Figure 7 Competitive inhibition and determination of cationic amino acid transporter systems...... 42

Figure 8 Arginine uptake in non-CF (a) and CF b) primary airway cultures 60 minutes after apical addition of 100 µM L-arginine...... 45

Figure 9 Apical to basolateral transport of 100 μM arginine in non-CF and CF primary airway cells...... 47

viii

List of Abbreviations

ANOVA: analysis of variance ASL: airway surface fluid ATB0,+ & B0,+: amino acid transporter B0,+, also called SLC6A14 AQP: aquaporin ATP: adenosine triphosphate b0,+: cationic and neutral amino acid exchanger system CAA: cationic (basic) amino acid Caco-2: human colonic cancer cell line Calu-3: human submucosal gland cancer cell line cAMP: cyclic adenosine monophosphate CAP protease: channel activating protein protease CF: cystic fibrosis CFBE41o-: immortalized cystic fibrosis bronchial epithelial cells CFBEdf: CFBE41o- cells stably overexpressing CFTR with a deletion of phenylalanine at position 508 CFBEwt: CFBE41o- cells stably overexpressing wild-type CFTR CFTR: cystic fibrosis transmembrane conductance regulator CPM: counts per minute ddH2O: double distilled water dF-508: CFTR protein containing a deletion of phenylalanine at position 508 ENaC: epithelial sodium channel FEV1: forced expiratory volume in one second, the standard measurement of lung function GABA: γ-aminobutyric acid the chief inhibitory neurotransmitter in mammalian cells GWAS: genome-wide association study HEPES: 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, a buffering agent HRPE: human retinal pigment epithelium cell line IBD: inflammatory bowel disease iNOS: inducible nitric oxide synthase kDa: kilo-daltons

Kt: Michaelis-Menten constant; substrate concentration at half maximal transport LeuT: Bacterial leucine transporter, a crystallized homolog of the SLC6 family mRNA: messenger ribonucleic acid mTOR: mammalian target of rapamycin NAA: neutral amino acid NKCC: Sodium-Potassium-Chloride co-transporter PCL: pericilliary liquid PKA: protein kinase A

PKC: protein kinase C PMA: phorbol-12-myristate-13-acetate RT-PCR: reverse transcriptase polymerase chain reaction SERT: a member of the SLC6 family, the serotonin transporter SNP: single nucleotide polymorphism SPLUNC1: short palate lung and nasal epithelial clone 1 TM: transmembrane UTP: uridine 5‟-triphosphate

Vmax: Michaelis-Menten constant, maximal substrate velocity WT: wild-type y+: cationic amino acid transport system y+L: cationic and neutral amino acid transporter system α-MT: alpha methyl-DL-tryptophan

Introduction

1 General Airway Physiology

The lung possesses a distally branching structure of increasingly smaller diameter airways that end in blind sacs called alveoli; the sites for gas exchange. The lumen of the respiratory tract is lined with pseudo stratified ciliated airway epithelial cells

(constituting the majority of airway cells), mucus secreting goblet cells, basal cells (a precursor cell), type I and II alveolar cells of the distal airways for gas exchange and surfactant secretion, respectively, Clara cells (an immune cell in the bronchioles and distal airways), and mucus and serous sub-mucosal glands. As the surface area available for gas exchange increases in the distal lung from 50 cm2 in the 3rd generation of bronchi, to 2 m2 in the 20-25th generations5, so does the potential for lung infection by way of inspiration of potentially contaminated air into a high surface area system that is abundant with nutrients, moisture, and heat. This therefore necessitates the establishment of an effective defensive system to repel attack. The immunological defenses begin with the airway surface liquid (ASL), which covers the surface of the luminal airway cells which consists of a mucus-dense layer resting on top of a more fluid pericilliary liquid (PCL)6. Particulates such as bacteria and other airway aggravants become deposited and trapped in this layer, then through the mechanical beating of cilia these invaders are propelled cranially to be swallowed or expectorated in a process that takes approximately 2-6 hours5. Antimicrobial agents such as lysozyme, lactoferrin, secretory leukocyte protease inhibitor and β-defensins7–9 are secreted concurrently into

the ASL by submucosal glands. Adequate ion and fluid exchange between epithelial

10,6 cells and the ASL is a critical regulator of ASL osmolarity and fluidity which are in B turn critical factors for the effectiveness of ASL clearance. The secretion of Cl- by the cAMP-activated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) present on the apical membrane of ciliated cells11,12 promotes the transepithelial iso- osmotic flux of water to hydrate the ASL13 through paracellular and transcellular routes

14 - (i.e. through aquaporins [AQP] 1,3,4, and 5 in the lung ), while secretion of HCO3 through CFTR likely plays a role in buffering ASL pH15. An additional Cl- channel is present on the apical membrane of airway epithelia, the Ca2+-activated Cl- channel,

CaCC (identified at the molecular level as TMEM16A16, however recent research indicates that this might not be the predominant CaCC in respiratory and intestinal epithelia17). This channel has been observed playing a role in fluid secretion in an acute manner in response to release of luminal nucleotides (ATP or UTP), compared to the more predominant contribution of CFTR to basal control of airway surface fluid levels; thus interplay between the two is responsible for liquid secretion18. The opposing process of water absorption is dependent on luminal Na+ absorption through the

Epithelial Sodium Channel (ENaC)13 and the iso-osmotic flow of water again paracellularly and transcellularly through aquaporins14. The open state of a transporter determines ion permeability across a membrane, but flux ultimately depends upon the presence of favorable electrical and chemical gradients to generate the forces for ion movement. The two major contributors for setting up these electrochemical gradients are present on the basolateral membrane of polarized respiratory epithelia and are the

Na+K+-ATPase and the Na+/K+/2Cl- co-transporter (NKCC) (Figure 1). The Na+K+-

ATPase pump exchanges 3 Na+ for two K+ in a process that makes the cytosol more negative than the airway lumen, thus providing an electrochemical gradient for Na+ absorption through ENaC. Activity of the Na+/K+/2Cl- co-transporter (NKCC)19 provides

Cl- for apical secretion. In addition to a number of factors that modify the activity of both

ENaC and CFTR (described below), in general the absorption of Na+ will increase the driving force for Cl- secretion and vice versa, thus promoting a balance of absorption to secretion which maintains the ASL at a hydration level sufficient for its immunological function. ASL volume is highly regulated by signaling, including intracellularly by ATP and PKA (via CFTR to achieve fluid secretion)20, sensation of extracellular channel activating protein (CAP) protease21 concentrations in the ASL (whereby dilution of CAPs causes ENaC-mediated water absorption), and mechanosensitive release of ATP through the movement of cilia and phasic shear stress from tidal breathing and cough22

(activating CFTR, CaCC, and water secretion). Extracellular ATP has been shown to inhibit ENaC-mediated current23, providing a further mechanism to ensure fluid absorption occurs exclusively of fluid secretion. Ion transport pathology leading to defects in ASL can lead to life-threatening infection and disease as seen in cystic fibrosis (CF).

Figure 1 Schematic of major fluid regulating ion transporters of the polarized airway epithelium. A polarized epithelial cell showing the localization of the predominant small ion transporters. Fluid absorption is dominated by ENaC-mediated Na+ absorption, and fluid secretion by CFTR-mediated Cl- secretion. Double-sided arrows indicate that substrates can travel downstream according to their concentration gradients. Water follows the transport of these ions iso-osmotically by paracellular and transcellular routes. Transcellular water flux occurs through the aquaporins (AQP) 1,3,4, and 5 localized to the apical or basolateral membrane as indicated. The Ca2+-activated Cl- channel CaCC contributes to a portion of Cl- secretion. The Na+/K+ ATPase is responsible for developing the Na+ gradient, while NKCC provides Cl- for CFTR and

CaCC. Water and complementary small ions follow the transport of Cl- or Na+.

2 Cystic Fibrosis

CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance

Regulator (CFTR) protein that prevents its functional expression on the apical surface of certain secretory epithelia24,25. The disease symptoms originate from defective Cl- and

- 26 27 HCO3 secretion in secretory organs such as the pancreas , sweat glands , liver (bile ducts28), vas deferens, intestines27, and lungs27 (Figure 2). Deficient fluid secretion in the pancreas can lead to hyper-concentrated protein secretions and protein precipitates leading to ductal plugging and pancreatic insufficiency due to organ failure from a very young age29. The result is failure to secrete digestive enzymes such as pancreatic lipase leading to malnutrition. Pancreatic fibrosis can accumulate and over time pancreatic β-cells are unable to secrete sufficient insulin causing CF-related diabetes30 in 12-34% of adult CF patients31. Insufficient CFTR-mediated Cl- secretion in the small intestine and colon32 can cause limited water secretion, viscous mucus accumulation, intestinal inflammation and in some severe cases obstruction of the intestinal lumen (i.e. meconium ileus (MI) in 15% of newborns or recurring distal intestinal obstructions in ~

33 - 10-20% of adult CF patients ). Limited secretion of HCO3 is also thought to play a role in the pathogenesis of MI. Mucins are normally secreted in the intestinal lumen in the form of a dense matrix which unpacks into a looser, properly hydrated network of mucus

- 34 with the assistance of HCO3 secreted through CFTR . In the acidic environment of the

CF intestine, this more dense mucus can block the lumen leading to MI and an atrophy of the colon distal to the site of obstruction35, termed a microcolon of disuse. The most commonly fatal organ disease in CF, however, remains progressive lung disease due to

severe recurring pulmonary infections. CFTR is thought to contribute in two primary ways to this pathomechanism, by defective secretion of Cl-, and by defective secretion

- of HCO3 with the major consequence of both being reduced bacterial killing. Defective

Cl- secretion disrupts the homeostasis of ASL fluid regulation resulting in an unchecked over-absorption of Na+ through ENaC 36. The result is a low-volume pericilliary liquid

(PCL) layer: the layer of fluid which bathes the cilia. Compared to the PCL which is as tall as the height of extended cilia in healthy airways (~7 µm), the mucus that rides on top of the PCL is much more viscous. When the PCL is dehydrated it does not fully surround the respiratory cilia10, effectively crippling the mucociliary ladder and allowing bacteria to establish hypoxic niches of growth within a layer of dehydrated viscous

37 - mucous (Figure 2). Reduced HCO3 secretion results in the acidification of the ASL, which has been shown to reduce bacterial killing38. As in the intestine, an acidified ASL environment additionally lacks the capacity to promote the fluidity of secreted mucins and contributes to the viscosity and stasis of airway mucus 34,39,40. Recent research has also identified the short palate nasal and epithelial clone 1 (SPLUNC1) protein as a pH- sensitive inhibitor of ENaC. Experimental alkalization of CF cultures prevented ASL hyperabsorption which was reversed when SPLUNC1 was knocked-down, indicating for

- the first time that reduced HCO3 secretion in the CF lung contributes to ASL dehydration.

Figure 2 Comparison of Non-CF and CF airway ion and fluid scenarios.

In non-CF airway epithelia (first panel) homeostasis exists between Na+ absorption and

Cl- secretion that maintains the PCL at least at the height of outstretched cilia for optimal ciliary beating (~7 µm, the height of outstretched cilia). A layer of mucus rests on top. When Cl- secretion is dysfunctional in CF (right panel) Na+ absorption overpowers the cells fluid secretory capacity and ASL height is reduced to minimum levels (~3 µm). Mucus becomes dehydrated and acidic and unable to be propelled by cilia or support normal bacterial killing.

Dehydrated ASL coupled with persistent mucus secretion promotes a static, inspissated, and mucus-dense layer, which prevents mechanical propulsion by cilia, thereby severely restricting its immunological capacity. A buildup of static mucus then promotes the formation of hypoxic niches that promote bacterial colonization and biofilm growth37. Infection with e.g. Staphylococcus aureus, Psuedomonas aeruginosa, and

Burkholderia cepacia 41 are common and contribute to chronic inflammation and fibrotic changes and decline in lung function.

2.1 Heterogeneity in CF

Approximately 70% of CF-causing alleles in Caucasian patients consist of the same mutation, a deletion of one base pair causing a reading frame shift and a deletion of phenylalanine at position 508 of the CFTR linear sequence (dF508-CFTR)42.

Expressing two copies of dF508-CFTR often leads to a severe phenotype (e.g. >95% of patients have insufficient exocrine pancreatic function43), however in other measures such as forced expiratory volume in one second (FEV1) in the lung, the genotype- phenotype correlation is highly variable44. Except for the strong correlation between pancreatic sufficiency and CFTR genotype45, there is very poor predictability for disease phenotype based on CFTR genotype alone. Similarly, only a subset of CF patients present with MI at birth (15%), or develop CF-related liver disease (up to 9%) or diabetes (12-34%)46,47,48. The lack of a clinical link between CFTR genotype and lung disease prognosis44 means that the most prevalent cause of modern CF patient mortality runs a largely unpredictable course. Variations in organ function between patients carrying the same mutation, as well as differences in pleiotropy between CFTR and the severity of organ systems affected make a strong case for the involvement of

both environmental and genetic modifying factors of the disease. Environmental influences such as dietary supplementation and antibiotic use have changed the course of CF and prolonged life spans dramatically, yet rates of complications like MI have remained largely unchanged through history. Even FEV1 scores were found to correlate better in monozygotic vs. dizygotic twins (with an overall heritability ranging from 0.54 to

149); a result of shared DNA being more important than a shared rearing environment.

Observations such as these exemplify the strong genetic modification of the disease50.

Understanding well the potentially numerous relationships between genotype and phenotype will allow us to better predict the course of disease in patients, and potentially offer treatments that can reduce or even prevent future symptoms from developing.

2.2 The Search for Modifying Factors of CF

The incredible heterogeneity in CF phenotypes, especially between patients expressing identical CFTR genotypes, has spurred on the search for genes external to CFTR which are capable of modifying disease outcomes. These searches first took a “candidate gene” approach where plausible pathways that were known to interact with the CFTR molecule were investigated for their possible role in CF. Replicate studies had identified a few probable modifiers of CF including mannose-binding lectin 2, transforming growth factor beta-1, interferon-related developmental regulator 1, and interleukin-851 which are all thought to modify lung disease severity by impairing the host‟s ability to resist infection. A calcium-activated chloride channel (CaCC) was also investigated and found to rescue a portion of Cl- current in CFTR knock-out mice, where an up-regulated CaCC offered improved survival compared to control52. This channel was localized to the

apical membrane in the same vicinity as CFTR, and thought to modify the disease by contributing to apical Cl- secretion. However, after progressing to a phase III clinical trial, the CaCC activator denufosol tetrasodium was not found to increase FEV1 significantly over placebo after 48 weeks of treatment53. This result agrees with research presented above which places the role of CaCC within acute fluid regulation, and not determination of basal hydration status. To sensitively detect further modifiers of CF, genome-wide search approaches were used to identify genes associated with increased severity of CF. These studies inherently require large sample sizes and so the North American CF Gene Modifier Consortium was formed between the Canadian

Consortium of CF Gene Modifiers, the Johns Hopkins University School of Medicine in

Baltimore, Maryland, and the University of North Carolina at Chapel Hill in the United

States who were able to combine data from over 3500 CF patients. The first GWAS undertaken using these data scanned the genome of dF508-homozygous CF patients in

2011 and found two loci associated with lung disease severity, EHF (a member of the epithelium specific Ets transcription factors) and APIP (Apaf-1-interacting protein)54.

EHF is a transcription factor expressed in tissues including the lung, thought to improve trafficking of dF508-CFTR55, and APIP is an inhibitor of apoptosis, predicted to worsen lung function when expression is increased.

Sun et al.1 recently published a hypothesis-driven genome-wide association study

(GWAS-HD) using the hypothesis that modifiers of CFTR are likely to be found at the apical membrane of epithelial cells. Furthermore, they specifically chose a severe CF outcome marker that is almost entirely genetically based, the presentation of meconium ileus (MI) at birth. This choice was made because MI exhibits high heritability

(exceeding 88%56), and occurs in utero before any significant environmental exposure such as seen with CF lung disease. MI still shows signs of genetic modification by non-

CFTR genes by the fact that only 15% of CF infants present with MI (24.9% in dF508

CFTR homozygotes57), and by evidence of discordance in MI between pairs of non-twin

CF siblings46,58. Sun et al.‟ found three apical membrane constituents which accounted for 17% of the variation in MI in North American CF patients, SLC6A14 (an amino acid

- - + + transporter), SLC26A9 (a Cl /HCO3 channel), and SLC9A3 (a Na /H exchanger). Their most significant association identified three single nucleotide polymorphisms (SNPs) in the promoter region of the SLC6A14 gene located on the X chromosome, the most significant being rs3788766 (p=1.28X10-12). Patients carrying this risk allele were found to be 1.5 times more likely to develop MI1. The SLC6A14 gene encodes the SLC6A14

(or ATB0,+) protein which encodes a neutral and cationic amino acid (NAA and CAA, respectively) transporter. Subsequent to finding SLC6A14 as being statistically significantly associated with risk of developing MI, Li et al. investigated the potential pleiotropic effects of SLC6A14 in two other CF affected organs, the lung and pancreas.

In order to detect a correlation between genetic expression and lung phenotype which is often highly influenced by environmental factors, Lei et al. specifically analyzed a pediatric population in their study. Specifically, within the Canadian CF population they assessed pleiotropy in 1) pediatric lung disease severity, 2) age at first P. aeruginosa infection, and 3) prenatal pancreatic damage2. The results of this study show that the

SLC6A14 SNP rs3788766 demonstrated pleiotropy for not only meconium ileus, but also for severe lung disease and age at first P. aeruginosa infection.

3 Mammalian Amino Acid Transport Systems

SLC6A14 is one of four known cationic amino acid transport systems of the plasma membrane of mammalian epithelial cells which includes, system b0,+, system y+, and system y+L (table 1). Usage of l-arginine as a model substrate for SLC6A14 as in these studies requires investigation of alternate mammalian cationic amino acid transporters.

Amino acid transporters are primarily identified based on substrate selectivity, ion dependency, patterns of cross-inhibition by other substrates, and Michaelis-Menten kinetic parameters59. Pioneering work in the 1960s by the lab of Halvor Christensen began the identification of cationic amino acid transporters using radiolabelled amino acids, a technique which still prevails today to study amino acid uptake physiology.

Christensen‟s group first identified system y+ in Ehrlich tumor cells60,61 and rabbit reticulocytes62, and it was later found to be the major influx route for cationic amino acids in all studied tissues of the human body63. It is also the only cationic amino acid uniporter of the group. Systems B0,+ and b0, + were first identified by Van Winkle et al. in mouse blastocysts64,65, and system y+L was found by Deves et al.66 using partial inhibition of lysine flux by neutral amino acids in human erythrocytes. B0,+ can transport cationic and neutral amino acids with NaCl, while systems y+L, and b0,+ exchange cationic for neutral amino acids.

K for K for Na+- t t Arg Leu Protein Gene System Depen Substrates Location transport transport -dent (mM) (mM) Ubiquitous (not liver CAT-1 SLC7A1 y+ no CAA 0.07-0.25 --- or lacrimal gland), Liver, skeletal CAT-2A SLC7A2 ND no CAA 2-5 --- muscle, pancreas Inducible in many cell 0.038- types (e.g. by LPS, CAT-2B SLC7A2 y+ no CAA --- 0.38 inflammatory cytokines) Thymus, ovary, testes, CAT-3 SLC7A3 y+ no CAA 0.04-.45 --- brain (neurons) CAT-4* SLC7A4 ------Brain, testes, placenta, Small intestine, SLC7A7 y+LAT- for CAA /NAA kidney, spleen, + y+L 0.34† 0.02 1/4f2hc NAA exchange leukocytes, placenta, SLC3A2 lung SLC7A6 Brain, small intestine, y+LAT2/ for CAA /NAA 0.12- + y+L 0.2-0.3 testis, parotids, heart, 4f2hc NAA exchange 0.14‡ SLC3A2 kidney, lung, thymus SLC7A9 Kidney, small b0,+ CAA /NAA + b0,+ no 0.08-0.2 0.3 intestine, liver, AT/rBAT exchange SLC3A1 placenta Blastocysts, small Yes, CAA or intestine, colon, lung, ATB0,+ SLC6A14 B0,+ with 0.08-0.15 0.01 NAA mammary, salivary, Cl- pituitary glands

Table 1 Descriptions of mammalian plasma membrane transporters of cationic amino acids. Positively charged amino acids are transported by four systems in mammalian cells.

Although their affinities for arginine are largely overlapping, their differential dependence on sodium (and chloride) for transport is a discriminating characteristic. Three of the four systems (all but b0,+) have been identified as contributing to transport in human airways. ND: not defined. *: no transport activity has been detected. †: Kt=93uM in the

+ + 67 absence of Na . ‡: Kt is independent of Na concentration. CAT proteins reviewed in , all transporters reviewed in68.

3.1 System B0,+ (SLC6A14)

The B0,+ system was technically first identified in 1969 in the rabbit ileum by the lab of B.

G. Munck69 however it was initially thought to be only a β-alanine carrier70–72. L. J. Van

Winkle in the lab of Halvor Christensen fully characterized a broad-scope transporter of zwitterionic and cationic amino acids in the mouse blastocyst, and named it the B0,+ system64. Through the use of sequence homology, electrophysiological and amino acid uptake functional studies in Xenopus oocytes, ATB0,+ was determined to be the protein responsible for system B0,+ amino acid transport in 1999 by Sloan et al.4. The Human

Genome Organization identifies human ATB0,+ as SLC6A14, the 14th member of the 6A solute carrier family73 which comprises transporters for neurotransmitters, amino acids, osmolites, and energy metabolites often with dependencies on NaCl for transport74.

SLC6A14 is a 645-amino-acid long protein with 12 putative transmembrane helices75 which function as an electrogenic amino acid transporter with a broad specificity for neutral (the 0 in B0,+) and cationic (the „+‟ in B0,+) amino acids (i.e. amino acid transport through SLC6A14 is current generating). Using ion-dependence uptake experiments, it is estimated that SLC6A14 co-transports Na+ and Cl- with a stoichiometry of 2 Na+ and

1 Cl- with 1 NAA or CAA in an apical-to-basolateral direction64,4,76 (Figure 3).

Physiological salt concentration gradients of 12 mM Na+ and 4 mM Cl- inside the cell to

145 mM Na+ and 116 mM Cl- in the plasma77 (as well as the resultant negative membrane potential of -30 mV to -50 mV) will lead to an inward flux of 1-2 positive charges and 1 AA0,+ with each transport cycle4. This has been shown to occur regardless of the charge on the amino acid. The coupling of NAA and CAA transport to the Na+, Cl-, and electrochemical4 gradient allows B0,+ to concentrate both NAA and

CAA‟s inside the cell at hundreds of times their external concentration78. The coupling of cationic amino acid transport to the NaCl gradient is termed secondary transport, as the process is powered by the electrochemical gradient of (primarily) Na+ which is set- up by the Na+/K+ ATPase residing on the basolateral membrane of all animal cells79,80.

The broad AA substrate affinity combined with the concentrative capacity of SLC6A14 is unique within all cationic amino acid transporters.

Figure 3 Schematic of selected transporters on a polarized airway epithelium. A polarized epithelial cell showing the localization of mammalian cationic amino acid transporters. There are four distinct transporters of cationic amino acids in mammalian cells, each with different combinations of affinities for amino acids and dependence on sodium or chloride for transport (as well as localization to the apical or basolateral surfaces of polarized epithelia). System B0,+, or SLC6A14, is the only member of this group which exhibits sodium-dependent transport of cationic amino acids.

3.1.1 The Expression of SLC6A14 in Human and Animal Tissues and Cell Lines

Expression of SLC6A14 in human tissues includes high mRNA and protein expression in the fetal lung and adult lung (cartilaginous and distal airways81), and expression in the salivary and mammary glands, with lower mRNA expression in the stomach, pituitary gland, colon, uterus, prostate, and testis4. In the pituitary gland SLC6A14 is hypothesized to play a role in hormone secretion via membrane depolarization caused by amino acid signalling81. SLC6A14 has also been found in a human colon adenocarcinoma cell line Caco-282, the human CF bronchial epithelial cell line

CFBE41o- (Saumel Ahmadi, personal communication), and the apical membrane of the human bronchial submucosal gland cell line Calu-383. B0,+ was first functionally localized to the apical membrane of polarized primary human bronchial epithelia by Galietta et al.84, a finding confirmed by the Gonska lab (ongoing studies). In primary human airway cells mounted in an Ussing chamber, Galietta et al. demonstrated a Michaelis-Menten constant for half-maximal transport (Kt) for arginine transport (in the presence of 100 µM amiloride, a blocker of the epithelial sodium channel; see Figure 1) of 80 ± 8.9 µM by measuring short-circuit current (Isc) when applying doses of arginine ranging from 1 µM to 1 mM84. These findings were supported following the cloning of B0,+ by Sloan et al.

4 who reported a Kt of 104 ± 35 µM for arginine uptake in Xenopus oocytes expressing human B0,+ using two electrode voltage clamp. In 2003, Sloan et al.81 found that mouse

ATB0,+ co-localized with beta-tubulin-IV found in cilia, supporting that mouse ATB0,+ is localized to the apical membrane of ciliated respiratory cells. In the distal lung, findings of wide expression suggested high expression in type I alveolar cells which constitute

95% of distal lung cells. Further, staining of ATB0,+ in the corner of alveolar sacs81, a

location and morphology matching that of alveolar type II cells, suggested expression in these cells as well. Inside of the lung, these features are thought to help SLC6A14 control luminal protein concentration81 as a means to reduce the amino acid “debris” which can influence airway compliance by interfering with the surface activity of pulmonary surfactant85. As an amino acid transporter, it is also possible that SLC6A14 plays a role in transporting nutrients not only into epithelial cells, but away from colonizing bacteria81 (this is especially relevant in diseases like CF)81. In times of need

(i.e. when the intestinal tract is stressed by cholera86, ulcerative colitis87, or IBD88),

SLC6A14 has been found up-regulated, and is hypothesized to play a role in fluid regulation during these disease processes.

Interestingly, human ATB0,+ obtained from human large airway and distal lung showed a difference in molecular mass (~70 and ~95 kDa respectively)81. This disparity was also seen between samples of protein collected from the mouse lung, stomach, and colon

(~95 kDa) versus mouse blastocysts and testis (~70 kDa) The fact that the mRNA is the same size (while protein size appears different) points to possible differences in post- translational modification, e.g. glycosylation or alternative splice products. Using

ScanProsite software, Sloan et al. was able to identify seven possible glycosylation sites on the second putative extracellular loop and one on the third4. It is unknown at this time if these different isoforms exhibit differences in function, or if the different isoforms detected by Western blot actually both represent SLC6A14 as this finding has not been repeated.

Similar to human ATB0,+, mouse ATB0,+ protein has been found in mouse stomach, testis, lung (trachea, bronchioles, alveoli, and bronchi89), colon, and blastocysts81.

Functional identification has also been successful in the bullfrog lung90 and rat alveolar epithelial cells91,92. It has additionally been located in ocular tissues such as the conjunctival epithelia, retinal ganglion cells, retinal pigment epithelium, and inner nuclear layer of the retina89, where it is being investigated as a transporter for the delivery of ophthalmic drugs.

3.1.2 The Regulation of System B0,+

There is very little information currently about the regulation of SLC6A14. Recent work has found that B0,+ surface expression increases in the presence of protein kinase C

(PKC) activator phorbol-12-myristate-13-acetate (PMA)93, which is contrary to the observed inhibition of other members of the SLC6 family by PKC. PKC has also been found to have a down-regulatory effect on systems y+ and y+L transport activity94. By scanning the amino acid sequence Sloan and Mager4 located a consensus sequence for casein kinase II binding, however evidence for regulation by this kinase has not yet been shown. The physiological concept of adaptive regulation95 discovered in system A

(a neutral amino acid transport system) and later found in system y+95, can also be predicted to exist for system B0,+, although this has not been confirmed. Adaptive regulation is a common method of amino acid transporter regulation, and has been identified in system B0, another member of the SLC6 family96. Adaptive regulation links substrate availability to transporter expression, whereby there is decreased transporter expression in the presence of abundant substrate (called repression), and increased transport expression in the presence of limited substrate (called de-repression)95.

Repression involves induction of synthesis of proteins involved in transporter degradation or inactivation upon abundant substrate availability97. There are two

phases to substrate-induced transporter de-repression, the acute phase and the chronic phase. During the acute phase reduced availability of substrate promotes the recruitment of preformed transport protein to the cell surface from intracellular pools.

Changes which occur during the chronic phase involve the induction of transporter gene expression to restore the intracellular transporter pools98. Adaptive regulatory functions maximize the efficiency of amino acid transport by conserving resources, and providing the cell with a maximal amino acid transport capacity once nutrients return.

Recent research has shown B0,+ uptake activity can be blocked by the selective non- transportable substrate alpha-methyl-DL-tryptophan (α-MT)99. This was discovered by pre-treatment of human retinal pigment epithelial (HRPE) cells transfected with mouse

ATB0,+ with various tryptophan derivatives followed by measurement of 3H-glycine uptake. Karunakaran et al. found a racaemic mixture of the D and L isoforms of alpha- methyltryptophan was able to block ~85% of glycine uptake at 2.5mM. They proposed

α-MT as a potential cancer therapeutic to be used to deprive tumors of nutrients99, and have recently patented it for this purpose100. They have additionally found that when

4F2hc and LAT1, making up the System L NAA transporter, were co-expressed in the

HRPE cell line, uptake of L-valine through this system could be blocked by α-MT101. α-

MT is therefore an effective blocker of SLC6A14 but may exhibit non-specific binding.

This limitation can be overcome in the radiolabelled uptake experiment by designing protocols to isolate Na+-dependent transporters (i.e. only SLC6A14). The Ussing chamber is a useful tool in this context as well; SLC6A14 is the only electrogenic transporter of CAAs on the apical membrane of respiratory epithelia.

3.1.3 Protein structure of SLC6 Transporters

The SLC6 transporter family comprises 20 Na+ (and some Cl-) -dependent symporters organized into 4 groups, the gabba-aminobutyric acid (GABA) transporters (e.g. transporters of GABA, taurine, and creatine), monoamine transporters (i.e. norepinephrine, serotonin, and dopamine), and two amino acid transporter groups (the glycine and proline transporters and system B0,+ in amino acid transporter group I, and epithelial and brain neutral amino acid transporters in group II) (reviewed in 74). These transporters all regulate extracellular solute concentration by powering uptake to the

Na+ gradient78. All members of the SLC6 family bind one, two or three Na+ ions during a transport cycle. Homologs of members of the SLC6A family are found in the genomes of all animals as well as in some bacteria102. Like SLC6A14, all SLC6 transporters are regulated by PKC however as mentioned previously, SLC6A14 is the only known SLC6 to be up- instead of down-regulated by PKC. They have also demonstrated regulation by other processes such as protein-protein interactions which can result in changes to transporter localization103.

The study of SLC6 biochemistry has been heavily aided by the crystallization of the

LeuT bacterial homolog from Aquifex aeolicus by Yamashita et al. in 200575. The common molecular structure of SLC6 family members is of 12 transmembrane domains, with the first 10 domains making up the core of the transporter and the final two possibly involved in transporter dimerization104. The substrate binding site S1 is located in transmembrane domains (TM) 1, 3, 6, and 8. One Na+ binding site (Na1) is located in domains TM 1, 6, 7, and the other (Na2) is between TM 1 and 8. The binding of 2 Na+ ions forms a dynamic outward-facing intermediate conformation that exposes the

primary binding site to amino acid/neurotransmitter105. Upon substrate binding, the external gate is closed and the substrate is occluded within the pore. The protein then changes to an inside-open conformation, releasing the substrate and ions into the cytosol106. The crystallized bacterial LeuT transporter is Cl- independent, so more elaborate studies have been done to try and uncover the common mechanism of Cl-- dependency of many SLC6 family members. Through site-directed mutagenesis,

107 homology models, and analyses of pKa‟s, Forrest et al. identified a likely location for

Cl- binding in the SERT (SLC6A4) transporter which was close (~5Å) to the Na1 binding site and approximately halfway across the lipid bilayer in residues from TM 2, 6, and 7.

Zomot et al.‟s108 findings, published at the same time as Forrest, are in support of this concept. In the Na+-dependent mouse SLC6A11 and human SLC6A3 proteins, Zomot et al. were able to mimic the presence of Cl- in a Cl--free solution by replacing an acidic amino acid with a basic amino acid using site-directed mutagenesis of a residue located near the Na+ binding sites, thereby suggesting this site for Cl- binding. By doing the reciprocal experiment, mutating a negatively charged amino acid to a positive at the same residue in the Cl--independent homologous bacterial proteins LeuT and Tyt1, they were able to confer Cl--dependent substrate transport. These findings place the Cl- binding site in close proximity to both Na+ binding sites near the midline of the transporter through the plasma membrane. The authors conclude that Cl- is likely required to counterbalance the forces from the Na+ ions, and the central location of the

Na+, Cl-, and substrate binding sites allows for substrate and ion access from both sides of the membrane.

3.1.4 Association of B0,+ with Disease

Increased SLC6A14 expression has been discovered in patients with pathologies involving fluid homeostatic disruption (e.g. cholera, ulcerative colitis) or altered metabolic function (e.g. cancer, obesity). SLC6A14 transcript was found to be up- regulated in rectal biopsies and mucosal specimens from the caecum and rectum in patients with ulcerative colitis compared to matched controls, where it was hypothesized to play a role in managing excessive fluid secretion87,109. Up-regulated SLC6A14 has also been linked by Flach et al. to cholera, another disease characterized by dehydrative water loss110. The authors demonstrated increased intestinal brush-border expression using immunostaining of intestinal tissue biopsies during acute cholera. The authors hypothesized that SLC6A14 was induced as a protective fluid absorption mechanism, whereby water is absorbed iso-osmotically through aquaporins (Figure 1) as a by-product of transport of 2 Na+ 1 Cl- and 1 amino acid. Since cholera toxin- induced secretory diarrhea is caused by CFTR-mediated fluid secretion111, and increased expression of CFTR is also found in ulcerative colitis112, it appears that

SLC6A14 and CFTR are involved in intestinal fluid regulation.

Ganapathy et al. showed that SLC6A14 mRNA and protein are up-regulated in estrogen receptor positive breast cancer113, colon cancer114, and cervical cancer115, similar to the amino acid transporter LAT1116. The pathophysiological reason for this is hypothesized to be due to the increased demand for amino acids for tumor growth, such as arginine and leucine. Leucine is a potent activator of the cell growth and proliferation regulator, the mammalian target of rapamycin (mTOR).

SLC6A14 has shown association with obesity in a genome-wide linkage scan in Finnish and Swedish families117. This is hypothesized to be caused by increased transport of tryptophan (either intestinally or across the blood brain barrier), a precursor to serotonin, which impacts appetite control. Other groups have struggled to replicate these findings however, and a recent meta-analysis has not found any link between this locus and body mass index118.

3.2 System y+L

System y+L, first described by Deves et al.66, may be discriminated from B0,+ by its

Na+ independence of cationic amino acid transport but Na+ dependence of neutral amino acid transport, and proposed basolateral localization in airway epithelia119,68. It is an obligatory exchanger and is responsible for the basolateral efflux of a cationic amino acid for the uptake of a neutral amino acid from the plasma. It is thought to be the major efflux route of cationic amino acids following the apical influx through SLC6A14120 in polarized respiratory epithelia. It is a heteromeric transporter consisting of the 4F2hc heavy chain which is encoded by

SLC3A2 and is required for proper targeting of the transporter to the plasma membrane. This is linked by a disulfide bridge to a light chain121: either y+LAT1 or y+LAT2 encoded by SLC7A7 and SLC7A6 respectively (as the pore forming unit).

Mutations in SLC7A7 leading to absence or reduced function of y+L has been implicated as the cause for lysinuric protein intolerance, a very rare and severe autosomal recessive inherited aminoaciduria122. This disease has wide ranging effects including failure to thrive as an infant, osteoporosis, developmental delay,

chronic renal disease and accumulation of a lipid-rich proteinaceous material in the alveolar space (alveolar proteinosis)123.

3.3 System b0,+

System b0,+ is another heteromeric transporter which functions as an obligate exchanger of cationic or neutral amino acids in epithelial cells of the kidney124, small intestine125, liver and placenta126. In these structures it contributes to the apical influx of cationic amino acids (especially cysteine) from the lumen121,127 for intracellular neutral amino acids. Transport is powered by the high intracellular concentration of neutral amino acids, the electrical potential across the apical membrane (cytosol negative), and the generation of a cystine concentration gradient by the intracellular reduction of cystine to cysteine128. mRNA and function of b0,+ have not been found in the lung119.

System b0,+ is composed of a light chain b0,+-AT (SLC7A9) and heavy chain rBAT

(rabbit b0,+ amino acid transporter-related protein) (SLC3A1). Autosomal recessive inherited mutations in rBAT cause cystinuria, a defect in cysteine and cationic amino acid transport in the small intestine and kidney129 leading to hyperexcretion of cysteine and dibasic amino acids in the urine and kidney calculi causing obstruction, infection, and renal insufficiency.

3.4 System y+

System y+, named for its characteristic substrate lysine, is a monomeric construct of one of the CAT (cationic amino acid transporter) proteins (CAT1, 2, 3, or 4) encoded by members of the SLC7 gene family (SLC7A1, 2, 3, and 4, respectively). System y+ is the most widely expressed cationic amino acid transporter, mediating the Na+-

independent influx of cationic amino acids in most non-epithelial cells. In polarized epithelial cells it has been localized to the basolateral membrane130,131,132. The resultant transport of cationic amino acids is electrogenic133 due to unitary transport of a positively charged amino acid. Transporter activity is stimulated by hyperpolarization of the membrane, and high concentration of substrate on the opposite membrane side

(trans-stimulation)121. At a resting potential of -50 mV y+ can maintain an 8-fold inward concentration gradient of CAAs at the plasma membrane, and membrane hyperpolarization increases this even more as represented by an increase in the Vmax for CAA influx133. Expression of y+ mRNA has been shown in the Calu-3 human respiratory submucosal gland cell line119 and by microarray analysis in mouse lung134.

The inducible form of system y+, CAT-2 can be upregulated in mouse macrophages in asthma135,134,136, which is hypothesized to play a role in providing arginine for production of nitric oxide by way of inducible nitric oxide synthase (iNOS). Nitric oxide is an important molecule in the lung for bronchodilation, regulation of inflammation, bacterial killing and regulation of ion flux137. Major discriminators of system y+ from system B0,+ are its Na+ and Cl- -independence of amino acid transport and its specificity for only cationic amino acids. Studies in mammalian cells as well as Xenopus oocytes show that

PKC activation causes a down-regulation of system y+ activity138.

Based upon these data, cationic amino acid transport in the lung is hypothesized to be attributable to the combined function of systems B0,+, y+, and y+L139. Current research indicates an apical localization of B0,+ 84 and basolateral localization of y+ and y+L139, yet the localization and relative contribution to CAA transport of all three of these transporters have not been definitively confirmed in primary human bronchial cultures.

Rationale

Cystic fibrosis is the most common deadly genetic disease in Caucasian populations140,

- - and respiratory infection caused by defective CFTR-mediated Cl and HCO3 transport is the highest cause of CF-related morbidity and mortality141. Mutations in CFTR have shown a surprisingly poor correlation to CF phenotype44 and so external genes are thought to play a significant role in modifying CF. Using a hypothesis-driven genome- wide association assay (GWAS-HD) approach, SNPs in SLC6A14, a NaCl-dependent transporter of basic and neutral amino acids4, have been recently identified as being statistically significantly associated with an increased risk of MI, increased severity of respiratory disease, and age of first P. aeruginosa infection in cystic fibrosis1,2.

However, the physiological consequence of the SNPs and any potential interaction of

SLC6A14 with CFTR remain unclear, despite its localization to the apical membrane of respiratory epithelia, similarly to lung CFTR. There is currently very little information regarding the role of SLC6A14 in the airway; but current hypotheses predict roles in epithelial nutrition, fluid regulation, infection control, and/or management of a low-amino acid concentration on the airway surface for gas exchange81,86. Alterations of apical membrane ion transport as occur in CF change the homeostasis of the airway, thus

SLC6A14 may behave differently in the CF lung. It is therefore important to study

SLC6A14 functional expression in the CF as well as the non-CF context to better illuminate SLC6A14‟s role in the airway.

Hypothesis and Aims

SLC6A14 serves as a major cationic amino acid transporter on the apical surface of CF and non-CF airway epithelia.

The specific aims of this graduate project are:

1) To develop a functional method for assaying expression of SLC6A14 in cultured airway epithelial cells and primary human airways.

2) To compare the functional expression of SLC6A14 in CF and non-CF cultured airway epithelial cells and primary human airways

Methods

1 Cell Line Culture

CFBE41o- cells were provided by Dr. Dieter Gruenert (University of California, San

Francisco). Untransfected cells as well as cells stably transfected with Δ-F508 (CFBEdf) or wild-type (CFBEwt) CFTR were used142. Cells were grown and maintained in 75 cm2 flasks with media changed every 2-3 days, and split upon 80-100% confluency to a dilution of 1:5. Eagles minimum essential medium was supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (stock solution contains 10000 IU penicillin, 10000ug/ml streptomycin). Transfected cell media also contains 0.3% 100 mg/ml hygromycin B as a selectivity reagent. For uptake experiments cells were plated at high density onto 24-well flat bottom tissue cultures plates. Media was changed every

2-3 days until cells showed confluency then every day for 5 days with experiments performed on the 6th day.

2 Primary Culture

Primary airway cells are obtained from bronchial tissue from CF and non-CF transplants thanks to collaboration between the Universities of Toronto and Iowa (Dr. Shaf

Keshavjee and Dr. Michael Welsh respectively). Cells are plated on a porous membrane (Millicell Millipore or Costar Transwell Clear) with a 0.4 μm pore size. This encourages cell polarization and differentiation and gives access to the apical and basolateral chambers143. Culture of cells with air on the apical side and media on the

basolateral side (an air-liquid interface) encourages the full differentiation of cells into a secretory and ciliated phenotype144.

3 Arginine Uptake Studies

All amino acids used are of the „L‟ stereoisomer. In cell culture experiments uptake kinetics of 1 μCi/ml of L-[2,3-3H]-arginine (specific activity 54.6 Ci/mmol) was examined during different time intervals, varying presence of competing arginine or other amino acids, or the presence or absence of buffer Na+ to determine the functional expression of various cationic amino acid transporters in these cells. At the beginning of each experiment cells were washed 2 times with 37ºC HEPES buffer at pH 7.4 followed by a

30 minute incubation with fresh HEPES solution to reduce intracellular amino acid pools and remove residual culture media. Cells were then exposed to the experimental buffer containing 1 μCi/ml of L-[2,3-3H]-arginine. At the end of each uptake buffer incubation, cells were washed 3x with ice-cold HEPES buffer containing 10 mM arginine to out- compete all uptake of L-[2,3-3H]-arginine, then cells are lysed on ice on a shaker for 15 minutes following addition of 200 μl 0.5 M NaOH. 100 μl of lysate was mixed with 1 ml of EcoScint scintillation buffer and the scintillation count was captured using a Beckman

Scintillation Counter (LS-6000IC).

3.1 Calculating Arginine Uptake Each additional amino acid added competes with L-[2,3-3H]-arginine for uptake into the cell and therefore causes a reduction in measured counts per minute (CPM, “C”) (see

3 Equation 1). The 10 mM arginine control ( ) detects how much L-[2,3- H]- arginine is transported into the cell in the presence of maximal uptake competition, either by diffusion or non-specific transport, and serves as a background CPM control.

This is subtracted from each CPM value. The resulting value is multiplied by the moles of arginine in a 100 μl sample of uptake buffer then divided by the CPM of that uptake buffer solution ( ), providing a ratio of moles arginine to CPM. This value is corrected for dilution in the scintillation buffer (e.g. 100 μl of sample is diluted in 1 ml of scintillant), divided by the time of uptake, and then normalized to mg protein in each well.

Equation 1 The determination of arginine uptake in cell lines.

( ⁄ ⁄ )

( ) [ ]

For the calculation of uptake into primary cultures, the cell lysate CPM (C) is multiplied by the ratio of moles arginine/CPM in the apical chamber at t=0. Similar to equation 1, this is multiplied by a dilution factor and then divided by the mg protein on the insert.

Equation 2 The determination of intracellular arginine uptake in primary cultures.

( ⁄ ⁄ )

[ ] ( )

Beginning at t=0 and every 10 minutes for 60 minutes total, 100 μl is removed from the apical and basolateral chambers (Equation 3). At each of the time points where t ≠ 0, the initial CPM in the basolateral chamber (Cinitial) is subtracted from the measured

CPM (C). This value is then multiplied by a ratio of moles arginine/CPM and divided by the area of the insert in cm2.

Equation 3 The determination of transepithelial arginine transport in primary cultures.

( ⁄ )

( )

3.2 Dose Response Experiments Following a 30 minute incubation in HEPES buffer, CFBE41o- cells were incubated with

1μCi/ml of L-[2,3-3H]-arginine +100 μM arginine for 5, 10, 20, 30, 40, 50, and 60 minutes to examine the time course of arginine uptake and determine the optimal time point for subsequent dose-response studies. In a separate set of experiments, uptake buffers were made containing 20, 50, 100, 200, 300, 400, or 500 μM or 10 mM arginine and 1μCi/ml L-[2,3-3H]-arginine in HEPES buffer. Each was exposed to CFBEwt or

CFBEdf cells for 15 minutes. Cell lysates from both experiment sets were collected and uptake values (V: nmol/min/mg protein) were fit to the Michaelis-Menten transport kinetics equation, where Y=substrate velocity (V, nmol/min/mg protein) and X=substrate concentration (μM). Kt is the Michaelis-Menten constant for concentration of substrate causing half-maximal transport, Vmax is maximal substrate velocity.

Equation 4 Michaelis-Menten transporter kinetics.

Y=Vmax*X/(Kt+X)

3.3 Competitive Inhibition Experiments

Using CFBEwt and CFBEdf cells, L-[2,3-3H]-arginine uptake was measured in the following conditions (all contain 100 μM arginine and 1μCi/ml L-[2,3-3H]-arginine: 1) control, 2) 0 Na+ buffer, 3) 0 Na+ buffer and 2 mM leucine, 4) 2 mM leucine, 5) 0 Na+ buffer and 2 mM leucine and 2 mM lysine. These conditions were chosen to specifically isolate the function of the four known cationic amino acid transporters (

Table 2). High concentrations of competing amino acids were chosen to maximally activate the intended transporters. Additionally, uptake of 100 μM arginine was examined in presence of 2 mM α-methyltryptophan (α-MT, a selective blocker of

SLC6A1499) in the presence and absence of Na+ (see section 5 Solutions page 35).

After the design and implementation of the protocols described here, a paper that undertook very similar experiments in neuronal cells was found. It seems appropriately honest to mention it here, Bae et al.145.

Contributing System-specific uptake Buffer Condition Transporters calculation 1 control y+; y+L; b0,+; B0,+ B0,+ = (1-2) 2 0 Na+ y+; y+L; b0,+ b0,+ = (2-3) 3 0 Na+, leucine y+; y+L y+L = (3-4) 4 Na+, leucine y+ y+ = (4-5) 5 Na+, leucine, lysine Passive diffusion

Table 2 Protocol utilized for examining competing cationic amino acid transporters.

Conditions and calculations used to separate the competing cationic amino acid transport systems for 100 μM arginine. (1) System B0,+ is the only Na+-dependent transporter of arginine and so its function was investigated by comparing uptake in the presence or absence of Na+. (2) System b0,+ is responsible for the competitive inhibition of arginine uptake by leucine in the absence of Na+. (3) Leucine will compete for the uptake of arginine through y+L in the presence but not the absence of Na+. (4 & 5)

Remaining transport is a combination of non-specific diffusion and transport through y+ that can be distinguished by using lysine.

3.4 Transepithelial Flux Experiments.

Human primary airway epithelial cells grown on 0.4 μm-pore inserts were mounted in an

Easy Mount Ussing Chamber (Physiologic Instruments) to allow separated access to

the apical and basolateral membrane. The cells were bathed in HEPES buffer maintained at 37°C and bubbled with 95% O2 and 5% CO2. Monolayer integrity was monitored throughout each experiment by calculating transepithelial resistance (Rte) using 1 mA pulses every 60s and Ohm‟s law (voltage [V] = current [I] * resistance [R]).

All measurements are recorded in open circuit mode in an Ussing chamber using

Ag/AgCl electrodes. Resistance of each insert was ≥ 100 Ω/cm2 with an average of 477

Ω/cm2.

Following a 15 minute equilibration period in warmed buffer, 100 μM arginine and

1μCi/ml L-[2,3-3H]-arginine ± apical Na+ or ± apical 2mM α-MT was added to the apical chamber. Transepithelial arginine flux was determined by removing 100 μl samples from the apical and basolateral chambers at t (min) = 0, 5, 10, 20, 30, 40, 50, 60 min. Finally, the primary cells were lysed with 0.5 M NaOH and arginine uptake and transepithelial transport was calculated as described above.

4 Cell Protein Assay

Total cell protein was determined using the microplate Bio-Rad protein assay (cat# 500-

0006) utilizing a Coomassie Brilliant blue G-250 dye. Protein detection is based on a shift in maximal absorbance from 465 nm to 595 nm upon protein binding. The concentration of protein in samples diluted 1:2 in ddH2O is determined using a bovine serum albumin standard curve.

5 Solutions

All cell-culture reagents (fetal bovine serum, penicillin/streptomycin, phosphate buffered saline, hygromycin-B) and media were purchased from Wisent Bioproducts (St-Bruno,

Quebec). All amino acids (lysine, leucine, arginine, and alpha-methyltryptophan) and buffer reagents were purchased from Sigma-Aldrich (Oakville, Ontario). HEPES buffer consists of (in ddH2O) 25 mM HEPES, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8

+ mM MgSO4 and 5 mM glucose brought to pH 7.4 with Tris-base. 0 Na HEPES buffer is made by replacing 140 mM NaCl with 140 mM N-methyl-D-glucamine-Cl (NMDG-Cl). L-

[2,3-3H]-arginine was purchased from American Radiolabeled Chemicals Inc. (MO,

USA).

6 Statistics

Data are presented as means ± standard deviation or ± 95% confidence intervals (for

Michaelis-Menten constants). n=3 for dose response and time course experiments.

Conditional uptake experiments were performed with 3-4 technical replicates and the mean was taken for further analysis. Primary culture experiments have n=4 for all conditions. To control for day-to-day variability in the magnitude of total arginine uptake, conditional uptake values were normalized to total arginine uptake from the same day.

Arginine uptake under different conditions in CFBEwt and CFBEdf cells was analyzed using a two-way ANOVA and a Tukey multiple comparisons test. Experiments on primary cells were analyzed in a paired fashion using a two-way ANOVA and Tukey‟s multiple comparison test. Data were analyzed using Graphpad Prism (v.6).

Results

1 Kinetics of L-arginine uptake

In order to establish optimal parameters for studying arginine uptake in the CFBE41o- cell line I first determined the time and the concentration at which cellular arginine uptake is linear. Determining cellular arginine uptake at different time points, the intracellular uptake of 100 μM arginine in CFBE41o- cells was in a linear range with an exposure time of 15 minutes (Figure 4). This time interval was used in subsequent studies. Secondly, using CFBE41o- cells stably transfected with WT or delta-F508

CFTR, uptake experiments were performed with increasing doses of arginine ranging from 20 μM – 500 μM. Arginine uptake followed Michaelis-Menten uptake kinetics in

CFBEwt and CFBEdF cells (Figure 5). Overall, the arginine uptake kinetics showed a statistically significant difference between CFBEdf and CFBEwt (A sum-of-squares F- test confirmed that the data could not be explained by a single kinetics curve, p<0.0001). In CFBEwt cells the half maximal concentration for arginine transport (Kt) was 98.9±13.2 μM and the maximal uptake velocity (Vmax) was 1.53±0.07 nmol/min/mg protein. CFBEdF cells displayed a statistically significant higher affinity for arginine uptake (Kt=70.5±13.2 μM) and a larger Vmax of 1.72±0.11 nmol/min/mg protein

(p<0.0001). A 100 μM arginine concentration was chosen for subsequent experiments based upon these results, which are consistent with previous literature3,4,146.

Figure 4 Time course of 100 μM arginine uptake in CFBE41o- cells.

CFBE41o- cells were exposed to 100 μM arginine and 1 μCi/ml of l-[2,3-3H]-arginine for between 5 and 60 minutes. 3H was measured in cell lysates. Points represent mean ±

SD, n=3.

Figure 5 Michaelis-Menten transport kinetics in CFBEwt and CFBEdf cells.

CFBEwt and CFBEdf cells exhibit significantly different intracellular Michaelis-Menten uptake kinetics for 20-500 μM arginine over 15 minutes. Points represent mean ±SD, n=3. Kt and Vmax are ± 95% CI.

2 Competitive Inhibition experiments

CFBEwt and CFBEdf cells were exposed to five different solutions containing different amino acid and Na+ combinations designed to probe for the functional presence of known cationic amino acid transporters (Table 2, Table 3).

2.1 B0,+ In the absence of Na+, 100 μM arginine uptake was significantly reduced in CFBEdf cells (p=0.004) and CFBEwt cells (p=0.04) indicating the functional presence of Na+- dependent arginine transporter SLC6A14 (Figure 6, Figure 7, Table 2). Functional

SLC6A14 in both cells is also supported by the statistically significant reduction in arginine uptake by α-MT in the presence but not absence of Na+ in both CFBEwt and

CFBEdf cells (Figure 6). By comparing the mean percentage of Na+-dependent arginine uptake for both cell types, contribution of SLC6A14 to total arginine uptake in both cell types was not statistically significantly different between CFBEwt (32±12%) or CFBEdf

(35±10%), p=0.7.

2.2 b0,+ The addition of 2 mM leucine (arginine + leucine –Na+) to a Na+-free buffer containing

100 μM arginine (arginine –Na+) did not significantly reduce arginine uptake in CFBEwt or CFBEdf cells (p>0.9 for both), suggesting that system b0,+ (the neutral and cationic amino acid exchanger) is not functionally present in these cells (Figure 7, Table 2).

Figure 6 Na+-dependent and α -MT-sensitive arginine uptake in CFBEdf and CFBEwt cells. CFBEwt and CFBEdf cells were exposed to 100 µM arginine and 1 µCi/ml 3H-arginine in one of 4 conditions for 15 minutes designed to elucidate the contribution of SLC6A14 to arginine uptake and test its sensitivity to 2mM α-methyl-DL-tryptophan. To control for intra-experimental variability, all values are normalized to control 100 μM arginine uptake measured in each experiment. The significant attenuation of arginine uptake due to α -MT and 0 Na+ in CFBEwt and CFBEdf cells, indicates the functional presence of system B0,+ in primary human airway cultures. No statistically significant difference between α -MT and 0 Na+ suggests inhibition of SLC6A14-mediated arginine uptake in both cases. CFBEwt n=4, CFBEdf n=5. *p<0.05, **p<0.01. Values presented as means

± SD.

Figure 7 Competitive inhibition and determination of cationic amino acid transporter systems.

CFBEwt and CFBEdf cells were exposed to 100 μM arginine in one of 5 different conditions with 1 µCi/ml 3H-arginine for 15 minutes, followed by cell lysis and counting of intracellular 3H-arginine. To control for intra-experimental variability, all values are normalized to control 100 μM arginine uptake measured in each experiment. There was functional evidence for system B0,+ and system y+ in both cell types, with no difference in system-specific uptake between cell types. *p≤0.05, **p≤0.01 CFBEwt n=3; CFBEdf n=4.

2.3 y+L Leucine competes for arginine uptake through system y+L in the presence (arginine + leucine) but not absence of Na+ (arginine + leucine –Na+) (Figure 7, Table 2). There was no statistically significant difference between arginine uptake in presence of leucine with and without Na+ in CFBEwt (p=0.62) or CFBEdf cells (p=0.57), so functional expression of this system cannot be confirmed.

2.4 y+ The addition of lysine (arginine + leucine + lysine) to buffer containing Na+ and leucine will compete with arginine transport via system y+ (Figure 7, Table 2). This system was functionally present in CFBEwt (p=0.02) and CFBEdf cells (p=0.005). There was no statistically significant difference in y+ activity between CFBEwt and CFBEdf cells. By comparing the mean percentage of system y+-mediated arginine uptake for both cell types, contribution of y+ to total arginine uptake in both cell types was not statistically significantly different between CFBEwt (32±17%) or CFBEdf (36±8%), p=0.7.

3 Primary Human Bronchial Culture Experiments 3.1 Intracellular Uptake of L-[2,3-3H]-arginine Primary cultures were mounted in an Ussing chamber with 100 μM arginine + 1 μCi/ml

L-[2,3-3H]-arginine applied to the apical side, and basolateral fluid was collected over 60 minutes. Cell lysates were collected at the end of each experiment. Uptake values are given in nmol/mg protein/60 minutes. Intracellular uptake of arginine (representing influx-efflux) (Appendix Table 4) was statistically significantly reduced with the removal of buffer Na+ in non-CF (58.58±11.54 to 28.78±11.73, p=0.0079), and CF cells

(76.19±28.79 to 24.64±17.20, p=0.017) (Figure 8). Similarly, α-MT statistically significantly reduced arginine uptake in both cell types (non-CF: 11.42±2.05, p=0.0007,

CF: 7.74±1.44, p=0.035). There was no difference in arginine uptake between the 0 Na+ and α-MT conditions (non-CF p=0.12, CF p=0.26).

Figure 8 Arginine uptake in non-CF (a) and CF b) primary airway cultures 60 minutes after apical addition of 100 µM L-arginine.

Uptake of arginine in either cell type was highly sensitive to the presence of Na+ or α-

MT. n=4 for both non-CF and CF. Values are means ± SD. *p<0.05, **p<0.01,

***p<0.0001

3.2 Transepithelial Transport of L-[2,3-3H]-arginine

Primary airway cells from non-CF and CF cells were mounted in an Ussing chamber to allow separation of the apical and basolateral chambers. Transepithelial arginine transport was assessed as basolateral appearance of arginine after application of 100

μM arginine and 1μCi/ml L-[2,3-3H]-arginine to the apical side. Removal of sodium statistically significantly reduced transepithelial arginine transport in non-CF and CF airway cells over the time course of 60 minutes (non-CF: p=0.03, CF: p=0.02). Addition of 2 mM α-MT to the apical side had a similar effect (non-CF: p=0.018, CF: p=0.002).

There was no difference in transport between the 0 Na+ and α-MT conditions in either cell type (non-CF p=0.87, CF p=0.23) (Figure 9). This inhibition pattern is characteristic of system B0,+ function at the apical membrane.

As per the cell culture results, non-CF and CF primary inserts did not demonstrate any statistically significant differences in arginine uptake. There was no difference in total intracellular arginine uptake between non-CF and CF cells (t-test; p=0.3) or intracellular

Na+-dependent arginine uptake as per B0,+ (t-test; p=0.14).Comparing baseline transepithelial transport of arginine in non-CF to CF over 60 minutes, there was no significant difference (p=0.25), nor was there a difference between cells in the 0 Na+

(p=0.3) or α-MT (p=0.6) conditions.

Figure 9 Apical to basolateral transport of 100 μM arginine in non-CF and CF primary airway cells.

Transepithelial arginine transport was reduced by the removal of Na+ and application of

α-MT supporting the functional presence of SLC6A14 in both (a) non-CF (n=4) and (b)

CF (n=5) primary cultures (accounting for ~50% of uptake in both cell types). Points are mean ± SD.*p<0.05.

Discussion

The human CF bronchial cell line CFBE41o- shows functional expression of a Na+- dependent arginine transport system, and these data were replicated on the apical membrane of primary human bronchial cells from non-CF and CF donor lungs. Since

SLC6A14, the recently cloned protein for the amino acid transport system B0,+, is the only known Na+-dependent transporter of cationic amino acids in the mammalian plasma membranee.g.59,64,104, these findings indicate the functional expression of

SLC6A14 in both of these airway epithelial cell systems. This is supported for the first time by the inhibition of arginine uptake by α-methyl-DL-tryptophan, an amino acid derivate which was previously shown to inhibit SLC6A14-mediated transport when over- expressed in Xenopus oocytes99. In CFBEwt and CFBEdf cells at 15 minutes of incubation, SLC6A14 accounted for ~25-45% of total arginine uptake as determined by

Na+-dependent and α-MT-sensitive arginine uptake (Figure 7). There was a similar pattern in primary human airway cells, where both Na+-dependent and α-MT-sensitive intra-cellular and trans-cellular arginine uptake accounted for ~50% of total arginine uptake accumulated over 60 minutes. These findings agree with those made by Rotoli et al. in the Calu-3 human bronchial submucosal gland adenocarcinoma cell line83, in which SLC6A14 was seen to be critical for transepithelial cationic amino acid transport, and was responsible for ~50% of apical arginine influx (as measured by 3H-arginine uptake using cells cultured on permeable supports). The description of SLC6A14 as a major amino acid transporter for arginine in primary airway cells is novel, and highlights the importance of studying SLC6A14 function in airway epithelia.

These data showing the functional presence of B0,+ in airway cells are in agreement with electrophysiological studies performed by Galietta et al.84 and Gonska (ongoing studies) in primary human airway cells, as well as functional and immunohistochemical investigations in excised human and mouse airway samples by Sloan et al.81. These findings agree with the properties originally described for system B0,+148 and by way of expression studies by Sloan et al. in Xenopus oocytes4 it can be concluded that the observed function matches that of SLC6A14 expression. There was also no difference in either Na+-dependent or total arginine uptake between non-CF and CF primary cells, or between CFBEwt and CFBEdf cells. This finding is not surprising as it agrees with the observations made by Galietta et al.84.

α-MT as an inhibitor of SLC6A14-mediated amino acid uptake is a subject of great interest by the Ganapathy lab101,149 due to its ability to effectively block the transporter in cancerous cells. With support from the Bear and Gonska labs in the form of unpublished electrophysiological data in primary airway cells, this research demonstrates the first published example of the usage of α-MT as a blocker and functional read-out of SLC6A14 in human airway epithelial cells. This compound offers a one-of-a-kind pharmacological read-out of SLC6A14 function that can be applied in numerous protocols to investigate the physiology and pathophysiology of SLC6A14 in the respiratory tract.

Overall kinetic dose-response curves for arginine uptake were statistically significantly different between CFBEwt and CFBEdf cells (Figure 5 Michaelis-Menten transport kinetics in CFBEwt and CFBEdf cells.Figure 5). These curves produced statistically significantly different Michaelis-Menten constants, with Kt values of 72.8±16 μM for

CFBEwt and 95±14 μM for CFBEdf, and Vmax values of 1.72±0.11 nmol/min/mg protein for CFBEwt and 1.53±0.07 nmol/min/mg protein for CFBEdf. This is in agreement with

0,+ 4 84 the 80 – 150 μM range of Kt‟s reported for the B system by Sloan and Galietta (Vmax values for arginine uptake through SLC6A14 have not been reported). The nature of these experiments, which were performed on flat plastic tissue culture plates, predicts that these uptake constants are representative of all transporters with an affinity for arginine in this concentration range, regardless of the subcellular localization. It is also well established that the mammalian CAA transporters have overlapping affinities for arginine (Table 1), and so it is difficult to explain the discrepant kinetic findings given only this data. Subsequent competitive inhibition experiments used optimal arginine concentration and incubation time data obtained from kinetic experiments to attempt to resolve the functional presence, and if possible relative contribution to arginine uptake, of each CAA transporter system.

Functional evidence for system y+ was found in both cell lines and contributed to approximately 30-50% of total arginine uptake, however systems y+L and b0,+ were not found in either cell. System b0,+ has not been functionally or molecularly identified in the human lung129,83 so its functional absence here as well is not surprising. As discussed earlier, subcellular localization is not specifically determined by this assay. It is possible that solutions delivered to cells plated on flat plastic are able to diffuse paracelluarly150,151, and uptake can be achieved by transport systems which have been described as basolaterally located in epithelia (e.g. y+L83 and y+139). System y+ is the most common entry route for arginine in most cells studied in human physiology139, thus it is no surprise it is functionally well expressed in this system. Lack of statistically

significant involvement of system y+L, could be due to a number of potential factors, which, when considering previous findings of system y+L in airway cells83,152, are likely a result of methodological limitation. This system is an exchanger which is dependent on intracellular Na+ and a neutral amino acid in exchange for influx of extracellular arginine to occur, it thus has more potential rate-limiting factors for each transport cycle compared to system y+ (a uniporter). If arginine succeeds in diffusing paracellularly to the basolateral membrane, it may be possible that the concentration reaching y+L is too small to cause statistically significant uptake as detected using these methods.

There are several limitations of the methods used. Despite being a powerful model, primary human airway epithelial cells are a limited resource and immortalized bronchial epithelial cell models were used. Cell line uptake was done using flat bottom tissue culture plates, thus it was impossible to explicitly distinguish between the apical and basolateral membrane transporters in the otherwise differentiated and polarized cells.

The use of these cell lines may also not necessarily represent in vivo physiology due to the consequences of immortalization or protein overexpression. Specifically in regards to amino acid transporters, immortalized (e.g. cancerous) cells may have an increased nutrient demand and thus altered activity of amino acid transporters not representative of native cells115. Overexpression of CFTR within these cell lines could also place increased demand on the cellular anabolic and catabolic pathways, especially increased degradation products overloading the endoplasmic reticulum153 in CFBEdf cells, or increased amino acid requirements for growth/synthesis in either overexpression cell line. It is likely that the small sample size of primary airway cells analyzed using the methods presented here was unable to detect any differences in SLC6A14 function in

non-CF and CF patients that may have resulted from the presence of risk SNPs identified by Sun and Li1,2 . Considering that only patients with severe CF (a portion of the CF population) were more likely to have certain risk SNPs in the SLC6A14 gene1, it will take larger sample sizes and likely stratification by presence of a specific SNP‟s to further elucidate differences in function that may result from SNP expression.

SLC6A14 was responsible for ~50% of arginine transport into and across primary respiratory epithelia. Combined with recent findings of SNPs in SLC6A14 being associated with MI, severe lung disease and age of first P. aeruginosa infection, questions then arise about what role an amino acid transporter could have in the airway, and how it could modify CF airway disease. It is likely that SLC6A14 serves a specialized role in the lung outside of only being a nutrient transporter, due to its ability to transport a broad range of amino acid substrates against the amino acid concentration gradient from coupling transport to the NaCl gradient81. Precedent is set by mutations in the gene encoding the system y+L amino acid transporter causing lysinuric protein intolerance, which is partially characterized by respiratory distress and alveolar proteinosis123. It is known that the airway surface liquid (ASL) of healthy patients contains a high concentration of free amino acids 154 a result of rapid endogenous protein turnover from macrophages and a preference of epithelial cells to utilize locally digested amino acids to synthesize new protein155. Higher levels of amino acids in the airway lumen are linked to an increased severity of respiratory disease including CF exacerbations156. Specifically, the amino acids arginine and lysine, both substrates for SLC6A14 (and y+L), are known to reduce the surface activity of pulmonary surfactant157 which can lead to small airway collapse. This provides an

explanation for findings by Li et al.2 who linked SLC6A14 to increased severity of CF respiratory disease and younger age of first P. aeruginosa infection. Infection and inflammation are very important components to CF pathogenesis, and a transporter such as SLC6A14 would be able to remove amino acid nutrients (even D-amino acids76, predominantly found in bacteria) from the airway lumen thereby helping to clear infection. As mentioned above, net transport of a positive charge by SLC6A14 would result in the paracellular and transcellular movement of water. SLC614 may therefore also play a role in water homeostasis in certain situations (e.g. cholera, or in CF). In this case, activity of SLC6A14 may lead to a decline in ASL height, leading to a reduction in respiratory health of the already dehydrated CF lung. Mucous plugging, air trapping, and a lack of bacterial clearance causing infection and inflammation from CF mutations are therefore potentially exacerbated by dysregulation in protein homeostasis in the

ASL as a result of SLC6A14 dysfunction, potentially leading to increased risk of respiratory distress and bacterial infection..

In summary, I have established a protocol based on radioisotope uptake that allows identification and quantification of the functional expression of SLC6A14 in lung epithelial cells. Using this protocol based on Na+ dependency and sensitivity to α- methyl-DL-tryptophan, I have demonstrated the functional expression of SLC6A14 in human airway epithelial cells. This amino acid transporter is of particular interest, since it has been associated with increased severity of CF disease in the intestine and lung.

These studies are some of the first to interrogate the functional role of SLC6A14 in cystic fibrosis. They pave the way for further studies into the role of SLC6A14 and provide a functional read-out of SLC6A14 function.

Future experiments should be directed at investigating the effect of SLC6A14 on bacterial infection, fluid regulation, its interaction with CFTR, and the functional consequence of the SNPs found by Sun et al.1. Investigations into the functional expression of cationic amino acid transporters would be strengthened by RT-PCR experiments to determine the expression of each of the cationic amino acid transporters y+, y+L, and b0,+ in primary airway cells and CFBE cells. This would lay groundwork for understanding the primary transporters for amino acid entry into airway cells, which has implications for CF but also other airway diseases such as asthma (i.e. arginine uptake into airway cells is necessary for NO production, an important factor in asthma pathology). Quantitative RT-PCR could additionally provide information about possible changes in expression as a result of SLC6A14 risk SNP expression in primary airway cells. Confirmation of SLC6A14 as the only Na+-dependent arginine transporter expressed in these cells should also be investigated using the comparison of the magnitude of 3H-arginine uptake ± shRNA designed to reduce SLC6A14 protein translation (thereby supporting the use of Na+-dependent arginine uptake methods).

These experiments are currently underway using the Caco2 intestinal adenocarcinoma cell line. This would also assist in identifying the presence of non-SLC6A14 specific targets of α-MT.

SLC6A14 could potentially interact with ASL-dependent bacterial killing. Microscope- based experiments to investigate the impact of SLC6A14 on fluid regulation, ASL viscosity/ciliary beat frequency, and the capacity of the ASL to host bacteria with and without functionally expressed SLC6A14 would be valuable to placing the importance of

SLC6A14 in the general picture of lung physiology, and for revealing potential avenues

for CF pathophysiology research and treatment. Information about interactions between

CFTR and SLC6A14 would be vital to the determination of its effect in CF, including the use of mouse knock-out studies and primary cell electrophysiological studies (currently in progress). These studies could begin to answer if activation of SLC6A14 modifies

CFTR-mediated Cl- secretion, and using mouse knockout models we will be able to investigate the effects of SLC6A14 knock-out systemically as well as its effect on CF phenotype when crossed with CFTR KO mouse models.

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Appendix

CFBEwt (n=3) CFBEdf (n=4) Arg Mean 1.280 1.225 SD 0.289 0.657 0 Na+ Arg Mean 0.847 0.769 SD 0.027 0.264 Arg + Leu Mean 0.495 0.516 SD 0.143 0.127 0 Na+Arg + Leu Mean 0.702 0.702 SD 0.198 0.172 Arg + Leu + Lys Mean 0.109 0.113 SD 0.048 0.062 Arg+ aMT Mean 0.543 0.699 SD 0.123 0.247 0 Na+Arg + aMT Mean 0.657 0.600 SD 0.075 0.084

Table 3.3H-arginine uptake data from CFBEwt and CFBEdf cells.

Values are in nmol/min/mg protein.

r 0 Na aMT CF (n=4) mean 76.1917371 24.6416806 7.74047552 SD 28.791542 17.1981298 1.44242409 non-CF (n=4) mean 58.5771559 41.4642301 11.419399 SD 11.5407631 11.7250329 2.04712879

Table 4 Primary culture intracellular 3H-arginine uptake data

Values in nmol/60 minutes/mg protein