Amino acid transporters as potential drug targets and their implications in toxicology

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AMINO ACID TRANSPORTERS AS POTENTIAL DRUG TARGETS AND THEIR IMPLICATIONS IN

TOXICOLOGY

Ahmed Montaser Master of Science thesis

University of Eastern Finland, School of Pharmacy Master´s Degree Programme in General Toxicology October 2018

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UNIVERSITY OF EASTERN FINLAND, Faculty of health sciences.

School of Pharmacy

Master’s degree in general toxicology.

AHMED MONTASER: Amino acid transporters as potential drug targets and their implications in toxicology.

Master’s thesis, 71 pages

Supervisors: Professor Jarkko Rautio, Associate Professor Jaana Rysä and M.Sc Jussi Kärkkäinen.

Keywords: Amino acid transporters, L-type amino acid transporter 1, LAT 1, transporter mediated toxicity, trans-stimulation assay.

ABSTRACT

Amino acid transporters play a pivotal role in the homeostasis of amino acids in our tissues.

They are mainly involved in the neurotransmission, absorption of amino acids and passage of amino acids across the biological barriers such as blood-brain barrier (BBB). Therefore, imbalance of their functions may give rise to serious health adverse effects. Disruption in the glutamine/glutamate cycle may manifest as several neurodegenerative and psychotic diseases.

Malfunction in the intestinal or renal (re)absorption of amino acids leads to phenotypes characterized by aminoaciduria such as hartnup disease, cystinuria or lysinuric protein intolerance. Downregulation of some transporters in placenta may lead to some developmental disorders. Dysfunction of some amino acid transporters were identified as risk factors in some chronic diseases such as cardiovascular diseases and diabetes.

Amino acid transporters are expressed in almost all our tissues. However, they display different patterns of expression, substrates selectivity and substrates affinity. Thus, some of them have been successfully exploited in the targeted drug delivery. For instance, L-type amino acid transporter 1 (LAT1) is expressed in brain at higher concentration than in any other tissue.

Therefore, it can be utilized to improve the specific uptake of neuropharmaceuticals into brain cells. Additionally, LAT1 and other amino acid transporters are selectively overexpressed in cancer cells to meet their increased demand of amino acids. Hence, this selective expression could be either inhibited and, consequently, cancer cells would starve to death or could be targeted to specifically deliver cytotoxic drugs. On the other side, some xenobiotics, environmental toxins and drugs may have undesirable access to the cells and, therefore, exerting toxicities. Collectively, thorough knowledge about amino acid transporters would permit the breakthrough discoveries of novel drugs and targeted drug delivery systems with minimal side effects. Trans-stimulation assay is being investigated in this study as a screening method of identifying the affinity of several prodrugs utilizing LAT1.

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ACKNOWLEDGEMENTS

I would like to emphasis my appreciation to the University of Eastern Finland (UEF) at the first place for giving me the opportunity to do my master’s degree. I would like also to thank all the professors at the department of Toxicology, UEF, who gave me lectures and tutorials that broadened my ability of critical thinking and the ability of conducting scientific researches. My special thanks to my thesis supervisors; Professor Jarkko Rautio for all the valuable comments on this thesis, guidance tips and support, and M.Sc. Jussi Kärkkäinen for all the practical guidance in the lab. I would like to thank also my family members, my friends and my fiancée for being by my side as a source of endless support and motivation.

Ahmed Montaser

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ABBREVIATIONS

ACE2: Angiotensin converting enzyme 2

AD: Alzheimer’s disease

ALS: Amyotrophic lateral sclerosis

AMPA: α-amino-3-hydroxy-5 methylisoxazolepropionic acid

APC: Amino acid-Polyamine-organoCation clans

ASCTs: Alanine, serine and cysteine transporters

ATP: Adenosine triphosphate

BBB: Blood brain barrier

BCH: Bicyclo[2.2.1]heptane-2-carboxylic acid, 2- amino

BMAA: β-N-methylamino-alanine

BSA: Bovine serum albumin

CATs: Cationic amino acid transporters

cDNA: Complementary DNA

CNS: Central nervous system

DMEM: Dulbecco's Modified Eagle Medium

DMG: N,N-dimethylglycine

DPBS: Dulbecco’s Phosphate-Buffered Saline

EAATs: Excitatory amino acid transporters

EEG1: Embryonic epithelia gene 1

ER^+:Estrogen receptor-positive

GABA: Gamma-Aminobutyric acid

HATs: Heterodimeric amino acid transporters

HBD: Hydrogen bond donner

HBSS: Hank's Balanced Salt Solution

HEPES: (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid)

HPLC/MS: High-performance liquid chromatography/ mass spectroscopy

HUGO: Human Genome Organization

KO: Knockout

LAT: L-type amino acid transporter

LSC: Liquid scintillation and luminescence counting

MCF-7: Michigan Cancer Foundation-7

MFS: Major facilitator superfamily

mRNA: Messenger RNA

mTORC: Mammalian target of rapamycin complex

MVM: Microvillus membrane

NMDAR:

^N-methyl-^D-aspartate receptors

NMR: Nuclear magnetic resonance

NO: Nitric oxide

PATs: Proton/amino acid transporters

PD: Parkinson disease

PET: Positron-emission tomography

QSAR: Quantitative structure-activity relationship

SAR: structure-activity relationship

SLC: Solute carrier

SNATs: Sodium-coupled neutral amino acid transporters

SNPs: Single nucleotide polymorphisms

TCDB: Transporter Classification Database

THPO: Tetrahydroisoxazolo[4,5‐c]pyridin‐3‐ol

VGAT: Vesicular GABA transporter

VGLUTs: Vesicular glutamate transporters

VIAAT: Vesicular inhibitory amino acid transporter

XA: Xanthurenic acid

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PART I/II: LITERATURE REVIEW 1. INTRODUCTION

Amino acids are essential for many cellular activities and therefore for cellular survival.

Homeostasis of amino acids inside our tissues is critical for the proper function of many processes; ranging from protein synthesis and as a source for metabolic energy to neurotransmission (Kilberg, Häussinger 1992). At the physiological conditions, most amino acids tend to be predominantly dipolar ions (zwitterions) and therefore lipid-insoluble. Hence, they cannot diffuse across the lipid bilayers of different membranes. There should be special kinds of membrane proteins to facilitate their transport through the plasma or intracellular membranes.

These special membrane proteins or transporters have drawn great attention because of their importance for regulating vital physiological processes and functions. Any abnormality in their function could be lethal or give rise to serious pathological conditions (Palacı́n, Borsani et al.

2001). For instance, some monogenic disorders, such as cystinuria, are caused due to deficit or lack of one or more amino acid transporters. Aminoaciduria is a common manifestation of any malfunction of those carriers such as lysinuric protein intolerance. Several neurological, nutritional and chronic disorders have been linked to malfunctions of one or more of those carriers. Examples of those disorders are Alzheimer disease (AD), Parkinson’s disease (PD), Hartnup disease and diabetes. Thus, these transporters can be potential drug targets to overcome such diseases.

Many chemical compounds might have similar structures and properties as the natural amino acid substrates and therefore may gain unwanted access to the cells through those transporters (molecular mimicry). Consequently, toxicological issues have arisen and should be taken into consideration. An obvious example of those compounds is gabapentin that could have an easy access through the placental barrier via L-type amino acid transporters (LATs) and exert embryo toxicity. On the other side, many researchers are trying to develop analogs and prodrugs that can be used to specifically deliver the active compounds into the target cells taking advantage of the physiological function of those transporters. Another approach that has drawn great attention recently is to deprive the cancer cells from important source of nutrition by inhibiting some over-expressed influx amino acid transporters.

Targeting specific amino acid transporter is a very promising strategy to improve the drug delivery and bioavailability with minimal off target effects. Tiagabine, a γ-aminobutyric acid

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(GABA) uptake inhibitor, is an example of anti-epileptic drug developed by the research of amino acid transporters (Iversen 2000).

In this thesis, I will review the characteristics, pharmacological and toxicological significance of the different amino acid transporters. In addition, I will review the different methods of studying those transporters while the focus will be placed on the L-type amino acid transporter 1 (LAT1).

2. TRANSPORT ACROSS BIOLOGICAL MEMBRANES

Translocation of exogenous molecules inside the cellular compartment is a key process to consider for determining their pharmacological actions and/or their toxicity. The movements of the molecules in and out of our cells are tightly regulated, since the cellular membranes have selective permeability and act as barriers that control the cellular uptake of various substances.

Some vital organs have a complex barrier structure that is highly selective towards certain substances such as blood-brain, blood-testis, blood-retina and blood-placental barriers. But how the cellular membrane acts as a selective barrier?

There are two widely investigated hypotheses about how xenobiotics can cross the cellular membrane and get access into the cells. One indicates that passive diffusion is the main mechanism of the cellular uptake of xenobiotics. It has been denoted by the term BDII; lipoidal phospholipid bilayer diffusion is important (Sugano, Kansy et al. 2010, Smith, Artursson et al.

2014). The other hypothesis strongly debates that there is not any evidence of lipoidal diffusion of xenobiotic and that the carrier-mediated transport is the rule rather than the exception. It has been denoted by the term PBIN; phospholipids bilayer diffusion is negligible (Kell, Oliver 2014, Kell 2015).

There is a plausible conclusion that both passive diffusion and carrier-mediated uptake coexist in the biological system (Sugano, Kansy et al. 2010). However, the quantitative contribution of each method should be identified case by case due to the huge variability and complexity of membrane permeation. Some cells under certain conditions may follow the first hypothesis, BDII, such as in the absorptive intestinal cells. Other types of cells under different conditions may follow the second hypothesis, PBIN, such as in case of hepatic and renal distribution of hydrophilic drugs. Thereby, one method could dominate under specific conditions as per the physiological function of the cell and the surrounding microenvironment.

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Even though, passive diffusion contributes significantly in the membrane permeation of drugs, lipophilicity is just one factor of many others. Carriers, cell geometry, molecular weight of permeates, temperature and the pH of the medium are also factors to be considered (Di, Artursson et al. 2012). In fact, little is known about the physiological role of several carriers.

Thus, there is a consequential underestimation of their roles in drug disposition (Rautio, Gynther et al. 2013). Up to date, nearly 400 influx transporters classified into 52 gene families have been identified (Hediger 2014). Since most of them have not been intensively studied, transport in some cases can be falsely attributed solely to passive transport.

According to the transport functions, membrane proteins can be classified, as shown in figure 1, into two main categories; 1) passive transport and 2) active transport. Passive transport proteins include ion channels and transporters of facilitated diffusion. Ion channels have the highest capacity of transport up to 10⁸molecules per second (Lodish, Berk et al. 1999). They transport water or specific types of ions which are mainly responsible for generating electric potential gradients across the cells. Facilitated diffusion is mediated via transmembrane proteins that translocate ions or molecules down their concentration gradient. Those membrane proteins do not require energy for their function. However, they are different than the free diffusion because the transport includes binding with the substrates, it follows a saturation state and it is relatively temperature-dependent (Friedman 2008). Active transporters are either primary that use energy to transport its substrates or secondary that use ion gradient or substrates exchange as a mode of transport. Primary active transporters are such as ATP pumps or ATP-binding cassette transporters (ABC transporters or efflux transporters). Secondary active transporters are influx transporters that couple the movement of an ion or molecule against its concentration gradient with the movement of another down its concentration gradient such as symporters and antiporters. Symporters transport molecules in one direction only and leads to net influx of its substrates such as Na⁺/amino acid symporters. Antiporters or exchangers transport substrates in exchange for other substrates (Bröer 2010). Symporters and antiporters can work co-operatively when the efflux substrates of one can act as the influx substrates for the other.

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Figure 1- Classification of membrane transport proteins according to function

A major emphasis recently is on the secondary active transporters or influx transporters. They are commonly known as solute carrier transporters (SLC family transporters). Solid understanding of their physiological roles and revealing their mechanism of transport would lead to breakthrough discoveries in not only novel drug design but also in targeted drug delivery.

A co-operation between several disciplines is needed to comprehensively understand the physiological and pathophysiological roles of transporters. Genomic tools such as transcriptomics, proteomics and genetic variations are valuable sources that should be considered. Bioinformatics is inevitable to handle all the genomics data. In silico and in vitro experiments shall be combined to predict the best interaction with the transporter in vivo.

2.1 Amino Acid transporters

Amino acid transporters belong to the influx transporters superfamily as 25 % of SLC transporters are reported to traverse amino acids (Fredriksson, Nordstrom et al. 2008). They have a vital role to maintain the homeostasis of essential and non-essential amino acids inside our bodies. Xenobiotics, pharmaceutical drugs or environmental toxins may structurally resemble amino acids (molecular mimicry). Thereby, they may gain access to cells through amino acid transporters. Therefore, identification of the characteristics of those transporters proved to be crucial from the pharmacological and toxicological perspectives. Up to date, there are 9 solute carrier families which are involved in traversing amino acids (Hediger 2014). These families are SLC 1, 6, 7, 16, 17, 32, 36, 38 and 48.

Membrane Transport

Passive

Ion channels Na⁺channels Faciliate

diffusers

Glucose transporters

Active

Primary Efflux

transporters

Secondary

Influx transporters

Symporters Antiporters

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Additionally, there are some regulatory proteins involving in the transport of amino acids such as in SLC3 family. They do not transport amino acids themselves, but they rather help other proteins to localize at the cellular membranes. Both proteins dimerize together to constitute a functional transporter such the heterodimeric amino acid transporters (HATs).

Throughout the literature, amino acid transporters have different types of classifications.

Firstly, the classification of the Human Genome Organization (HUGO) Gene Nomenclature Committee (HGNC) where all transporters of amino acids, glucose or ions are classified into different families according to their amino acid sequence. Members of the same family typically share about 20 – 25 % of their amino acid sequence as well as sharing similar substrate(s) (Schlessinger, Matsson et al. 2009). They are denoted by SLC followed by the family number.

Each family may include several members denoted by the letter (A) followed by a number, SLC1A1 for instance. Secondly, amino acid transporters are classified mainly under two phylogenetic clusters as shown in figure 2. α-Families that contain transporters of the major facilitator superfamily (MFS) and β-families that contain transporters of the amino acid- polyamine-organoCation (APC) clans (Schiöth, Roshanbin et al. 2013). Thirdly, classification according to their substrates such as anionic, cationic and neutral amino acid transporters.

Additionally, they are further classified according to the transport characteristics and substrates selectivity such as system A, system N, system L, system ASC, etc., as briefly summarized in table 1. In addition, there are other important parameters for their classifications such as Na⁺- dependency, pH sensitivity, polarity, and mode of transport.

Table 1- Main amino acid transport systems

Transporter System ASC

System A System L System N System y⁺L

Preferred substrates

L-Ala, L-Ser, L-Cys & L-

Gln

L-Ala, L-Gln, L-Gly L-Pro

& MeAIB

L-Leu, L-Ile, L- Tyr, L-Try, L-Val,

L-Phe, L-Met, L- Gln & BCH

L-Gln, L-Asp

& L-His ^L-Arg, L-Ser

Transport mode

Exchange or

net flux Net flux Exchange (1:1). Facilitator Exchange or net flux

pH-sensitivity insensitive ^Usually Sensitive Usually insensitive

Highly

sensitive Sensitive

Na⁺-

dependence ^Dependent ^Dependent Independent Dependent

Dependent according to

substrate

specific

inhibitors ^-- ^MeAIB

BCH

(competitive) -- --

Examples ^ASCT1,2 ^SNAT1,2,4 ^LAT1,2 ^SNAT3,5,7 ^y⁺^{LAT1, y}⁺^LAT2

System ASC: acronym for alanine, serine and cysteine. System A: acronym for alanine. System L: acronym for leucine. System N: indicating nitrogen containing amino acids. MeAIB: alpha-(methylamino)isobutyric acid. BCH: Bicyclo[2.2.1]heptane-2-carboxylic acid, 2-amino, L- Alanine (L-Ala), L-Serine(L-Ser), L-Cysteine (L-Cys), L-Glycine(L-Gly), L-Proline(L-Pro), L-Isoleucine(L-Ile), L-Tyrosine(L-Tyr), L- Tryptophan(L-Try), L-Valine(L-Val), L-Phenylalanine(L-Phe), L-Asparagine(L-Asp), L-Histidine(L-His).

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Figure 2- Phylogenetic clusters of amino acid transporters

Hereunder we are going to have a general view about all solute carrier families classified according to HGNC system that include one or more amino acid transporter. Focus will be mainly placed on their physiological role, expression, substrates selectivity, substrates affinity, pharmacological applications and toxicological implications.

2.1.1 Solute carrier 1 family

According to the similarities of the amino acid sequence, influx transporters are classified into subfamilies. Despite the members of the same subfamily have quite similar sequence identity, they display some differences in the transport functions and substrates affinity.

Figure 3- Classification of SLC 1 amino acid transporters

EAAT: Excitatory Amino acids transporters, ASCT: Alanine Serine Cysteine Transporters, SLC: Solute carrier Transporters family.

Solute carrier subfamily 1 includes mainly two categories of transporters; excitatory amino acids transporters (EAATs) and alanine, serine and cysteine transporters (ASCTs).

Am ino a cid trans po rter s

α-familes SLC16

SLC17 SLC43

β-families

SLC32 SLC36 SLC38

Others

SLC1 SLC6 SLC7

SLC 1

EAATs

SLC 1A3 (EAAT 1) SLC 1A2 (EAAT 2) SLC 1A1 (EAAT 3) SLC 1A6 (EAAT 4) SLC 1A7 (EAAT 5) ASCTs

SLC 1A4 (ASCT 1 ) SLC 1A5 (ASCT 2)

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Atomic structures for SLC 1 family transporters have not been identified so far. Therefore, a prokaryotic aspartate transporter homolog GltPh has been crystalized that possess about 23%

sequence similarity with ASCTs and 36% similarity with EAATs (Scopelliti, Ryan et al. 2013).

It is used as a comparative model to study the transport functions of SLC1 transporters. This model could be utilized in predicting some substrates of the transporters. Thereafter, structure- activity relationship (SAR) studies could be conducted to identify the binding motifs of the transporter. Ultimately, suitable candidates could be further tested experimentally, and a pharmacophore model of the transporter could be generated.

2.1.1.1 Excitatory amino acids transporters (EAATs)

EAATs are responsible for the reuptake of the neurotransmitter glutamate from the synapses to the parenchymal cells (neurons and astrocytes) in the central nervous system (CNS) but also to a lesser degree in periphery (Kanai, Clémençon et al. 2013). They uptake L-glutamate (L-Glu) against high concentration gradient by translocating three sodium ions (Na⁺) and one proton (H⁺) inside the cells while effluxing one potassium ion (K⁺) outside the cell for each substrate.

EAAT1, EAAT2 and EAAT3 are mainly expressed in the brain and account for the uptake of almost all glutamate from the synaptic cleft (Kim, Lee et al. 2011). EAAT1 and 2 are expressed in glial cells and have the highest role in clearing up excess glutamate in and around glutamergic synapses and thus, they prevent the excitotoxicity and the death to the surrounding neurons (Kanai, Clémençon et al. 2013). EAAT3 is mainly expressed in neurons of the CNS as well as in other peripheral organs such as skeletal muscles, intestine, liver, kidney and placenta.

EAAT4 is another neuronal transporter largely expressed in cerebellum. EAAT5 is primarily expressed in retina.

EAAT1 selective expression in dementia cases showing Alzheimer-type pathology is a direct indicator of the contribution of this transporter in Alzheimer disease (AD) (Scott, Pow et al.

2002). Thus, EAAT1 substrates might be either toxins that down-regulate or block this transporter or therapeutics that up-regulate their expression. By the aid of chemical libraries and using the human embryonic kidney cells (HEK293) expressing EAAT1, allosteric selective inhibitors of EAAT1 have been identified (Jensen, Erichsen et al. 2009, Hansen, Erichsen et al.

2016). Thereby, structure-activity relationship (SAR) studies would help us to predict the toxicity of other xenobiotics exploiting this transporter causing different neurodegenerative diseases. While EAATs are expressed only in the abluminal side of the BBB (Hawkins, Viña 2016) , toxins should have access to the brain first in order to exert their adverse effects and that should be taken into account.

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EAAT2 is a critical transporter for glutamate uptake as it is expected to clear up about 90% of the total extracellular glutamate to the astrocytes (Kim, Lee et al. 2011). Ceftriaxone and beta- lactam antibiotics have been found to enhance EAAT2 expression and therefore, they relieve glutamate-induced neuropathic pain and hyperalgesia (Chelini, Brogi et al. 2017). Indeed, EAAT2 is a crucial drug target as it regulates the level of the neurotransmitter glutamate in the synapses. Enhancing EAAT2 action would have a beneficial role in many neurodegenerative diseases clearing up the excess glutamate. On the other hand, demolishing its action would lead to serious neurotoxicities.

A compound from a spider venom shows a neuroprotective effect by enhancing the glutamate uptake (Fontana, Guizzo et al. 2003). The purification of this compound, parawixin1, has paved the way for further investigations about its mechanism of action. Parawixin1 turned out to enhance glutamate uptake through EAAT2 transporter by targeting a step in the transport cycle different than the substrate inward and outward binding sites. This finding opened up a new area of developing new drugs that act allosterically on the transporter (Fontana-Andréia, de et al. 2007). As a result, a recent study managed to develop effective and selective allosteric modulators of EAAT2 that are awaiting the clinical translation (Kortagere, Mortensen et al.

2017). Indeed, translation of those modulators into therapeutics would lead to breakthrough discoveries of treating not only acute excitotoxicity but also several neurodegenerative diseases such as AD, PD, amyotrophic lateral sclerosis (ALS), epilepsy and traumatic brain injury (TBI).

Moreover, kainic acid and dihydrokainic acid are structurally similar compounds to glutamate.

They showed inhibition of EAAT2 (Arriza, Fairman et al. 1994). Domoic acid, a kainic acid analogue, is a naturally occurring toxin that causes amnesic shellfish poisoning (ASP).

Therefore, it is anticipated that one of its toxic mechanisms could be by inhibiting EAAT2.

In the CNS, EAAT3 takes part in the uptake of glutamate from synapses (Bjørn-Yoshimoto, Underhill 2016). In addition, it is the main transporter for the neuronal uptake of cysteine and therefore, it plays a significant role in the synthesis of glutathione; a potent antioxidant. Hence, EAAT3 has a crucial role preventing the oxidative stress in the CNS that might arise from the high metabolic rate of neurons.

Both EAAT4 and EAAT5 exert properties of glutamate-gated chloride conductance in both pre- and post-synapses while the glutamate uptake is independent from the chloride ion (Cl^-) current (Fairman, Vandenberg et al. 1995, Arriza, Eliasof et al. 1997). Although the molecular function of the Cl^- current has not been understood, it is suggested to play a role in the physiological

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excitability of Purkinje cells and retinal neurons due to EAAT4 and EAAT5, respectively (Arriza, Eliasof et al. 1997, Tse, Chung et al. 2013, Tse, Chung et al. 2014). Unlike the other group members, the double function as a carrier and ion channel is a distinct property of EAAT4

& 5. However, the transporter should function as either a glutamate transporter or a Cl^- ion channel at a specific time point (Fahlke, Kortzak et al. 2016).

2.1.1.2 Alanine, serine and cysteine transporters (ASCTs)

Alanine, serine and cysteine transporters (ASCTs) have very close structure similarities with the glutamate transporters of EAATs family (Utsunomiya-Tate, Endou et al. 1996). Both ASCT1 & 2 are expressed in brain, preferentially in astrocytes, but also to some extent in neurons (Kanai, Clémençon et al. 2013). ASCT1 is expressed in glial cells, skeletal muscles and pancreas. ASCT2 is, however, highly expressed in peripheral tissues such as lung, intestine, kidney and testis. ASCT2 is also over-expressed in several cancer cells.

ASCTs were first identified by Christensen and colleagues and given the name ASC due to their preference for neutral amino acids such as L-alanine (L-Ala), L-serine (L-Ser), L-cysteine (L-Cys) (Christensen, Liang et al. 1967). Unlike EAATs, they transverse neutral amino acids in exchange for intracellular amino acids (antiporters). There are two members in this category that share 57% sequence identity with each other and about 40% with the other family members (EAATs) (Kanai, Clémençon et al. 2013, Scopelliti, Heinzelmann et al. 2014). Despite the huge sequence similarity between the two members, they are different in function and even in substrates’ selectivity. ASCT2 has broader substrate selectivity with high affinity towards L- glutamine (L-Gln) and low affinity towards D-Ser, L-methionine (L-Met) and L-leucine (L-Leu).

ASCT1 transports neutral amino acids in a Na⁺- dependent manner with uncoupled Cl^- conductance current (Scopelliti, Ryan et al. 2013). It provides neuronal cells with their metabolic needs with high selectivity towards L-Ala, L-Ser and L-Cys. ASCT1 helps in releasing L-Ser from glial cells, where it is synthesized and stored, in exchange for another extracellular substrate (Sakai, Shimizu et al. 2003). L-Ser is reported to play a pivotal role in neuronal development and function. Many psychotic disorders such as congenital microcephaly, psychomotor retardation, cognitive dysfunction and seizures have been linked to reduced L-Ser function in the brain and therefore, speculating the role of ASCT1 transporter in those diseases (Savoca, Ziegler et al. 1995). Additionally, L-Ser is a precursor for neuromodulators such as glycine and D-Ser which are important for the N-methyl-D-aspartate receptors (NMDAR) activation (de Koning, Snell et al. 2003). Therefore, indirect contribution to the pathophysiology of schizophrenia has been suggested (Foster, Farnsworth et al. 2016).

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Genetic studies have linked several mutations in SLC1A4 gene with several neurological diseases supporting the aforementioned pathophysiological role (Srour, Hamdan et al. 2015, Damseh, Simonin et al. 2015).

ASCT2 has high affinity to L-Gln and expressed extensively in some cancer cells (Hassanein, Hoeksema et al. 2012, van Geldermalsen, Wang et al. 2015). Thus, a role in providing the glutamine-addicted cancer cells with its life elixir (L-Gln) has been outlined. Selective inhibition of ASCT2 has been a matter of great interest for many researchers in terms of developing anti-cancer agents. Firstly, a commercially available compound L-γ-glutamyl-p- nitroanilide (GLPN) was found to exhibit ASCT2 inhibition but with low affinity in the millimolar range (Esslinger, Cybulski et al. 2005) followed by structural modification in a way to improve the affinity. Thereafter, substituted benzylproline showed, surprisingly, considerate inhibition affinities to ASCT2 despite that L-Pro is not a substrate itself (Singh, Tanui et al.

2017). Hence, benzylproline derivatives along with GLPN derivatives constituted a valuable starting point in a way to develop the best therapeutic candidate.

Solute carrier 3 family includes two glycosylated protein subunits; 4F2hc (SLC3A2) and rBAT (SLC3A1) (Fotiadis, Kanai et al. 2013). They do not function as transporters themselves but as regulatory heavy subunits that dimerize with the light subunits of SLC7 transporters to form the heterodimeric amino acid transporters (HATs). 4F2hc dimerizes with most of the light chains of SLC7 family via covalent disulfide bond (Chillarón, Roca et al. 2001). rBAT subunit dimerizes with only SLC7A9 via non-covalent interaction. Their principal function is to help trafficking the functional light subunits into the cellular membrane.

Many amino acid transporters are included under this family as shown in figure 4 (Hediger 2014). These transporters are involved mainly in either i) neurotransmitters reuptake, ii) amino acid uptake into specific cells or iii) (re)absorption of amino acids from the gut and kidney tubules (Rudnick, Kramer et al. 2014). They are Na⁺- and Cl^--dependent transporters that previously were named as neurotransmitter transporters (NNTs) because they were mainly devoted for neurotransmission. Thereafter, other functions such as the (re)absorption of amino acids from the intestine and kidney, were identified for the orphan members of the family (Bröer 2006).

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Figure 4 - Classification of SLC 6 amino acid transporters

2.1.3.1 Uptake of neurotransmitters

Glycine transporter 1 (GLYT1) is expressed in glial cells that surround both inhibitory and excitatory synapses (Rudnick, Kramer et al. 2014). Glycine transporter 2 (GLYT2) is expressed in presynaptic inhibitory glycinergic neurons and displays higher affinity than GLYT1 in the CNS. High affinity L-Proline (L-Pro) transporter (PROT) is localized exclusively at glutaminergic nerve terminals (Schulz 2011). Broad neutral and basic amino acid transporter (ATB^0,+) is expressed in lung epithelia, colon and intestine; albeit with low levels (Sloan, Mager 1999). ATB^0,+ is also over-expressed in several cancer cells (Karunakaran, Ramachandran et al. 2011). Broad neutral amino acid transporter 2 (B⁰AT2) is mainly expressed in neurons particularly in hippocampus (Santarelli, Namendorf et al. 2015). Neurotransmitter transporter 4 (NTT4) is a vesicular protein that is expressed in the glutamatergic and some GABAergic neurons in the CNS (Bröer 2006).

GLYT1 is a high affinity glycine transporter that helps in the reuptake of glycine from both the glycinergic and glutamergic synapses (Bradaïa, Schlichter et al. 2004). GLYT1-deficient mice died in the first postnatal day after experiencing severe respiratory depression and reduced motor functions (Gomeza, Hulsmann et al. 2003). This was explained by the increased inhibitory tone at the glycinergic synapses during the early postnatal life. On the other hand, glycine is an important co-agonist, along with L-Glu and D-Ser, for the activation of NMDAR at the glutamergic synapses. Therefore, modulating glycine level at the glutamergic synapses

SL C 6

Neurotransmitters uptake

SLC6A5 (GLYT2) SLC6A9 (GLYT1) SLC6A7 (PROT) SLC6A14 (ATB^0,+)

SLC6A15 (B⁰AT2)

SLC6A17 (NTT4) SLC6A16 (NTT5)

(Orphan)

Amino acid uptake

SLC6A18 (B⁰AT3) SLC6A19 (B⁰AT1) SLC6A20 (SIT1)

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has emerged as a potential solution to cognitive impairment and psychotic disorders related to NMDAR hypofunction (Zeilhofer, Acuña et al. 2018). The selective inhibition of GLYT1 would lead to extracellular increase in glycine that in turn augments NMDAR actions relieving some psychotic disorders. Within this concept, several natural substrates and their derivatives have been investigated to experimentally prove this hypothesis. For instance, sarcosine and N,N-dimethylglycine (DMG) were found to be GLYT1 inhibitor that paved the way for further investigations in controlling the negative symptoms of schizophrenia (Lee, Lin et al. 2016).

Also, bitopertin and several other synthetic drugs have been developed to inhibit GLYT1 (Harsing, Juranyi et al. 2006, Umbricht, Alberati et al. 2014). Bitopertin and sarcosine showed positive results in small clinical trials up to phase II; however, they failed to prove significant improvement in the large phase III study (Dunayevich, Buchanan et al. 2017). Indeed, the theory has been identified and established but the translation into effective therapeutics is still missing.

GLYT2 regulates the glycine level in the synaptic cleft of glycinergic neurons (Liu, Lopez- Corcuera et al. 1993). It transports glycine from the synapses back into neurons where the vesicular inhibitory amino acid transporter (VIAAT) takes it up into the vesicles. Therefore, GLYT2 plays a critical role in glycine neurotransmission. It helps not only in initiating the vesicular glycine packaging preceding its release into synapses but also in terminating the signal at the synaptic cleft. GLYT2-deficient mice died within two weeks after birth after experiencing severe muscular spasticity (Gomeza, Ohno et al. 2003). This was explained by the hypoglycinergic signal at the inhibitory synapses. However, the transient blockage of GLYT1 and GLYT2 enhances the inhibitory tone and decreases the nociceptive conductance (Zeilhofer, Acuña et al. 2018). Therefore, they have been considered a promising drug targets to treat the neuropathic pain. An emerging approach tested the effect of some endogenous lipids and their derivatives on GLYT2. N-arachidonyl-glycine, N-oleoyl glycine and oleoyl L-carnitine showed selective inhibition of GLYT2 that may lead eventually to therapeutics for neuralgia (Carland, Handford et al. 2014).

PROT is believed to reuptake the neuromodulator L-Pro from the synapses of glutamenergic neurons (Pérez-Arellano, Carmona-Álvarez et al. 2010). Receptors for L-Pro have not been identified so far. However, L-Pro is used for glutamate synthesis. Therefore, a role in regulating the glutamate neurotransmission was outlined (Rudnick, Kramer et al. 2014). PROT knockout (KO) mice showed behavioral alterations such as improved learning ability, increased anxiety and decreased locomotor activity (Schulz 2011). However, the exact physiological role of

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PROT has not been unraveled so far. Enkephalins, peptides involved in nociception, showed inhibition of PROT in vitro, both in rat synaptosomes and transfected HeLa cells (Fremeau, Caron et al. 1992, Galli, Jayanthi et al. 1999). Therefore, a role of this transporter in analgesia may be anticipated.

Amino acid transporter with broad substrate selectivity (ATB^0,+) is an energy coupled transporter with several driving forces ( Na⁺, Cl^- and membrane potentials) (Karunakaran, Ramachandran et al. 2011). It transports a wide variety of substrates including several neutral (⁰) and basic (⁺) amino acids in a unidirectional manner. All essential amino acids as well as L- Gln and aspartate can be concentrated inside the cell via ATB^{0, +}. It is able to concentrate its substrates inside the cell higher than any other amino acid transporter with approximately 1000 times higher than the extracellular medium (Coothankandaswamy, Cao et al. 2016). Its upregulation in several cancers, such as in colon, pancreatic, cervical and estrogen receptor – positive (ER⁺) breast cancers, have been detected (Bhutia, Babu et al. 2014). Thus, it constitutes a crucial source of delivering nutrients and signaling molecules such as L-Gln and L-Leu to cancer cells. SLC6A14 gene silencing or inhibition with α- methyl-L-Try decreased the proliferation of (ER⁺) breast cancer cells and pancreatic cancer cells in in vitro and in vivo studies (Bhutia, Babu et al. 2014, Coothankandaswamy, Cao et al. 2016). Indeed, ATB^0,+ is a very promising drug target to develop anti-cancer drugs. Investigations about the physiological role of ATB^0,+ are still not complete. A possible role in luminal protein removal in lung epithelia was suggested (Sloan, Grubb et al. 2003).

The broad neutral amino acid transporter 2 (B⁰AT2) is a Na⁺/amino acid symporter that co- transports its substrates with Na⁺ in a 1:1 stoichiometry manner (Bröer, Tietze et al. 2005). It transports large neutral amino acids with high affinity, roughly in the following order; L-Met >

L-Ile > L-Leu > L-Pro and with low affinity, roughly in the following order; L-Ala > L -Phe > L

-Gln. B⁰AT2 and PROT were identified to be responsible for L-Pro uptake in synaptosomes (Rudnick, Kramer et al. 2014). Genome wide association studies (GWAS) revealed single nucleotide polymorphisms (SNPs) in B⁰AT2 as a risk factor for some psychotic disorders such as major depression (Santarelli, Namendorf et al. 2015). Furthermore, a correlation between L- Pro uptake and glutamate transmission has been established explaining the role of B⁰AT2 in psychotic disorders.

NTT4 is another neurotransmitter transporter that is believed to be inactive while localization at the synaptic vesicles (Bröer 2006). However, once the vesicle is fused and attached to the

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synaptic terminal, NTT4 is able to uptake some neutral amino acids in a Na⁺-dependent manner.

Selective uptake of proline, glycine, leucine, alanine and glutamate was observed (Bröer 2006, Parra, Baust et al. 2008). Although, stereoselectivity was not reported or studied, NTT4 prefers most probably L-isomers as it resembles B⁰AT2 structurally. Homozygous mutation in SLC6A17^-/-gene correlated with some cognitive disabilities (Iqbal, Willemsen et al. 2015).

Proline and glycine affects glutamate synthesis and NMDAR activation respectively. Thus, NTT4 dysfunction may manifest as intellectual and psychological disorders.

2.1.3.2 Uptake of Nutrient amino acids

The broad neutral amino acid transporter 1 (B⁰AT1) is a luminal transporter expressed in the small intestine and kidney proximal tubules (Rudnick, Kramer et al. 2014). It requires association with other membrane proteins for its functional expression such as angiotensin- converting enzyme 2 (ACE2) and its analogue collectrin in the intestine and kidney tubules respectively. Sodium/imino acid transporter 1 (SIT1) follows the same pattern of expression and membrane protein association as B⁰AT1. The broad neutral amino acid transporter 3 (B⁰AT3) is a renal amino acid transporter expressed also in the proximal tubules. There is a suggestion that B⁰AT3 may no longer be functional in human due to a frequent SNP encodes for a stop codon.

B⁰AT1 is a putative major neutral amino acid transporter in the intestinal epithelia (Jando, Camargo et al. 2017). It transports all neutral amino acids with high affinity to branched chain amino acids (BCAAs) and L-Met in a sodium-dependent manner. Mutation in SLC6A19 gene causes Hartnup disorder; an autosomal recessive metabolic disorder characterized by the loss of neutral amino acids (re)absorption (Kleta, Romeo et al. 2004). One hypothesis suggests that protein-restriction diet or decreased amino acids absorption from intestine would induce the release of some hormones such as fibroblast growth factor 21 (FGF21) and glucagon-like peptide 1 (GLP-1) that control glycaemia (Jiang, Rose et al. 2015). In addition, B⁰AT1-deficient mice have showed improved glycemic control. Therefore, B⁰AT1 is believed to be a drug target to control the hyperglycemia in diabetic patients. However, more studies should be conducted to evaluate the benefit/risk ratio of B⁰AT1 inhibition in vivo.

SIT1 traverses imino acids such as L-Pro as well as N-methylated amino acids such as MeAIB and sarcosine in a Na⁺ and Cl^--dependent manner (Takanaga, Mackenzie et al. 2005). L-Pro is considered the major substrate for SIT1. L-Pro homeostasis is crucial as it is a precursor of many vital endogenous compounds such as glutamate, ornithine (Orn), citrulline, and arginine.

However, absorbed L-Pro in the intestinal epithelial is quickly degraded and is not available for

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extra-intestinal tissues. SIT1 malfunction may lead to a form of iminoaciduria. Neonates may experience a transient hyper-excretion of proline due to immature SIT1 (Lasley, Scriver 1979).

Two main categories are classified under this family as shown in figure 5 (Fotiadis, Kanai et al.

2013). One is the cationic amino acid transporters (CATs) that facilitates the diffusion of basic amino acids such as L-arginine (L-Arg) and therefore, it plays an important role in nitric oxide (NO) synthesis in certain cells. The other category is the heterodimeric amino acid transporters (HATs), which consist of functional light subunit of SLC7 family and regulatory heavy subunits of SLC3 (4F2hc or rBAT) family. Both subunits are linked together via disulfide bond(s).

Figure 5- Classification of SLC 7 amino acid transporters

2.1.4.1 CATs

Cationic amino acid transporters (CATs) were previously known as system y⁺ or Ly⁺ referring to lysine as its first model substrate when it was first identified and ⁽⁺⁾ for basic amino acids (Christensen, Handlogten et al. 1969). CAT1 is a ubiquitous transporter with the exception of

SLC 7

Cationic transporters (CATs)

SLC7A1 (CAT1)

SLC7A2 (CAT2)

SLC7A2A (CAT2A)

SLC/A2B (CAT2B) SLC7A3 (CAT3)

SLC7A4 (CAT4) SLC7A14

Heterodimeric transporters (HATs)

Dimerize with 4F2hc (SLC3A2)

SLC7A5 (LAT1) SLC7A8 (LAT-2) SLC7A7 (y⁺LAT1) SLC7A6 (y⁺LAT2)

SLC7A11 (xCT)

SLC7A10 (Asc1) Dimerize with rBAT

(SLC3A1) SLC7A9 (b^0,+AT)

Un-identified dimerisation.

SLC7A15 (arpAT) SLC7A13 (AGT1)

SLC7A12 (Asc2)

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liver cells (Cui, Zharikov et al. 2005). It is mainly expressed in the plasma membranes including the luminal and abluminal sides of the endothelial capillaries in the BBB (Tachikawa, Hirose et al. 2018). CAT2B is expressed in macrophages and T-cells while CAT2A is expressed in muscle and liver cells (Jungnickel, Parker et al. 2018). In addition, CAT1 and CAT2 have been detected in human skin cells. CAT3 is selectively expressed in the brain (Cui, Zharikov et al.

2005) and placenta (Steinhauser, Wing et al. 2017). CAT4 and SLC7A14 are orphan members in this group. Meanwhile, CAT4 is localized at the plasma membranes (Wolf, Janzen et al.

2002), SLC7A14 is expressed in brain and retina (Jin, Huang et al. 2014). Their transport functions have not been identified so far.

There are huge similarities in the amino acid sequence between all the group members of CATs as well as they have common substrates, but with different affinities (Closs, Boissel et al. 2006).

CATs transport L-isomers of basic amino acids with preference towards L-Arg, L-lysine (L-Lys) and L-ornithine (L-Orn) in a Na⁺-independent manner. CATs can function either as exchangers or uniporters. CAT1 follows preferably the trans-stimulation mode of transport while CAT2B and CAT3 are less dependent, and CAT2A is relatively insensitive to the exchange mechanism.

The main physiological role for CATs is to regulate L-Arg levels (Jungnickel, Parker et al.

2018). L-Arg is a precursor and the rate-limiting molecule in the synthesis of NO and L-Orn. In addition, L-Arg is a key regulator of the mammalian target of rapamycin complex 1 (mTORC1).

Therefore, CATs are believed to play vital roles in the vascular system, inflammation and cell growth. However, the site of expression determines their functional endpoint.

CAT1 provides most cells with L-Arg, L-Lys, L-Orn and L-His required for cell differentiation and proliferation (Shima, Maeda et al. 2006). CAT1 is upregulated in colorectal cancer cells that accumulates L-Arg required for cancer cell survival and growth (Lu, Wang et al. 2013). In addition, hematopoietic stem cells could not differentiate into erythroid cells in vitro in the absence of L-Arg. Thus, CAT1 is reported to be essential for the differentiation of red blood cells (Shima, Maeda et al. 2006). Silencing the translation of SLC7A1 gene by micro-RNA 145 (miRNA-145) was found to have a correlation with hypertension (Wang, Jin 2018). In addition, SLC7A1^-/- KO mice died within the first 12 hours after birth with severe anemia (Perkins, Mar et al. 1997) while the heterozygote littermates showed no abnormalities. Therefore, CAT1 is a promising drug target to treat some types of cancers and also to resolve some vascular disorders.

CAT2 displays two splice variants of two isomers with distinct expression pattern; CAT2A and CAT2B. CAT2B has higher affinity for L-Arg in the micro molar range (µM) than CAT2A

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(milli-molar range, mM) (Closs, Boissel et al. 2006). SLC7A2^-/- KO mice showed no structural abnormalities, but several biochemical alterations in the immune response of macrophages and T cells were detected in the lung. Activated macrophages require high supply of L-Arg for the activity of arginase and NO synthase. Therefore, CAT2B was identified as a regulator of inflammation in the lungs of mice (Niese, Chiaramonte et al. 2010, Fotiadis, Kanai et al. 2013).

CAT3 is believed to have a major role in embryogenesis and fetal development due to its abundant expression in placenta (Fotiadis, Kanai et al. 2013). One study identified some gene variants in SLC7A3 rendering psychological disorders such as autism and epilepsy (Nava, Rupp et al. 2015). However, limited evidence is available for both hypotheses.

2.1.4.2 HATs

Heterodimeric amino acid transporters (HATs) are composed of two subunits linked together via disulfide bond(s) (Fotiadis, Kanai et al. 2013). The two subunits are 1) heavy subunit of SLC3 family that has a regulatory role, and 2) functional light subunit of SLC7 family. The heavy subunit is responsible for the localization of the functional light subunit at the cellular membrane. The concomitant expression of both subunits is inevitable for the function of the transporters. HATs transport amino acids with broad selectivity in an obligatory exchange manner.

L-type amino acid transporter 1 (LAT1) was first identified and cloned from rat C6 glioma cells (Kanai, Segawa et al. 1998). Expression in cancer cells, therefore, was evident. LAT1 is also expressed considerably in the BBB, bone marrow, thymus and placenta. L-type amino acid transporter 2 (LAT2), however, is highly expressed in several tissues such as kidney, placenta, brain, prostate, testis and ovaries (Pineda, Fernández et al. 1999, Fotiadis, Kanai et al. 2013).

System y⁺L -type amino acid transporter 1 (y⁺LAT1) is expressed mainly in the basolateral membrane of the kidney tubules while system y⁺L -type amino acid transporter 2 (y⁺LAT2) is expressed, additionally, in several other tissues such as brain, heart and testis. Extracellular cysteine transporter (xCT) is expressed in cancer cells, brain and macrophages. The heterodimer alanine, serine and cysteine transporter 1 (Asc1) is a neuronal transporter expressed mainly in the brain. It is also expressed peripherally in the lung, intestine and placenta. The heterodimer broad neutral and cationic amino acid transporter (b^0,+ AT) is expressed in intestine, liver, kidney and placenta. The aromatic amino acid-preferring transporter (arpAT) is expressed mainly in the brain and intestine. The aspartate/glutamate transporter 1 (AGT1) is expressed in the S3 segment of proximal tubules in kidney. The heterodimer alanine, serine and cysteine

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transporter 2 (Asc2) is expressed in kidney, lung, skeletal muscles and placenta (Chairoungdua, Kanai et al. 2001).

LAT1 transports branched chain and aromatic neutral amino acids such as L-Leu and L-Phe respectively (Kanai, Segawa et al. 1998). LAT2, however, has broader substrate selectivity. It transports small neutral amino acids such as L-Ala. In placenta, LAT2 is expressed in both apical and basal membrane of the trophoblast (Gaccioli, Aye et al. 2015). Thus, LAT2 constitutes the most valuable delivery system of small and large neutral amino acids from the mother to the fetus. In kidney, physiological reabsorption role has been outlined. LAT2 malfunction, therefore, may be attributed to aminoaciduria, and consequently, lead to nutritional imbalance. However, LAT2 KO mice did not show neither amino acids depletion nor defects in placental function, indicating that other transporters may compensate (Fotiadis, Kanai et al. 2013, Ohgaki, Ohmori et al. 2017). β-N-methylamino-alanine (BMAA), that is a toxin produced by cyanobacteria and that can be accumulated in terrestrial and aquatic food chain, is a substrate for LAT2 (Andersson, Ersson et al. 2017). Therefore, BMAA could be transferred from mother to fetus via placenta or infants via milk through mammary glands causing embryotoxicity or toxicities to the neonates.

System y⁺L transporters were firstly identified as basic and neutral amino acid transporters in the plasma membrane of erythrocytes (Deves, Chavez et al. 1992). The transport of the basic amino acids is reported to be Na⁺-independent while the transport of neutral amino acids is reported to be Na⁺-dependent, although not completely (Kanai, Fukasawa et al. 2000). Na⁺ and Lithium (Li⁺) were reported to increase the affinity of y⁺L transporters to the neutral amino acids but not to the basic counterparts. Additionally, y⁺LAT1 showed enhanced transport of neutral amino acids in the presence of H⁺. Mutation in SLC7A7 gene encoding y⁺LAT1, has been linked with lysinuric protein intolerance; a cationic aminoaciduria disorder being reported particularly in the Finnish population (Torrents, Mykkanen et al. 1999) . The basolateral expression of y⁺LAT1 in kidney and intestine indicates its role in the (re)absorption of cationic amino acids. Taken together, y⁺LAT1 is suggested to mediate an obligatory exchange of the cationic intracellular amino acids for the extracellular neutral amino acids (Fotiadis, Kanai et al. 2013). SLC7A7^-/- KO mice displayed neonatal death due to intrauterine growth restriction.

Thus, y⁺LAT1 plays a significant role in the function of the placenta and any defect in the transporter may lead to fetal growth retardation (Sperandeo, Annunziata et al. 2007).

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y⁺LAT2 shows the same mechanism of transport as y⁺LAT1. However, it prefers the efflux of intracellular L-Arg in exchange with extracellular L-Gln. Therefore, it is identified as L-Arg/ L- Gln exchanger (Bröer, Wagner et al. 2000, Fotiadis, Kanai et al. 2013).

xCT is a Na⁺-independent exchanger of extracellular L-Cysteine (L-Cys) with intracellular L- Glu (Van Liefferinge, Bentea et al. 2016). Apparently, xCT has two functions. One is to provide cells with L-Cys needed for glutathione synthesis while the second is to release L-Glu.

Augmented function of xCT would lead to increase in the intracellular anti-oxidant glutathione and increase extracellular release of the neurotransmitter L-Glu. xCT is over-expressed in several cancer cells. Increase in intracellular L-Cys leads to enhanced glutathione synthesis.

Thereby, it protects cancer cells from oxidative damage and promote cell growth. Additionally, increasing attention has been placed recently on L-Glu extracellular release and potentiation of oncogenic effects (Bhutia, Babu et al. 2015). On the other hand, defective xCT transporter is attributed to decreased glutathione production and consequently decreased protection against oxidative stress in microphages (Nabeyama, Kurita et al. 2010). It, thus, enhances the effect of reactive oxygen species (ROS) and cell death. xCT inhibition proved to suppress tumorigenesis (Lewerenz, Hewett et al. 2012). Collectively, xCT is making the balance between cellular protection and extracellular glutamate neurotransmission. Therefore, disruption to this process may promote cancer or neurodegeneration such as in multiple sclerosis (Merckx, Albertini et al. 2017).

Asc1 transports small neutral amino acids with high affinity, not only the L-isomers such as L- Ala and L-Gly, but also some D-isomers amino acids such as D-Ser, in a Na⁺-independent manner (Nakauchi, Matsuo et al. 2000). KO studies in mice showed that Asc1 is the primary transporter of D-Ser in brain (Rutter, Fradley et al. 2007). Unlike the other HATs, Asc1 transporter acts mostly as an exchanger but also acts as a facilitate diffuser (Fotiadis, Kanai et al. 2013). The prominent role of this transporter is to regulate the concentration level of glycine and D-Ser in synapses. Glycine and D-Ser are putative glutamate co-agonists of the NMDAR.

Moreover, Asc1 is localized in brain areas specialized for cognitive functions. Therefore, Asc1 constituted a valuable drug target for treating NMDARs hypofunction implicated in diseases such as impaired cognitive functions, AD and schizophrenia (Kutchukian, Warren et al. 2017).

b^0,+ AT is an antiporter that ensures the influx of cationic amino acids and L-Cys in exchange of the efflux of neutral amino acids (Fotiadis, Kanai et al. 2013). It has affinity in the (µM) range for its extracellular substrates and in the mM range for its intracellular substrates.

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Moreover, b^0,+ AT expression in the brush-border membranes in intestine and kidney suggests its absorptive role of cationic amino acids and L-Cys.

The renal reabsorption of cationic amino acids and L-Cys to the blood stream requires a transport system at both the apical and basolateral membranes of renal tubules. Therefore, a co- operation between two amino acid transporters is suggested to ensure reabsorption of L-Cys and cationic amino acids (Bauch, Forster et al. 2003). Firstly, b^0,+ AT exchanges intracellular neutral amino acids with extracellular (renal lumen) amino acids such as L-Cys and cationic amino acids. Cationic amino acids in turn act as efflux substrates for the basolateral transporter y⁺LAT1 in exchange for extracellular (blood stream) neutral amino acids. This process ensures reabsorption of L-Cys and cationic amino acids. However, the neutral amino acids excreted in the renal lumen during this co-operative process are indeed influx substrates for other transporters such as LAT2. Mutations in SLC7A9 gene or SLC3A1 gene, encoding rBAT heavy subunit, lead to loss of transport function that manifest as cystinuria (Palacin, Fernandez et al.

2001).

In primates, the SLC7A15 gene encoding for arpAT is no longer active (Fernandez, Torrents et al. 2005). However, studying the pseudogenes is still a matter of research whether they have endogenous functions. The mouse arpAT , co-expressed with either 4F2hc or rBAT, transports

L-Tyr, L-Dopa, L-Ala, L-Cys, L-Arg, L-Gln and L-Ser with low affinitiy and in a Na⁺- independent manner (Fotiadis, Kanai et al. 2013).

Asc2 and AGT1 transporters belong to HATs where the coupled heavy chain subunits have not been completely revealed yet. It is still arguable if there might be additional subunits other than 4F2hc or rBAT sorting them on the plasma membrane. Nonetheless, recent study managed to reconstitute AGT1 transporter along with rBAT subunit into proteoliposomes to study the transporter’s activity (Nagamori, Wiriyasermkul et al. 2016). Consequently, another reabsorption system of L-Cys has been suggested and two transporters are involved and believed to work in harmony (Nagamori, Wiriyasermkul et al. 2016). They are AGT1/rBAT and EAAT3 that co-localized in a close proximity in the proximal tubules to support each other’s function. EAAT3 facilitates the diffusion of L-Asp, L-Gln and L-Cys from the tubular lumen into the cytoplasm of the epithelial cells in the proximal tubules. L-Asp and L-Gln work as efflux substrates for the AGT-1 in exchange with L-Cys. b^0,+ AT-rBAT and AGT1-rBAT are partners in the reabsorption of L-Cys from the S1 and S3 segments of proximal tubules respectively.