Transport of amino acids in the kidney.

Transport of Amino Acids in the Kidney Victoria Makrides,1 Simone M.R. Camargo,1 and Franc¸ois Verrey*1 ABSTRACT Amino acids are the building blocks of proteins and key intermediates in the synthesis of biologically important molecules, as well as energy sources, neurotransmitters, regulators of cellular metabolism, etc. The efficient recovery of amino acids from the primary filtrate is a well-conserved key role of the kidney proximal tubule. Additionally, renal metabolism participates in the whole body disposition of amino acids. Therefore, a wide array of axially heterogeneously expressed transporters is localized on both epithelial membranes. For transepithelial transport, luminal uptake, which is carried out mainly by active symporters, is coupled with a mostly passive basolateral efflux. Many transporters require partner proteins for appropriate localization, or to modulate transporter activity, and/or increase substrate supply. Interacting proteins include cell surface antigens (CD98), endoplasmic reticulum proteins (GTRAP3-18 or 41), or enzymes (ACE2 and aminopeptidase N). In the past two decades, the molecular identification of transporters has led to significant advances in our understanding of amino acid transport and aminoacidurias arising from defects in renal transport. Furthermore, the three-dimensional crystal structures of bacterial homologues have been used to yield new insights on the structure and function of mammalian transporters. Additionally, transgenic animal models have contributed to our understanding of the role of amino acid transporters in the kidney and other organs and/or at critical developmental stages. Progress in elucidation of the renal contribution to systemic amino acid homeostasis requires further integration of kinetic, regulatory, and expression data of amino acid transporters C 2014 into our understanding of physiological regulatory networks controlling metabolism. American Physiological Society. Compr Physiol 4:367-403, 2014.

Introduction Amino acids are central metabolites of the body, which are used not only as building blocks of proteins, but also as substrates for energy metabolism, precursors of biologically important molecules, neurotransmitters, and for many other functions. The 20 proteogenic amino acids have a common chemical structure with both a carboxyl and an amino group attached to a central carbon (α-Carbon). The nature of the side group (side chain) attached to the α-Carbon, however, strongly differs between amino acids. Two special cases among the 20 proteinogenic amino acids are glycine and L-proline. Glycine has a hydrogen atom on its α-Carbon instead of a side chain so that it lacks chirality, in contrast to the other proteinogenic amino acids that are all L enantiomers. L-Proline is circular, with the end of its side chain linked to the nitrogen atom; therefore, this molecule is actually an imino acid. With molecular weights of between 57 (Glycine) and 186 (tryptophan) daltons, and the hydrophilic nature of their carboxyl and amino groups, amino acids require transport proteins to cross lipid membranes. Due to their differing side chains their metabolic pathways also differ and furthermore, different amino acid transporters are required for transport across membranes. Actually of the approximately 50 known human amino acid transporters, nearly all display differing amino acid selectivity.

Volume 4, January 2014

Essential vs. nonessential amino acids As regards nutrition and fluxes of amino acids in the body, it is important to consider that our body can only synthesize about half of the proteinogenic amino acids. Amino acids that must be taken up from food are called “essential” (His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val), whereas those the body can produce from other sources are called “nonessential.” This distinction is not absolutely unambiguous since some amino acids are only essential in special conditions (conditionally essential) such as during growth (for instance, Arg, Cys, and Tyr) and/or their production depends on the availability of an essential amino acid (e.g., Tyr from Phe).

Overall Picture of Body Amino Acid Metabolism/Fluxes A simplified schema of the main fluxes of amino acids is shown in Figure 1. Ingested proteins are the source of body * Correspondence

to [email protected] of Physiology and Center of Integrative Human Physiology, University of Zurich, Zurich, Switzerland 1 Institute

Note: Victoria Makrides and Simone M.R. Camargo contributed equally to this work. Published online, January 2014 (comprehensivephysiology.com) DOI: 10.1002/cphy.c130028 C American Physiological Society. Copyright

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Transport of Amino Acids in the Kidney

Comprehensive Physiology

up a large amount of amino acids and play a central role in their metabolism such that the liver can be considered as the sink for many of them. As regards the disposal of amino acid nitrogen, periportal hepatocytes are the major site of incorporation via the ornithine cycle into urea that is excreted by the kidney. Additionally the pericentral hepatocytes can incorporate nitrogen into glutamine for use by the kidney to produce ammonia that may serve for the renal excretion of protons. As regards the carbon chains, hepatocytes represent the major site of their utilization, in particular for the production of glucose via gluconeogenesis.

Extracellular amino acids as a bottleneck of AA fluxes

Figure 1

The role of the kidney in total body amino acid homeostasis. This schematic representation of the main amino acid fluxes through the body shows that amino acids absorbed in the intestine reach the systemic circulation via the liver. Following a single meal the amount ingested may exceed the quantity found in the extracellular space by several fold. Therefore, the rapid cellular uptake and metabolic use of amino acids play central homeostatic roles. Liver, muscles, intestine, and kidney are major sites of amino acid metabolism. Additionally, to prevent their loss the kidneys reabsorb approximately 50 g/day of amino acids from the primary urine.

amino acids. They are digested into small peptides and single amino acids that are absorbed mainly by the small intestine enterocytes. Transepithelial transport by enterocytes is analogous to the reabsorption of amino acids occurring across the proximal kidney tubule epithelial cells (discussed in detail in this review) and is mediated largely by the same transport proteins. Transepithelial transport involves the transport across two membranes: first, the uptake from the (intestinal/proximal tubule) lumen across the apical membrane and then the basolateral efflux to the extracellular space and the blood stream (206). In small intestine enterocytes, approximately half the amino acids are taken up in the form of di- and tri-peptides via the peptide transporter PEPT1 (SLC15A1) and hydrolyzed intracellularly into single amino acids (75, 159). Furthermore, the intestine metabolizes a large amount of amino acids, both some of the absorbed ones and others that reach the intestine via the blood flow and are taken up by intestinal enterocytes via the basolateral membrane. Important metabolic activities of the intestine for amino acids concern in particular glutamine and glutamate, and also some essential amino acids as threonine, leucine, lysine, and phenylalanine (26). Amino acids reaching the blood flow from the gut are transported via the portal vein into the liver lobules where they circulate through the sinusoids along periportal and then pericentral hepatocytes before arriving at the central veins of the lobules and ultimately the hepatic veins. Hepatocytes take

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Amino acids that leave the liver via the hepatic vein reach the systemic circulation, which is in equilibrium with the extracellular space. Importantly, the extracellular space, including blood plasma, represents a bottleneck for body amino acids. Indeed, the total amount of amino acids in this space (∼12 L for a 70 kg human) during the postabsorptive phase amounts to approximately 4 g, whereas in western countries the amino acid uptake during a single meal may be 10 to 30 times this amount. From these numbers, it can be deduced that amino acids need to be taken up and metabolized rapidly. Importantly, aside from incorporation into proteins (anabolism), there is no amino acid storage within the body. As indicated in Figure 1, amino acid metabolism is not limited to the liver, but takes place in many tissues, in particular also in intestine, muscles, and kidney.

Role of kidney for amino acid reabsorption to avoid loss and in the metabolism of specific amino acids The major role of the kidney tubule, as regards amino acids, is to prevent their loss in the urine; a function that is evolutionarily old and well-conserved. Amino acids are not retained at the level of the glomerulus. Their daily filtered load corresponds to approximately 50 g (450 mmol) of which the vast majority is reabsorbed by the proximal tubule. Additionally, filtered and locally produced di- and tri-peptides are also reabsorbed from the primary urine and hydrolyzed intracellularly to single amino acids before subsequent basolateral export. The plasma and thus the ultrafiltrate concentration of free amino acids range from the low μmol/L levels to nearly 1 mmol/L (Table 1). Most amino acids are reabsorbed to ∼99.5% and thus their fractional excretion is below 0.5% of the filtered load. Notable exceptions in humans are serine, glycine, histidine, and taurine for which a higher fraction is lost in the urine (Table 1). Additionally, for several amino acids the fractional excretion is variable and tends to increase with filtered load (187, 196). A substantially higher plasma amino acid concentration and thus filtered load is observed during the absorbtive phase. This is an effect that may be amplified by the increase in renal plasma flow and glomerular filtration rate (GFR) induced by a high protein meal. The increased GFR may be



Table 1


Plasma and urine concentration and fractional excretion of amino acids in human adults

Amino acid

Plasma concentration μmol/L (range)1

Urine concentration (mmol/mol creatinine)2

Fractional excretion (%)3

Alanine

146-494

9-98

0.44

Arginine

28-108

0-8

0.77

Aspartic acid

2-9

5-50

1.96+

Asparagine

26-92

10-65

1.54$

Citrulline∗

10-58

1-22

Cystine

24-54

2-12

Glutamic acid

6-62

0-21

0.68

Glutamine

340-798

11-42

0.71

Glycine

100-384

17-146

2.4

Histidine

68-108

49-413

6.52

Isoleucine

39-90

30-186

0.51

Leucine

98-205

1-9

0.26

Lysine

119-243

2-16

0.92

Methionine

13-37

2-53

0.66

Ornithine∗

36-135

1-5

0.32$

Phenylalanine

42-74

1-5

0.69

Proline

97-297

3-13

0.047$

Serine

78-166

0-9

2.19

Taurine∗

18-95

18-89

6.55+

Threonine

92-197

13-587

1.04

Tryptophan

25-65

6-74

Tyrosine

26-78

3-14

1.38

Valine

172-335

3-36

0.2

1 (173). 2 (203),

3 (196). + (176).

24 h collection.

$ (116). ∗ Nonproteinogenic.

mediated by glucagon, NO or the tubuloglomerular feedback (148, 165). Differences in the fractional excretion of individual amino acids may result from the variation in expression levels, kinetics, ion dependence, and axial localization of the selective amino acid transporters along the proximal tubule of the nephron. The fact that amino acid reabsorption along the nephron is axially heterogeneous and takes place essentially in the proximal tubule (Fig. 2) was first suggested from clinical observations in Fanconi’s syndrome, a condition characterized by glucosuria, phosphaturia, and aminoaciduria (29). The hallmark experiments confirming the role of proximal tubules were carried out using micropuncture and microinjection (57, 175, 177). These experiments demonstrated that approximately 80% of filtered amino acids are reabsorbed in the first half of the proximal convoluted tubule (S1 segment)


whereas less than 1% remains in the tubular fluid at the end of the straight portion (S3 segment) of the proximal tubule (174). Except for taurine, the later tubular segments do not substantially participate in the reabsorption of amino acids (10, 57, 83, 178). A limitation of these functional experiments is that they were perfomed on superficial nephrons. Therefore, differences in the absorptive properties of deep nephrons cannot be excluded (176). The proximal kidney tubule performs other important functions in the disposition of amino acids in addition to mediating reabsorption. As described in the section on amino acid metabolism, this tubular segment plays a central role for the production of ammonia from glutamine. This function is strongly upregulated in acidosis, leading to an increased basolateral glutamine uptake (126). Also, the proximal tubule is a major site for the production of L-arginine from citrulline.

369



cystinuria (Fig. 3B). For an exchanger to participate in the net transport of amino acids the expression of another transporter that can recycle (directionally transport) the exchanged substrate(s) must be expressed in the same membrane (Fig. 3B). In this case, B0 AT1 imports neutral amino acids which can be exchanged by b0,+ AT-rBAT. Whereas the function of the amino acid transporters in the luminal membrane is essentially to take up all amino acids present in the primary urine, the basolateral transport machinery must also preserve an adequate intracellular amino acid concentration. Thus, basolateral amino acid export appears to be controlled by the essential amino acid equilibrium which is maintained between cytosol and extracellular space. Indeed, nonessential amino acids quit the cells apparently only via obligatory exchangers while the recycling essential amino acids can leave the cell via facilitated diffusion pathways (uniporters) (Fig. 3B). The efflux pathway of anionic amino acids from renal proximal tubule epithelial cells has not yet been reliably identified.

From Transport Systems to Transporters Lessons from classical “transport systems”

Figure 2 Scheme of nephron segments involved in amino acid reabsorption. Amino acids are freely filtered at the glomerulus and approximately 99.5% are reabsorbed in the proximal tubule (PT, blue color), mainly in the first and second segments (S1 and S2). Notable exceptions in humans are serine, glycine, histidine, and taurine, which lose a higher fraction in the urine.

The epithelial cells of the proximal tubule are equipped with a wide array of transport proteins on both cellular sides. On their apical pole they display a dense so-called “brush border” of long microvilli that strongly increase the luminal surface, similar to the brush border of small intestine enterocytes. The surface of their basolateral pole is also strongly enlarged, in this case by lateral membrane infoldings. The general scheme of transepithelial transport of amino acids involves the secondary or tertiary active uptake through the luminal membrane followed by the mostly passive efflux toward the extracellular space across the basolateral membrane (Fig. 3A). The apical amino acid transporters are all symporters which use Na+ or H+ influx to drive the influx of their substrate amino acids, with the exception of one transporter that functions as obligatory exchanger (antiporter). The major luminal transporter for neutral amino acids is B0 AT1 (SLC6A19). It functions as a Na+ symporter and defects in its function cause Hartnup disorder. The single luminal antiporter called b0,+ -rBAT (SLC7A9-SLC3A1) takes up cationic amino acids or L-cystine (disulfide dimer of L-cystein) in exchange for intracellular neutral amino acids. Analogous to B0 AT1, this transporter is also expressed in the small intestine and its defect causes an amino aciduria, in the case of b0,+ AT-rBAT

370

The development of new technologies during the second half of the last century allowed researchers to assay amino acid transporter biochemical characteristics. Specifically, it became possible to determine transport selectivity, stereospecificity, ion-dependence (sodium, chloride, potassium, or proton), thermodynamic properties (equilibrative or concentrative), and mechanisms. The availability of radio-labeled amino acids and sensitive analytical quantification methods such as paper and liquid chromatography were especially crucial. The most widely employed experimental systems were initially based on erythrocytes and in vitro cell and tissue culture models. Due to their anatomical and functional characteristics, transport studies often used small intestine and kidney tissues. For example, brush border membrane vesicles (BBMVs), which allow the measurement of luminal amino acid transports in the presence of defined solutes, were produced from intestinal enterocytes and renal proximal tubules. Based on the described characteristics the identified transports were classified as “transport systems” (46, 127, 190). “Transport systems” were named with letters corresponding to the main/first described amino acid substrate, for example, system A (L-alanine), N (L-asparagine), L (L-leucine), and ASC (L-alanine, L-serine, and L-cysteine). The Na+ -dependent systems (except systems L and T) were indicated by capital letters and lowercase letters denoted Na+ -independent systems (e.g., b0,+ ). If charged amino acids were transported, a plus or a minus symbol (e.g., system y+ L and X− AG ) were used to identify the transport of basic or acidic amino acids, respectively. The systems with a broad amino acid selectivity (for example B0 , B0,+ , or b0,+ ) were indicated with letter B. Transport system substrate specificity and ion dependence are shown in Table 2.


(A)

Secondary / tertiary active transporters1 + obligatory exchanger2

AA

AA

Facilitated diffusion transporters3 + obligatory exchanger2

AA

Uniporters3 Symporters1

for essential AA´s

AA+Na+

AA

AA

AA

AA

AA

AA AA

Cystine,AA+ Antiporter2

Antiporter2

Epithelial cell Tubulus lumen

Extracellular space

(B) SLC1A1

EAAT3

AA–+Na++H+

Various AA transporters with specialized metabolic and/or housekeeping functions

K+ AA–

Dicarboxylic aminoaciduria

SLC36A 1/2

PAT1/2

Gly,Ala,Pro+H+

SLC6A20

Gly,Ala,Pro

AA–

Pro

AA0

SIT1

Pro+Na+

?

TAT1 and LAT4

AA– SLC16A10 and 43A2

AA0

Tmem 27 4F2hc

SLC6A19/18 AA0+Na+ Hartnup disorder

B0AT1/3

AA0

AA0

SLC3A2

AA0 LAT2

SLC7A8

4F2hc

SLC3A2

y+LAT1

SLC7A7

Tmem27

SLC7A9

0,+

b

AT

Cystine,AA+ SLC3A1

AA0 Cys,AA–

AA+

rBAT

Lysinuric protein intolerance

SLC15A1/2

Tubulus lumen

PEPT1/2

Hydrolysis

Cystinuria

di-/tripeptides+H+

AA0+Na+

AA Epithelial cell

Extracellular space

Figure 3 Cellular model for the reabsorption of amino acids across a proximal tubule cells. Panel A shows a schematic representation of the luminal and basolateral amino acid transporters. Most amino acids are transported across the luminal membrane by symporters that use the electro-chemical gradient of Na+ or H+ for driving the influx of amino acids. Neutral amino acids may be recycled to the lumen by the exchanger b0,+ AT-rBAT (SLC7A9-SLC3A1) to allow the uptake of cationic amino acids and cystine. The basolateral efflux of most nonessential neutral amino acids and of cationic amino acids is mediated by antiporters. Essential amino acids taken up in exchange for cationic amino acid efflux may recycle to the extracellular space via selective antiporters. Panel B depicts most of the known renal transport proteins; their protein and human gene names are indicated. In mice most of the transporters are expressed mainly in the early segments of the proximal tubule (S1 and S2). However, SIT1 (SLC6A20) is expressed along the entire proximal tubule, and B0 AT3 (SLC6A18) and EAAT3 (SLC1A1), are expressed in the later proximal tubule (S2 and S3). Key: AA0 neutral amino acid, AA− anionic amino acid, AA+ cationic amino acid, TMEM27 (collectrin).


Table 2


Overview of amino acid and oligopeptide Transport Systems and corresponding Solute carrier families (SLC) Transport systems

Solute carrier families

Transport systems

Ion dependence Substrate

Type SLC

Gene and protein name

− XAG

Na+

S/A

SLC1A3 EAAT1, GLAST

Negative charge

SLC1

ASC

Na+

Neutral

NA

NA

Neurotransmitter Na+ /Cl−

Neurotransmitters

A

Na+ /Cl−

Osmolites, GABA

7 AAT

SLC1A2 EAAT2, GLT1 SLC1A1 EAAT3, EAAC1 SLC1A6 EAAT4, EAAT5

SLC3

S

SLC3A1 rBAT SLC3A2 4F2hc

2 accessory proteins

SLC6A2 NET, NAT1, NET1

S

SLC6A3 DAT1, DAT SLC6A4 SERT, 5-HTT SLC6A1 GAT1 SLC6A13 GAT2, GAT3 SLC6A11 GAT3, GAT-B, GAT-4 SLC6A12 BGT1 SLC6A6 TauT SLC6A8 CT1, CRTR

creatine

Na+ /Cl−

Creatine

S

GLY

Na+ /Cl−

Glycine

S

SLC6A5 GlyT2 SLC6A9 GlyT1

B0,+ , ATB0,+ B0

Na+ /Cl− Na+

Neutral/Cationic Neutral

S

SLC6A14 ATB0,+ SLC6A15 B0 AT2, v7-3, NTT7-3 SLC6A16 NTT5

Na+ /Cl−

Family members

SLC1A4 ASCT1 SLC1A5 ASCT2, ATB0

SLC6 BETA

Transport scheme

S

9 AAT 2 orphans ∗∗ 2 pseudogenes 7 neurotransmitter 2 other solutes

SLC6A17 NTT4, XT1 SLC6A18 XT2, B0 AT3 IMINO

Na+ /Cl−

Imino

S

y+

None

Cationic

U

SLC6A19 B0 AT1, XT2s1 SLC6A7 PROT SLC6A20 SIT1, XT3s1, Xtrp3 SLC7A1 CAT-1 SLC7

SLC7A2 CAT-2 SLC7A3 CAT-3 SLC7A4 CAT-4

L

None

Neutral

A

y+L

None

Cationic

A

SLC7A5 LAT1 SLC7A8 LAT2 SLC7A6 y+LAT2

b0,+

Na+ None

Neutral Cationic Neutral

A

SLC7A7 y+LAT1 SLC7A9 b0 ,+AT

x−

None

Anionic

A

SLC7A11 xCT SLC7A13 AGT-1

asc

None

Neutral

A

SLC7A10 Asc-1

Peptide

H+

Oligopeptide

S

SLC15A1 PEPT1 SLC15A2 PEPT2 SLC15 SLC15A3 PHT2 SLC15A4 PHT1

372

12 AAT 2 orphans∗∗ 2 pseudogenes

4 peptide transporters



Table 2


(Continued) Transport systems

Solute carrier families

Transport systems

Ion dependence Substrate

Type SLC

T

None

U

Aromatic AA

Gene and protein name

SLC16 SLC16A10 TAT1

Transport scheme

Family members 1 AAT 7 orphans∗∗ 6 other solutes

Glu

H+

Glutamate

Ves. A

SLC17A6 VGLUT2 SLC17A7 VGLUT1 SLC17 SLC17A8 VGLUT3

3 vesicular AAT 2 orphans∗∗ 4 other solutes

GABA

H+

GABA, glycine

Ves. A

SLC32 SLC32A1 VIAAT, VGAT

1 vesicular AAT

IMINO

H+

Imino

S

SLC36 SLC36A1 PAT1, LYAAT1

3 AAT

SLC36A2 PAT2, tamodorin 1 SLC36A3 PAT3

1 orphan∗∗

SLC36A4 PAT4, LYAAT2 A

Na+

Neutral

N

Na+ /H+

Neutral

S/A

SLC38 SLC38A1 SNAT1, ATA1, NAT2, SAT1 SLC38A2 SNAT2, ATA2, SAT2 SLC38A4 SNAT4, ATA3, NAT3, PAAT SLC38A3 SNAT3, SN1

6 AAT 5 orphans∗∗

SLC38A5 SNAT5, SN2 SLC38A7 SNAT7 L

None

Neutral

U

SLC43 SLC43A1 LAT3 SLC43A2 LAT4

2 AAT 1 orphan∗∗

SLC43A3 EEG1 44 Amino acid and oligopeptide transporters ∗ Based on http://www.bioparadigms.org/slc/intro.htm. ∗∗ orphans, or orphan transporters, correspond to genes

with not yet attributed function; AAT, amino acid transporter(s); Ves., vesicular; S,

symporter; A, antiporter; U, uniporter.

Molecular indentification and SLC families In the late 1980s, the first transporters were molecularly identified starting a new era in the transporter field. The molecular characterization of transporters was made possible by the use of the complementary techniques of DNA cloning and cellular overexpression in systems such as Xenopus laevis oocytes (22, 86, 95, 139, 205). Since the transporter systems were originally named based on their substrate and ion specificity initially the “old” nomenclature rules were applied to the newly molecularly identified proteins (46) resulting in multiple names for the same transporter (Table 2). To address this problem, the human genome orga-


nization (HUGO) introduced a new classification and nomenclature scheme for naming transporter genes as “Solute Carriers” (SLC). Transporters were grouped in families based on sequence homology (86). Among the 52 SLC families currently listed (http://slc.bioparadigms.org), there are ∼50 genes grouped in 11 families that code for proteins, which are vesicular, or plasma membrane localized amino acid, oligopeptide, and/or neurotransmitter transporters (Table 2). In addition to the more detailed functional characterizations accomplished using cellular overexpression systems, the availability of cDNA allowed for measurement of mRNA gene expression; first by Northern blot analysis, and more recently

373


using real time quantitative RT-PCR and microarray chips. Specific antibodies could now also be generated for localizing transporter proteins at the tissue, cellular, and subcellular levels. Furthermore, the genetic defects underlying hereditary amino acid transport-related diseases, such as hyperaminoacidurias like Hartnup Disorder (104, 169) could now be identified. The diseases and the known mutations causing them will be discussed in a later section on aminoacidurias.

Transporter mechanisms, kinetics Solute carriers transport substrates by a complex process known as the “Alternating Access Model,” which was originally proposed in 1966 (93). It is depicted in Figure 4A and B (68, 93). This model hypothesizes allosterically regulated conformational changes in the transporter results in the protein sequentially opening the substrate binding site to alternating sides of the membrane. Substrate(s) movement through the membrane is accomplished without substantially changing the relative position of the substrate binding site in the membrane. Furthermore as for other enzymes, there are a defined number of substrate binding sites, that is, transport is saturable. Importantly, substrate binding is ordered and binding sites are exposed to only one side of the membrane at any given time. Carriers are subclassified as uniporters (U), symporters (S) (or cotransporters), or antiporters (A) (or exchangers) according to the number, and relative direction of the movement of the solutes (Fig. 4C). For the simplest type of carrier, the uniporter (U), or facilitated diffuser (e.g., TAT1 transporter) the chemical gradient of the transported solute determines the direction of net solute flux. This means that facilitated diffusers are unable to concentrate substrate. In contrast, symporters and antiporters can be concentrative. This is accomplished by tightly coupling the transport of an amino acid or peptide substrate to the energetically favorable concentration and/or electrical gradient (driving force) of another substrate. For example, B0 AT1 and PEPT1 are symporters that use Na+ or H+ electrochemical gradients across the membrane to drive the import of neutral amino acids and oligopeptides, respectively. Several transporters from the SLC7 family, for example, LAT2-4F2 and b0,+ -rBAT, are antiporters, which mediate the obligatory 1:1 exchange of amino acid substrates. Antiporters (obligatory exchangers) can use substrate concentration gradients between the extracellular and intracellular compartments as a driving force (121). Antiporter transport is described in more detail in the section “Cooperation of amino acid transporters.” Table 3 summarizes the transport mechanism, substrate selectivity and affinity for solute carrier proteins expressed in the kidney. As can be observed, some transporters with similar substrate selectivity differ in their apparent affinity for the same substrate, as well as mechanism of transport. For example, the apparent affinity for the neutral amino acid L-isoleucine varies 25-fold between the obligatiory exchanger, LAT2-4F2 (apparent Km of 40 μmol/L), and Na+ symporter, B0 AT1 (1 mmol/L Km ).

374


Transgenic animal models The molecular identification of transporter genes along with the development of techniques for the genetic modification of animals has lead to the generation of transgenic animal models (Table 4). Furthermore, genetic modifications can now be tailored. For example, the total or partial gene ablation, or the up- or downregulation of gene expression spatially and/or temporally in specific subsets of cells and/or at desired developmental stages can be engineered (88). Since amino acid transporters potentially supply essential compounds for many tissues and/or during critical developmental stages the ability to fine-tune expression is particularly important. These models have proven to be valuable tools for the in vivo analysis of the physiological roles, function, and regulation of accessory proteins, and amino acid and oligopeptide transporters. Therefore, their availability has provoked a resurgence in the use of classical physiological techniques for example, urine analysis, determination of amino acid fractional excretion, microperfusion, isolated tubule perfusion, etc. An overview of current transgenic rodent models for acessory protein, amino acid, and oligopeptide transporters is found in Table 4. Revelant results for renal function from experiments using transgenic models are described briefly in the sections on specific transporters.

3D structure The structure-function relationships (such as protein interactions, solution accessibility of residues, tertiary and quaternary structure, allosteric binding domains, etc.) of amino acid transporters and other solute carrier family members can be probed using variety of structural manipulations (70, 94, 161). Figure 5 shows representative data from two such experimental strategies. Figure 5A and B illustrate the chimeric rBAT/SLC3A1 and 4F2/SLC3A2 proteins produced to delineate the domains of each accessory protein interacting with their respective Slc7 family partners (i.e., b0,+ AT with rBAT and LAT1 with 4F2hc). A more detailed description of the interactions between the SLC3 and SLC7 “Heterodimeric Amino acid Transporters” is in the section on “Accessory proteins.” Figure 5C demonstrates the “cysteine scanning method,” which by systematically replacing amino acids with cysteines tests for the formation of di-sulfide bridges, that is, the accessibility of the mutated residue to reagents or drugs. This approach was used to investigate the substrate binding site of the neurotransmitter transporter SERT (Slc6a2). The determination of high-resolution three-dimensional (3D) cystal structures for several bacterial homologues of mammalian transporters has resulted in further insights in amino acid transporter structure-function. To date solved structures include homologues of: (i) the sodium-dependent glutamate transporters (SLC1 family), namely the aspartate transporter from Pyrococcus horikoshii (GltPh , PDB 1XFH); (ii) the sodium-dependent neutral amino acid transporter B0 AT1/SLC6A19 (SLC6 family), namely the leucine



(A)


Symporters

(B)

Antiporters Outward face

1

2

6

1

2

4

3

5

4

3

Inward face

Substrates Ion

(C) Solute gradient number of substrate

Example

Uniporter 1 solute In direction of solute gradient

Solute

Solute Na+ or H+

Symporters 2 or more solutes Against solute gradient

Antiporters 2 solutes Against solute gradient of at least 1 substrate

Solute Solute

In

TAT1, LAT4

B0AT1, PEPT1

LAT2-4F2hc, b0,+AT-rBAT

Out

Figure 4

Transport mechanisms: symporters, antiporters, and uniporters. Solutes are transported by secondary active transporters by an alternated access mechanism. The number of steps and solutes involved depends on the transport mechanism (uniporter, symporter, or antiporter). Transport mechanisms are classified based on the number of substrates simultaneously transported and the relative direction of substrate movements. (A and B) Alternative access mechanism for symporters and antiporters. (A) Symporters: Upon substrate (red circle) and ion (blue square with red center) binding to the open outward-facing state (2), the substrate-ion-bound transporter changes to the occluded and inward-facing states (172) releasing substrates. A transition between the unbound inward- and outward-facing conformations is required to renew the transport cycle (4 and 1). For antiporters (B), substrate binding to the outward-facing state (blue diamond) results in the transition to the inward-facing states (172) and substrate release. Restoration of the outwardfacing state (4 to 1) requires the binding of a substrate to the inward-facing transporter state (red circle) (5) to allow the transporter to return to the outward-facing conformation and release the substrate (6). (C) Chart of each of transporter mechanism. Uniporters, move one solute along the solute concentration gradient; Symporters, translocate two or more substrates in the same direction, one solute (e.g., AA) eventually against its concentration gradient and the other solute (e.g., Na+ ) along its concentration gradient; Antiporters, translocate two substrates in opposite directions against the gradient concentration of one of the substrates. For symporters and antiporters, the presence of both substrates involved in the transport cycle is required for the translocation. All three mechanisms are utilitzed by amino acid and oligopeptide transporters. TAT1 (SLC16A10) is an example of an aromatic amino acid uniporter. B0 AT1 (SLC6A19) is a symporter, cotranslocating neutral amino acids with sodium. PEPT1 (SLC15A1) is another symporter that cotransports peptides and protons. The heterodimeric amino acid transporter LAT2-4F2hc (SLC7A8- SLC3A2) is an antiporter that exchanges neutral amino acids.


375


Table 3


Renal amino acid transporter mechanism, substrates, ion dependence, stoichiometry and apparent affinities1

Protein name2

SLC name3

Transport system

Ion dependence and stoichiometry

Substrates

Transport type

Apparent affinity (mmol/L)

Apical surface localization B0 AT1 B0 AT3

SLC6A19 SLC6A18

B

Neutral AA Neutral AA

1 Na+ : 1 AA 1 Na+ : 1 Cl− : 1 AA

S S

Ile 1 (35) Ile 0.2 (180)

b0,+ -rBAT

4 SLC7A9-SLC3A1

b0,+

R,K,ornithine, cystine

—

A

Cystine 0.04 (145)

PAT2

SLC36A2

IMINO

P,G,A, MeAIB

1 H+ : 1IA

S

Gly 0.5 (41)

SIT1

SLC6a20

P, MeAIB

2 Na+ : 1 Cl− : 1AA

S

Pro 0.13 (106)

L or D-D, L-E

3 Na+ : 1 1K+ (-A)

S/A

Glu 0.04 (186)

di- or tri-peptides

1-2H+ : Pep

S

Gly-Sar 0.3 (113) Gly-Sar 0.07 (154)

Taurine, hypotaurine, and β-alanine

2 Na+ : 1 Cl− : 1AA

S

Tau 0.04 (185)

Leu 0.04 (121)

EAAC1/EAAT3

SLC1a1

X−

PEPT1 PEPT2

SLC15A1 SLC15A2

Peptide

Taut1

SLC6A6

AG

H+ (-S);

Basolateral surface localization LAT2-4F2

SLC7A8-SLC3A2

L

Neutral AA, but not P L,I,M,F,BCH

—

A

—

U

LAT4

SLC43A2

y+ LAT1-4F2

SLC7A6-SLC3A2

y+ L

K,R,Q,H,M,L

1 Na+ : 1 NAA (-S); CAA (-A)

A

Leu 0.02 (146)

TAT1

SLC16A10

T

F,Y,W

—

U

Phe 30 (151)

S/A

Gln 1.5 (18)

A?

Asp 0.025 (120)

A

Ala 0.023 (73)

Na+ : 1AA H+ (-A)

SNAT3/SN1

SLC38A3

N

Q,N,H

1 1

AGT1/XAT2

SLC7A13-SLC3A2

x−

L-D, L-E

—

(-S);

Unkown subcellular localization Asc1-4F2

SLC7A10

Asc2

SLC7A12&

asc

G,A,S,C,T

—

S,G,A,T,L,F,Y

—

Ser 0.003 (40)

1 (22, 34, 97, 139, 193, 204, 206), AA, amino acid; IA, imino acid; CAA, 2 predominant alias. 3 SLC names designated by the HUGO gene nomenclature committee.

cationic amino acid; Pep, peptide.

transporter from Aquifex aeolicus (LeuTAa , PDB 2A65) and; (iii) the mammalian peptide transporters (SLC15 family), the peptide transporter from the bacterium Shewanella oneidensis (PEPT(So), PDB 2XUT) (132, 211, 213). As illustrated in Table 5, the 3D structures broadly share an inverted repeat motif, although the exact number and orientation of repeats is specific for each SLC family. The substrate binding site is situated between the repeats and located approximately in the center of a vertical axis through the cell membrane. This is consistent with the alternating access model in which transporter protein conformational changes result in the exclusive

access of the substrate binding site to either the inner or outer membrane surface without its net movement in the membrane. The structures of mammalian transporters modeled based on their homology to prokaryote SLCs can be combined with data derived from mutagenesis and other structural studies to achieve a more comprehensive understanding of the structure-function relationship of these molecular machines. Currently, the research focus is on questions such as identifying ion binding sites, and analyzing conformational changes occurring during the transport cycle, [for review L. Forrest (68, 69)].

376



Table 4


Accessory protein, amino acid, and oligopeptide transporter transgenic animal models

Protein/gene

Substrates∗

Tissue expression∗

Phenotypes

References

EAAT3 (Slc1a1)

L-Glu, D/L-Asp

brain (neurons), intestine, kidney, liver, heart, placenta

High urinary L-glutamate and aspartate

(142)

EAAT1 (Slc1a3)

L-Glu, D/L-Asp

Brain (astrocytes, Bergmann glia), heart, skeletal muscle, placenta

Increased novelty-induced locomotor hyperactivity, abnormal social behavior characterized by reduced initiation of social interactions, poor nesting, and impaired pairwise visual discrimination learning.

(99)

rBAT (Slc3a1)

Heterodimerizes with light subunit b0,+

Kidney, small intestine, liver, pancreas

High urinary cystine and dibasic amino acids

(63, 67, 144)

4F2hc (Slc3a2)

Heterodimerizes with Slc7 family light subunits y+ Lat1, y+ Lat2, Lat1, Lat2, xCT, and associates with β-integrins

Ubiquitous

Total knockout is early embryonic lethal. Deletion from lymphocytes and vascular smooth muscle decreased proliferation, altered adaptive humoral immunity or vessel repair respectively. Overexpression in gastrointestinal epithelium induces barrier dysfunction, cell proliferation and tumorigenesis

(37, 38, 66, 199)

Gastrointestinal epithelial knockout reduces resistance to inflammatory disease and colitis GLYT2 (Slc6a5)

Glycine

Brain (glycinergic neurons, Golgi cells, brain stem, cerebellum), spinal cord

Impaired high-affinity glycine uptake in brain areas with glycine-mediated transmission.

Taut (Slc6a6)

Taurine

Brain, retina, liver, kidney, heart, spleen, pancreas, placenta, skeletal muscle, lung

Decreased plasma taurine, increased urinary taurine, severe retinal degeneration, impairment of reproduction and reduced total exercise capacity

(87, 90)

CT1 (Slc6a8)

Creatine

Ubiquitous

Learning and memory deficits

(182)

GLYT1 (Slc6a9)

Glycine

Brain, retina, liver, spleen, kidney, pancreas, uterus, stomach, lung, placenta, intestine

Severe motosensory and respiratory deficits, Early postnatal death.

(80)

B0 AT2 (Slc6a15)

Large, neutral amino acids

Brain (amygdala, putamen, corpus callosum), kidney

Low L-leucine accumulation in synaptosoms.

(56)

Kidney (proximal tubule)

Abnormal renal excretion of several neutral amino acids especially glycine

(149, 180)

Lacks renal B0 AT1 expression. Massive neutral aminoaciduria without glucosuria or phosphaturia.

(52, 118)

Lacks intestinal B0 AT1 expression.

(36)

B0 AT3

(Slc6a18)

Neutral amino acids

Coll (Tmem27)

Associates with renal B0 AT1, B0 AT3

Ace2 (Ace2)

Associates with intestinal B0 AT1

B0 AT1 (Slc6a19)

Neutral amino acids

(81)

Premature death

Modest behaviorial effects

Impaired intestinal neutral amino acid transport. Intestine (duodenum, jejunum, ileum), stomach, kidney, liver, prostate

Reduced body-weight. Impaired neutral amino acid transport in Brush-border vesicles.

(19)

(continued)


377


Table 4


(Continued)

Protein/gene

Substrates∗

Tissue expression∗

Phenotypes

References

CAT-1 (Slc7a1)

Cationic amino acids

Ubiquitous

Reduced birth weight Lethal at birth

(143)

CAT-2 (Slc7a2)

Cationic amino acids

CAT-2A: liver, skeletal muscle, pancreas, CAT-2B: inducible in many cell types

Reduced NO production

(133)

y+ LAT1 (Slc7a7)

Cationic amino acids (Na+ indep.), large neutral L-amino acids (Na+ dep.)

Small intestine, kidney, spleen, leucocytes, placenta, lung/basolateral in epithelial cells

Intrauterine growth retardation and low survival rate

(189)

LAT2 (Slc7a8)

Neutral L-amino acids, T3, T4, BCH

Small intestine, kidney, lung, heart, spleen, liver, brain, placenta, prostate, ovary, fetal liver, testis, skeletal muscle

High urinary neutral amino acids

(17)

b0,+ (Slc7a9)

Cationic amino acids, large neutral amino acids

Kidney, small intestine, liver, placenta

High urinary cystine and dibasic amino acids

(63, 67, 144)

Asc1 (Slc7a10)

Small neutral amino acids

Brain, CNS, lung, small intestine, heart, placenta, skeletal muscle, kidney

Severe brain phenotype with tremors and seizures.

(163, 210)

Early postnatal death.

xCT (Slc7a11)

Cystine (anionic form), L-glutamate

Macrophages, brain, retinal pigment cells, liver, kidney

High plasma cystine

(166)

PEPT1 (Slc15a1)

Di- and tri-peptides, protons, β-lactam antibiotics

Small intestine, kidney, pancreas, bile duct, liver

Reduced intestinal absorption of glycylsarcosine

(89, 131)

PEPT2 (Slc15a2)

Di- and tri-peptides, protons, beta-lactam antibiotics

Apical surface of epithelial cells from kidney and choroid plexus; neurons, astrocytes (neonates), lung, mammary gland, spleen, enteric nervous system

Increase glycylsarcosine clearance

(137, 171)

TAT1 (Slc16a10)

Aromatic amino acids, T3, T4

Kidney, intestine, muscle, placenta, heart

Increased urinary and plasma, muscle and kidney aromatic amino acid concentrations

(119)

MCT12 (Slc16a12)

Creatine

Kidney, retina, lung, muscles, testis

High urinary creatine excretion

(1, 39)

SNAT3 (Slc38a3)

Glutamine, histidine, alanine, and asparagine

Liver, skeletal muscle, kidney, pancreas

Lethal postnatal day 10-14

C.Wagner, personal communication; (160)

LAT4 (Slc43a2)

L-BCAAs, amino alcohols

Placenta, kidney, peripheral blood leukocytes, small intestine

No functional data is yet available.

F. Verrey, personal communication

∗ Based

on http://www.bioparadigms.org/slc/intro.htm.

Regulation of Transporter Expression, Localization, and Function Partner proteins For their appropriate function many amino acid transporters require physical or functional interaction with one or more partner proteins. The proteins associating with transporters act to either guarantee appropriate transporter insertion in the

378

membrane, modulate transporter activity, or increase substrate supply. The interacting or cooperating proteins are members of various protein classes such as cell surface antigens (CD98), endoplasmic reticulum (ER) proteins (GTRAP3-18 or 41), amino acid transporters (TAT1 and LAT2-4F2hc or ASCT2 and LAT1-4F2hc), enzymes (ACE2, aminopeptidase N), etc. The specific mechanisms of interaction will be discussed in the following sections.




(A)

(B)

rBATΔ117

B44B

1.00 0.75 0.50 0.25

BB4B B44B rBATΔ117

Not inj

0.00

4F2hc rBAT 444B 44BB 44B4 4BB4 BBB4 BB44

TM Cytoplasmic tail

L-arginine influx rate (relative to rBAT)

Extracellular domain Neck

BB4B

BB44

BBB4

rBAT

4F2hcΔ133

4F2hcΔ117

4BB4

44B4

44BB

444B

4F2hc

1.25

(C)

Figure 5

Chimeras and cysteine scanning experimental strategies to study the structure function relationship of solute transporters. Functional heterodimeric amino acid transporters (HATs) are formed by SLC7 transporters interacting covalently with accessory proteins of the SLC3 family, rBAT (SLC3A1) or 4F2hc (SLC3A2). To date rBAT has only been confirmed to interact with one SLC7 family member, b0,+ (SLC7A9); while 4F2hc interacts with several members including LAT1 (Slc7a5). (A) Schematic representation of chimeras and truncations of rBAT and 4F2hc. To define the domain(s) of rBAT and 4F2hc involved in the recognition and interaction with the Slc7 transporters, chimeras, and truncations of rBAT and 4F2hc were constructed and in combination with b0,+ and LAT1 functionally assayed. The chimeras were prepared by combining the following protein regions: cytoplasmic tail, transmembrane domain, neck, and glycosidase-like domain. rBAT is represented in blue and 4F2hc in yellow. (B) L-Arginine transport by b+0 AT coexpressed with various chimeras. When coexpressed with b0,+ AT, chimeras containing rBAT cytoplasmic tail (BB44, BB4B) with or without the transmembrane domain (BBB4) transport L-arginine indicating a role in the interaction between rBAT with b0,+ AT [adapted, with permission, from (70)]. (C) Schematic representation of targeted SERT residues. “Cysteine scanning” or the systematic introduction of cysteine or lysine mutations is another method for studying the relationship of specific residues to transporter function. To probe conformational changes occurring during the transport cycle the targeted residues are evaluated for cytoplasmic or extracellular accessibility and/or impact on transport function or protein expression. Rudnick and collaborators [adapted from reference (161) with permission] extensively studied the serotonine transporter (SERT/ SLC6A2), which is responsible for reuptake of 5-hydroxytryptamine (5-HT, Serotonin) in the postsynaptic membrane and is a target for antidepressants such as Fluoxetine (Prozac). The scheme for SERT is based on the LeuT model (discussed in the text). Residues studied by cysteine scanning are labeled in red. Combining the structural predictions based on the bacterial homologs and the cysteine scan, the authors suggested several possible protein conformations during the transport cycle that allow substrate binding and dissociation from both sides of the membrane.

Accessory proteins Heavy chains and the heterodimeric amino acid transporters (SLC3 family) Heterodimeric amino acid transporters (204) are formed by an amino acid transporter or light chain (SLC7 family members) and a partner protein from the SLC3 family, rBAT, or CD98/4F2hc (SLC3A1 and SLC3A2, respectively).


CD98/4F2hc or rBAT are type II glycoproteins, and for historical reasons are also called “heavy chains.” To date, only b0,+ AT (Slc7a9), has been shown to interact with rBAT, while several SLC7 family members are known to interact with 4F2hc [LAT1, LAT2, y+ LAT1, y+ LAT2, xCT, and ASC1 (SLC7A5, 8, 7, 6, 11, and 10, respectively)]. The SLC7 and SLC3 family member proteins are covalently linked via a disulfide bridge (205) (Fig. 6A and B). The interaction is

379


Table 5


3D structure of bacterial homologs amino acids and solute transporters

Bacterial transporter and SLC mammalian homolog

Secondary structure and repeats

Sodium-dependent glutamate transporter

3D

Reference (213)1

GltPh

SLC1

Oligopeptide–proton symporters PepTSO

(132)

SLC15

Na+ /Cl− -dependent

(211)

leucine transporter leuT

SLC6

Sodium galactose transporter vSGLT

(59)

SLC5

Na+ -betaine transporter

(156)

BetP

1 From [Faham S, Watanabe A, Besserer GM, Cascio D, Specht A, Hirayama BA, Wright EM, and Abramson J. The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+ /sugar symport. Science 321: 810-814, 2008]. Reprinted with permission from AAAS. Otherwise all figures were reprinted with permission from the cited references (numbers: 213, 132, 211, 156).

necessary for the correct targeting of the transporter-complex to the plasma membrane. When heterologously overexpressed in the absense of b0,+ AT, rBAT is sensitive to endoglycosidase H and degradation by the ER-mannosidase-dependent pathway (7, 48). However, when rBAT is coexpressed with

380

b0,+ AT most of the protein is found in a complex as b0,+ rBAT. The fate of the b0,+ -rBAT complex was suggested to be defined by a trafficking signal on b0,+ . Once the b0,+ -rBAT complex forms, it trafficks out of the ER, is fully glycosylated, and reaches the plasma membrane as an active complex.




(A)

(B) SLC3

SLC7

rBAT (SLC3A1)

b0,+ (SLC7A9)

4F2hc (SLC3A2)

LAT1 (SLC7A5) y+LAT2 (SLC7A6)

Extracellular

Accessory protein (SLC3)

y+LAT1 (SLC7A7) Disulfide bridge

LAT2 (SLC7A8) asc1 (SLC7A10)

H110

xCT (SLC7A11)

C158

COOH C109

Catalytic subunit (SLC7)

NH2 NH2

COOH

Cytosolic

Figure 6

The heterodimeric amino acid family. The HAT family is formed by glycoproteins from the SLC3 (rBAT and 4F2hc) family and amino acid transporters from the SLC7 family. (A) Chart of known SLC3 transporter partners. 4F2 interacts with several SLC7 transporters while rBAT is only known to interact with b0,+ AT. (B) Schematic representation of covalent interaction between SLC3 and SLC7 members. The SLC3 heavy subunits (pink), which are type II membrane glycoproteins with an intracellular NH2 terminus and a single transmembrane domain, and the SLC7 transporters [light subunit (blue)] are linked by a disulfide bridge (yellow) with conserved cysteine residues (e.g., cysteine 158 for the human xCT and cysteine 109 for human 4F2hc). [Adapted, with permission, from (140).]

If the b0,+ AT C-terminal signal is deleted or mutated, the b0,+ -rBAT complex still forms but is inactive (164). Transgenic animal models with either rBAT or 4F2 deletions have been used to investigate their in vivo roles (Table 4). For more details about the effects of mutation on rBAT function, see b0,+ -rBAT activity in the section on luminal proximal tubule transporters. Briefly, although a knock out of the Slc3a1 gene coding for rBAT results in a relatively mild phenotype, the total knockout of the Slc3a2 gene expression (4F2) is embryonic lethal (199). This may be due to disruption of essential signaling pathways induced by 4F2 interactions with β1 and β3 integrins in addition to its amino acid transporter interactions. The physiological role of 4F2/ Slc3a2 has been further probed in tissue specific contexts (e.g., intestine, lymphocytes, and vascular smooth muscle cells) using conditional transgenic animals (30).

Modulating interacting proteins (GTRAP3-18 or 41, negative modulators of Slc1 family members) The Slc1 family members 1 and 6, which encode the transporters EAAT3/EAAC1 and EAAT4, are glutamate and aspartate transporters expressed in epithelial cells (kidney and intestine) and brain. They have been shown to be modulated by GTRAP3-18 or 41 (Glutamate TRansport Associated Protein). GTRAPs are structurally homologous to the Ras superfamily but lack a GTP-binding consensus motif. GTRAP3-18 is an integral ER membrane protein with four transmembrane domains, and cytosolic N and C termini (162), which interacts with the carboxy-terminal end of EAAT3/EAAC1. GTRAP318 and EAAT3 are expressed in the same tissues (32) and the interaction of EAAT3 in the ER with GTRAP3-18


oligomer protein may regulate function by delaying trafficking of EAAT3 from the ER to the Golgi and consequently reducing EAAT3-mediated glutamate transport (162).

Neutral amino acid transporters associate with TMEM27 and ACE2 Solute transport and the renin angiotensin system Expression and/or function of SLC6 family neutral amino acid transporters have recently been shown to rely on the expression of two members of the renin angiotensin system (RAS), collectrin/TMEM27 and the angiotensin-converting enzyme 2 (ACE2). Both genes are localized on the X-chromosome (48, 215). ACE2 shares approximately 40% sequence identity with the ACE. It is a carboxypeptidase localized to the apical membrane in polarized cells including kidney proximal tubules (212). ACE2 is not inhibited by classical ACE inhibitors (captopril and enalapril) (85) and also functions as receptor for SARS coronavirus (91, 108). TMEM27 or Collectrin is a nonenzymatic homologue of ACE2 (215) expressed in kidney proximal tubule brush border membranes and is essential for the expression of several amino acid transporters (52, 118). In the tmem27 knockout mice, the kidney expression of B0 AT1/Slc6a19 and B0 AT3/Slc6a18 is strongly downregulated resulting in a urinary loss of massive amounts of amino acids (52, 118). Tmem27 gene ablation induces abnormal protein expression of amino acid transporters in proximal tubules with reduced transport function. Not only does the knockout affect expression of B0 AT1 and B0 AT3, additionally expression of the imino transporter SIT1 (Slc6a20) and the SLC1 L-glutamate and L-aspartate transporter EAAT3/EAAC1 (slc1a1) was shown to be abolished

381



(A)

(B)

Residues

Chromosome

ACE2

805

Xp22

S

F

G

P R

(D) B0AT1

(E) B0AT1

Collectrin

P

Protein

P

(C)

ACE2

bm F or AA NA+

N

F

C

AA AA

(F)

(G) Merge

Tmem27 collectrin

222

Xp22

C

AA NA+

In

Out

(J)

(K)

(L)

(H)

ace2 WT KO +/y

–/y

(I)

Merge

SIT1

ACE2 coll WT KO +/y

–/y

***

(M) βA

βA

60 L-proline transport (pmol/h/oocyte)

Kidney

Small intestine

Merge

N

40

20

0

hSIT1

hSIT1 + hACE2

hACE2

Interaction of SLC6 family members with the Renin-Angiotensin system proteins. (A) Schematic representation of B0 AT1 interaction with RAS members ACE2 and TMEM27. ACE2 and TMEM27/Collectrin are type I integral membrane proteins with N-terminal (N), transmembrane, and short C-terminal (C) domains. ACE2 has one zinc-binding motif (HEMGH) (bm) in the extracellular domain and is a carboxipeptidase with the consensus sequence P()1-3 P/+. The bradikinin-(1-8) peptide depicted (reversed) is one of the best in vivo targets of ACE2 (84). (B-J) ACE2 and Tmem27/collectrin tissue specific colocalization with B0 AT1. Representative immunofluorescent images of mouse tissue sections labeled with antibodies against B0 AT1 (red) in small intestine (C) and kidney (E), and against Ace2 in small intestine (B) and Tmem27/collectrin in kidney (D). Panel (F) shows colocalization of B0 AT1 and Ace2 in small intestine enterocytes (G) the colocalization of B0 AT1 with Tmem27/collectrin in the proximal tubules. (H and I) Ace2 and Tmem27/collectrin knockout ablates B0 AT1 expression in the small intestine or kidney, respectively. ACE2 and TMEM27 are encoded by X chromosome-linked genes. Western blots of small intestine (H) and kidney (I) tissue lysates from wild-type vs ace2−/y and coll−/y knockout mice probed with anti-B0 AT1 and anti-β-actin (loading control) antibodies (36, 52). (J-M) SIT1 colocalizes and functionally interacts with ACE2. SIT1 (SLC6A20) colocalizes with ACE2 in human small intestine (J, K, and L) and its coexpression with ACE2 in Xenopus laevis oocytes stimulates SIT1 transport (M).

Figure 7

and/or strongly reduced (52, 118). The association between TMEM27 and B0 AT1 may occur through noncovalent interactions, in contrast to the covalent bonds formed between 4F2hc or rBAT and SLC7 members. B0 AT1 and TMEM27 colocalize in vivo in the kidney proximal tubule, and the surface expression of B0 AT1 in vitro is increased by the presence of TMEM27 suggesting a chaperone function (Fig. 7A). However, the strong effect on function implies that the association

382

may also impact transporter function, possibly by increasing the transport rate. Using the ACE2 knockout mouse model (48), we demonstrated ACE2 partners with B0 AT1 in small intestine (36). As shown in Figure 7B, B0 AT1-ACE2 colocalize on the apical membrane of enterocytes. In ACE2 knockout mice, expression of B0 AT1 is completely abolished in the brush border of small intestine, but normal in kidney (Fig. 7C). The reverse



situation is observed in the kidney of Tmem27 knockout animals Taken together the data confirm the tissue specific interaction of B0 AT1 with TMEM27 in kidney and with ACE2 in the intestine. As for TMEM27, the mechanism of interaction between B0 AT1 and ACE2 is also not known. Based on the analysis of several B0 AT1 mutants, we postulated that the absence of ACE2 may cause a decrease of transporter insertion or stability at the membrane. The physiological relevance of this interaction for amino acid transport may arise from the activity of the ACE2 carboxypeptidase in providing amino acid substrates for the associated transporter (Fig. 7A) (60, 107). This theory would explain the coexpression of B0 AT1 and ACE2 in the small intestine, since the carboxy amino acids released by ACE2 cleavage are mostly neutral amino acids (49). However in contradiction, a catalytically dead ACE2 mutant was found to still promote B0 AT1 function (36). Alternatively, it is also possible that expression of the transporter is in some way beneficial for ACE2 expression or function. However, contrary to this hypothesis the ACE2 enzyme is appropriately localized to membranes in organs in which the amino acid transporter is not expressed. Furthermore, the Slc6a19 knockout model (B0 AT1) has normal levels of ACE2 and Tmem27 consistent with transporter ablation having no impact on RAS system expression (19, 25). The cell surface expression of B0 AT3 (Slc6a18) and SIT1 (Slc6a20) is modulated by the same accessory proteins as B0 AT1 (Slc6a19) in vivo (157, 180). Surprisingly, exogenous expression of B0 AT3 with TMEM27 in Xenopus laevis oocytes yielded different results depending on the study. In experiments carried out by our group, the expression of B0 AT3 with TMEM27 (mouse or human orthologs) did not result in a substantial increase of transport function or membrane expression unlike observed with ACE2 (180). In contrast, Vanslambrouck and colleagues showed a greater than tenfold increase in mouse B0 AT3 activity with the coexpression of Tmem27 (202). The mechanism for the protein interaction and the reason of the discrepancy mentioned above are not known. The transporter SIT1 (Slc6a20) colocalizes with ACE2 in the small intestine (Fig. 7J–L). As was shown for B0 AT1, coexpression of the human SIT1 transporter with ACE2 increases transport rate (Fig. 7M). In the Ace2 knockout mice we observed a decrease in Na+ -dependent, pH independent L-proline transport (unpublished results). These data suggest SIT1 also interacts with Tmem27 and ACE2.

Cooperation of amino acid transporters The functional cooperation of symporters or uniporters with antiporters not only increases the supply of substrates to be transported but may also result in amino acid transport activities that single antiporters alone cannot perform. Indeed, antiporters do not sustain the directional transport of an individual amino acid unless there is another amino acid available for exchange. The exchanged amino acid required by the antiporter is provided by another transporter that recycles the amino acid through the membrane. Cooperation



between transporters was first demonstrated for the neutral amino acid antiporter (obligatory exchanger) LAT2-4F2hc (SLC7A8-SLC3A2) and the aromatic uniporter (facilitative diffusion transporter) TAT1 (SLC16A10). These transporters are both expressed in the basolateral membrane of proximal tubule cells. Using the Xenopus laevis oocyte expression system, it was shown that LAT2-4F2hc can efflux intracellular amino acids to an amino acid free buffer. However, only if a second transporter is coexpressed that recycles an amino acid for uptake by LAT2-4F2 in exchange for intracellular amino acid efflux (Fig. 8A, C) (150). This experiments nicely demonstrated that the net transport of a substrate by an antiporter (here the efflux of glutamine) can be controlled by the parallel recycling pathway provided, in this case, by the low-affinity aromatic amino acid uniporter TAT1. The exchanger LAT2-4F2hc (and also the other basolateral antiporter y+ LAT1-4F2hc (SLC7A7-SLC3A2) can indeed reuptake with high affinity the aromatic amino acids effluxed via TAT1 in exchange for the efflux of cytoplasmic neutral amino acids. However, in proximal kidney tubule TAT1 is apparently not the sole recycling pathway for vectorial amino acid reabsorption via LAT2-4F2hc and y+ LAT1-4F2hc. Since amino acid reabsorption was preserved in Tat1 knockout mice, unless the mice were subjected to a high protein diet (119). Another candidate for amino acid recycling in proximal kidney tubule is LAT4 (SLC43A2). LAT4 is an essential amino acid uniporter that was also shown to cooperate with LAT2-4F2hc in the Xenopus laevis oocyte expression system (150). The cooperation of amino acid antiporters with other amino acid transporters was also demonstrated in the context of mTORC1 signaling and cellular growth. In this case, the import of L-leucine via the antiporter LAT1-4F2hc (SLC7A5-SLC3A2) and the activation of mTORC1 were shown to depend on the import (= recycling) of nonessential amino acids (in particular LGln) via ASCT2 (SLC1A5). At high intracellular concentrations L-Gln can be effluxed via LAT1-4F2hc and thereby drive the exchange of L-leucine to activate mTORC1 (72, 134). Consequently, the functional interactions between amino acid transporters upstream of mTOR result in the import of essential amino acids only in the presence of the high intracellular concentrations of nonessential amino acids required for growth and proliferation.

Reabsorption of Amino Acids and Oligopeptides Amino acid and peptide reabsorption by proximal tubule epithelial cells Amino acids and peptides are nearly 100% reabsorbed by the proximal kidney tubule, mostly in the early convoluted segments S1 and S2 and to a lesser extent in the more distal straight segment (S3). A sequential distribution of lowand high-affinity transporters along the apical membranes of the proximal tubule lumen is one mechanism suggested to

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(A)

(B)

AA

NI

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NI

LT

NI

LT

NI

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*AA

4F2hc Lysate IP-4F2hc WB: LAT2

Lysate IP-4F2hc WB: TAT1

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Extracellular amino acid concentration [μM]

30

Nonaromatic LAT2 substrate with enhance transport in presence of TAT1

25

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*

NI LAT2-4F2hc LAT2-4F2hc-TAT1 TAT1

20 15 10

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*

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Ile A la Va l M et Pr o Tr p Ty r Ph e

ly

sn Th r Se r H is A rg Ly s Le u

A

ln

lu

G

G

G

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sp

0

Figure 8

Functional interaction between LAT2-4F2 and TAT1. (A) Schematic representation of the functional interaction between LAT2-4F2 and TAT1. LAT2-4F2 recycles aromatic amino acids effluxed by TAT1 in exchange for efflux of neutral amino acids, which are not TAT1 substrates. (B) TAT1 does not physically associate with 4F2hc or LAT2. Coimmunoprecipitation was performed using lysates of biosynthetically 35 S-met labeled oocytes coinjected with 4F2hc, LAT2, and TAT1 cRNAs (LT) or noninjected oocytes (NI) and analyzed by autoradiograph. Western blot (WB) of total lysates, or lysates immunoprecipitated using anti-4F2 (IP-4F2hc) antibodies detected LAT2 but not TAT1 coimmunopreciptated with 4F2hc. (C) Coexpression of TAT1 and LAT2-4F2hc stimulates efflux of LAT2-4F2 substrates that are not TAT1 substrates (e.g., L-Gln). The amount of amino acids accumulating in the extracellular bath of oocytes with and without expression of LAT2-4F2 and/or TAT1 was analyzed by UPLC [for details, see (150)]. The efflux of LAT2-4F2 substrates (yellow) such as L-glutamine, asparagine, serine and alanine was increased in the presence of TAT1. However, TAT1 (blue) substrate concentrations were not altered by coexpression of 4F2hc with TAT1. [Figure adapted from reference (150) with permission].

optimize recovery from the ultrafiltrate (Fig. 9). This organization is thought to improve the reabsorption of residual solutes in later segment regions including those that have diffused back into the tubular fluid via a relatively leaky paracellular pathway. Such an arrangement is well-described for glucose reabsorption where expression of the high affinity Na+ -Glucose cotransporter SGLT (SLC5A1) is highest in the S3 proximal tubule segment downstream of the peak expression of the low-affinity SGLT2 (SLC5A2) transporter. Analogously, the low-affinity transporters for neutral amino acids, B0 AT1 (SLC6A19) and small oligopeptides, PEPT1 (SLC15A1) are found highly expressed in early segments. While the high-affinity transporters, B0 AT3 (SLC6A18) and PEPT2 (SLC15A2) are localized to later proximal tubule regions (51, 157, 180, 202, 209). A few amino acids, such as glycine and histidine and the nonproteinogenic amino acid, taurine, are not completely recovered from the urine. This loss

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is thought to result from the competitive inhibition of uptake since some amino acid transporters, such as B0 AT1, accept multiple substrates. The amino acids taken up across the luminal membrane of the epithelial tubular cells are effluxed into the extracellular space across the basolateral membrane. Since normal cellular function requires adequate intracellular amino acid concentrations, basolateral transporters also control intracellular content in addition to releasing amino. Importantly, tubular cells maintain relatively stable amino acid gradients between the cytosol and the extracellular fluid. Therefore, the intracellular concentration levels of many nonessential amino acids are kept much higher than their extracellular concentration. Some amino acid concentration differences are surprising. For example, the anionic amino acids glutamate and aspartate that carry a negative charge potentially providing a driving force for efflux, are present at high cytosolic and low




(A)

Proximal tubule segments G S3 S1

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References 52

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References

B0AT1/SLC6A19

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B0

AT3/SLC6A18

157

SIT1/ SLC6A20

144

b0,+/

SLC7A9

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References

S1

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References

y+LAT1/ SLC7A7

119

LAT2/SLC7A8

158

AGT/ SLC7A13

119

TAT1/SLC16A10

101

SNAT3/ SLC38A3

125

LAT4/SLC43A2

43

Figure 9 Expression of amino acid and oligopeptide transporters in kidney by region. (A) Scheme of proximal tubule. The proximal tubule segments extend from the kidney cortex to the outer medullary stripe (OM) of the medulla. The renal arcuate artery (red line) and vein (blue line) demarking the separation between the cortex and medulla are indicated. The glomerulus is indicated with (G). The proximal convoluted (S1) and (S2), and the proximal straight (S3) segments are separated with straight dotted lines. (B-E) Reported expression of accessory proteins and transporters in proximal tubule segments S1 to S3. Panels B and C give expression data for apical, and panels D and E for basolateral localized proteins. (continued over leaf)


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Distal tubule regions Medullla

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OMCD

References 215 43 ESBL Databases

Collectrin /TMEM27 ACE2 4F2hc/ SLC3A2 (H) Transporter

Medulla L o o p T AL

Cortex T AL

DCT

CNT

Medulla CCD

References

OMCD 202 11 146 40 43 43 43

EAAT3/ SLC1A1 TAUT/SLC6A6 Asc1/ SLC7A10 Asc2/ SLC7A12 AGT1/ SLC7A13 SNAT3/ SLC38A3 LAT4/ SLC43A2

Figure 9

(Continued) (F) Scheme of distal nephron regions. The arcuate artery and veins separating cortex and medulla are indicated as in panel A, and the outer and inner medulla is separated by curved dotted lines. The thick ascending limb, distal convoluted tubule (DCT), connecting tubule (CNT) and collecting duct are indicated. (G and H) Reported expression of accessory proteins and transporters in distal nephron regions. The data shown for transporter and accessory protein expression in distal segments are incomplete and reflect cases in which the level of expression reported suggests a significant physiological role. No subcellular localization data are provided. Where no protein data are available, the mRNA expression is represented. For all panels (B-E, G, and H) the data are represented using an arbitary scale of 0 to 100% expression—white being no reported expression with increasing expression indicated by darker colors. The relative expression of different genes is not represented. For all genes, a reference is given for the expression data represented.

extracellular concentrations (65, 121). On the other hand, cationic amino acids whose positive charge could drive influx, in many cells, display intracellular concentrations similar to plasma levels. For amino acids with higher intracellular concentrations the transmembrane gradients might be largely explained by the fact most are not substrates for basolateral uniporters. Therefore, they cannot leave the cell just along their concentration gradient by facilitated diffusion. They exit the cell only via antiporters with asymmetric substrate affinities such as LAT2-4F2hc that displays much lower intracellular than extracellular affinities. Additionally, directional transport via these antiporters requires the recycling of exchange substrates through parallel pathways such as provided by TAT1. TAT1 mediates the facilitated diffusion of aromatic amino acids and thereby controls, along with other transporters, the amount of non-essential amino acids transported (efflluxed) via the antiporters LAT1-4F2hc and y+ LAT14F2hc.The pathway(s) that may mediate the basolateral efflux of anionic amino acids from proximal kidney tubule cells has not yet been identified. Further work is required to better understand the controlled efflux of amino acids through the basolateral membrane of transporting epithelia and other cells. The following sections briefly describe known important apical and basolateral transporters. The information is also

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summarized in several Figures and Tables that present: (i) a scheme of the reabsorption of amino acids along the nephron (Fig. 2); (ii) a cellular model for the apical and luminal distribution of transporters in proximal tubules (Figures 3B, 10G); (iii) transporter expression in various nephron regions (Figures 9, 10); (iv) the correspondences between transporter systems and molecular identity (Table 2); (v) the substrate specificity and mechanism of action for important renal transporters (Table 3) and; (vi) an overview of transgenic animal models reported to date (Table 4).

Luminal transporters Neutral amino acids B0 AT1 (SLC6A19) is the major luminal transporter responsible for neutral amino acid reabsorption by the proximal tubule. Mutations in the human gene coding for B0 AT1 cause the neutral aminoaciduria Hartnup disorder (for more details see “Neutral aminoacidurias”). The electrochemical Na+ gradient provides the driving force for a low affinity (Km ’s in the low millimolar range), high capacity cotransport of a broad range of proteogenic neutral amino acids (14, 35). Exogenous expression in Xenopus laevis oocytes coupled with two-electrode voltage clamp experiments demonstrated




Figure 10 Transporter basolateral versus apical membrane localization in kidney proximal tubule segments. (A-I) Representative immunofluorescence images from labeled mouse kidney tissue sections of apical membrane amino acid transporters. Tissue sections were stained as follows: (A) B0 AT1, (B) B0 AT3, and (C) SIT1 are labeled in red, and in (A-C) the basolateral membrane transporter, 4F2hc, is labeled with green (157). For panel (D), collectrin is labeled in green and shown without a labeled basolateral transporter (180). (E and F) The opposing axial distribution along proximal kidney tubule of mRNA for the HAT catalytic subunits, b0,+ AT, and rBAT, its glycoprotein partner. In situ hybridization of kidney section shown at a low magnification to demonstrate the individual HAT subunit mRNA gradients from cortex (c) to the medullary outer strip (os) and inner stripe (is) kidney regions (145). (G) Scheme of the apical versus basolateral localization of transporters expressed in kidney proximal tubules. Direction of transport and transporter mechanism is represented by arrows. Evidence exists for the expression of an as yet unidentified basolateral symporter and facilitative diffusion transporter(s) indicated as unlabeled transporters. (H-I) Representative immunofluorescence images from stained mouse kidney tissue sections of basolateral localized amino acid transporters. The basolateral localized (H) SNAT3 (126) is labeled in red, (I) TAT1 (151) is labled in green, (J) LAT2 localization and (K) y+ LAT1 transporters are labeled in red and (L) 4F2hc is labeled in green (126).

B0 AT1 mediates the electrogenic transport of neutral amino acids. Cotransport of amino acid substrates and Na+ generates a saturable, reversible, inward current with Michaelis-Menten kinetics. For example, at a membrane holding potential of −50 mV, the Km for Na+ is ∼15 and ∼1.2 mmol/L for Leu. B0 AT1 cotransports one Na+ per neutral amino acid. As for some SLC6 family members, Li+ can partially substitute for Na+ ; however, B0 AT1 transport is not Cl− dependent unlike many SLC6 family members. Furthermore, B0 AT1 does not transport any of the chemically related nonamino acid substrates accepted by other SLC6 family members (20, 35). B0 AT1 mRNA is highly expressed in kidney proximal tubule and small intestine. In both mice and humans B0 AT1 protein


is expressed in the brush border membrane of early proximal kidney tubules (S1) (107, 157). In the intestine B0 AT1 interacts with the angiotensin converting enzyme 2 (ACE2). In kidney although there is some expression of ACE2, B0 AT1 apparently only interacts with the accessory protein collectrin (TMEM27), which is not expressed in intestine. TMEM27 is a type I transmembrane protein that through a noncovalent mechanism(s) modulates B0 AT1 expression. Expression of B0 AT1, TMEM27, and ACE2 are developmentally regulated and are highest during the early postnatal period (202). The collectrin null mouse (Tmem27−/y ) lacks expression of renal B0 AT1 while the B0 AT1 intestinal expression is normal. Furthermore, TMEM27 knockout animals display a massive

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neutral aminoaciduria without glucosuria or phosphaturia consistent with TMEM27 being a kidney specific obligatory B0 AT1 interacting protein (52, 103, 118). Conversly, the ACE2 knockout mouse lacks intestinal B0 AT1 expression but retains normal renal B0 AT1 levels, and displays impaired intestinal neutral amino acid transport (36, 181). The data from the B0 AT1 knockout are consistent with B0 AT1 playing the major role for Na+ -dependent neutral amino acid absorption. The knockout eliminated both renal and intestinal B0 AT1 although normal expression of both Tmem27 and ACE2 accessory proteins remained (25). On normal protein diets (20% protein), B0 AT1 null mice had lower body weight than wild-type littermates, additionally both low- and high-protein diets provoked a rapid weight loss by knockouts. Small intestine or kidney proximal tubule BBMVs prepared from knockout mice tissue lacked Na+ -dependent neutral amino acid uptake. Nor did null mice compensate for the loss of B0 AT1 transport with an increased BBMV oligopeptide or other neutral amino acid transporter activity. B0 AT3 (SLC6A18), which is expressed at high levels on apical kidney proximal tubule membranes of mice, is highly related to B0 AT1 both in structure and function. The human SLC6A18 gene is localized in tandem with the SLC6A19 gene on chromosome 5p15 and the two protein products share ∼50% identity (157). Mouse B0 AT3 transports a broad range of neutral amino acids (Ala, Met, Val, Ile > Gly, Ser, Leu) with a high affinity (micromolar range) in a Na+ - and Cl− dependent manner (180). Consistent with a role in reabsorbing residual tubular amino acids, that is, those not reabsorbed in early segments by B0 AT1 and/or that have reentered the lumen via a paracellular route, B0 AT3 expression is highest in later S2 and S3 proximal tubule segments (141, 180). Knockout of slc6a18 expression, which did not stimulate changes in the expression of any other amino acid transporters, did result in a broad neutral aminoaciduria (especially for glycine, glutamine, alanine, and methionine) (149, 180). However, plasma amino acids were not significantly altered in the knockout model. The first characterization of a B0 AT3 null mice (mixed strain background) displayed a glycine supplementation-responsive elevation in blood pressure not found in the subsequent fully C57BL6 backcrossed (to 10 generations) B0 AT3 null mouse strain (149, 180). Additionally, for humans, it is not likely that B0 AT3 plays a role in blood pressure control and probably also not for amino acid transport since nearly half of the Japanese population has a single nucleotide nonsense polymorphism in Slc6A18 with no associated phenotype (58). B0 AT3, like B0 AT1 requires the accessory protein TMEM27 for renal expression and as for B0 AT1 Tmem27−/y animals do not express B0 AT3 in kidney proximal tubules (52, 180, 202).

Imino and small neutral amino acids SIT1 (SLC6A20) is a symporter, cotransporting with high affinity (micromolar range) L-proline, α-(methylamino)-

388


isobutyric acid (MeAIB), methyl-proline, or hydroxyproline with Na+ and Cl− . The SIT-1 mRNA is highly expressed in small intestine, kidney, lungs, spleen, testis, and brain, and the protein has been shown to localize to the luminal brush border membranes of the entire kidney proximal tubule and the small intestine (157). SIT1 interacts with the accessory protein Tmem27, and tmem27−/y animals have a strongly reduced kidney expression of Sit1 (52). Coexpression experiments in Xenopus laevis oocytes confirmed that SIT1 can functionally interact with both the accessory proteins TMEM27 and ACE2. Consistent with this in human small intestine brush border membranes SIT1 was found colocalized with ACE2 (Vuille-dit-Bille, Camargo and Verrey, Fig. 7D, E). As yet, no genetically modified animal model for SIT1 has been reported. PAT1 (SLC36A1) and PAT2 (SLC36A2) cotransport H+ and small neutral amino acids or imino acids with a 1:1 stoichiometry by Na+ -independent electrogenic transport that is influenced by membrane potential (15). Initially, since the H+ gradient across the luminal membrane of proximal tubule cells depends on the activity of the Na+ /H+ exchanger NHE3, PAT transport was incorrectly described as Na+ -dependent. PAT1 mRNA is highly expressed in small intestine, colon, kidney, and brain and PAT2 is found in lungs, heart, kidney, testis, muscles, and spleen. The PAT transporters accept a similar range of substrates, for example, L- and D-proline, hydroxyproline, glycine, sarcosine, L, D, and β-alanine, D-serine, D-cysteine, betaine, MeAIB, and GABA. However, PAT1 prefers glycine, proline, and alanine, which are transported with a low affinity and similar maximum velocity. Typical substrate Km values for PAT1 are 2 to 10 mmol/L and PAT1 activity is increased at acidic pH. PAT2 transports proline, glycine, and alanine with Km values of ∼0.1 mmol/L (proline) to ∼0.6 mmol/L (glycine). Additionally, unlike PAT1, PAT2 transports glycine with a significantly higher maximum velocity than proline and alanine and is considered to be a high affinity IMINO transporter (15). Additionally, PAT2 demonstrates weaker pH dependence and is more active at neutral and alkaline pH than PAT1. Also PAT2 prefers L- over D-alanine but transports L- and D-serine with equal affinity and has a tenfold lower affinity for GABA than PAT1 (100). Similar to SIT1, PAT1 recognizes the amino acid analog MeAIB. However, SIT-1 can be distinquished from PAT1 by its substrate specificity, particularly the lack of glycine transport and its ion dependency (92, 195). In human kidney PAT2 protein localizes on early proximal tubule segments (24). However, the kidney expression of PAT1 protein, which has been localized on intestinal enterocytes at the brush border membranes, has only been detected intracellularly in subapical membrane regions in proximal tubule cells (202). B0 AT1, B0 AT3, PAT2, and SIT-1 together are responsible for proline and glycine reabsorption in kidney proximal tubules; mutations in all four genes have been linked to iminoglycinuria (24). No transgenic animal models for either PAT1 or PAT2 have been reported.



Cationic amino acids and L-cystine b0,+ AT (SLC7A9) and the accessory protein rBAT (SLC3A1) are covalently linked to form what is commonly known as a heterodimeric amino acid transporter (see “Partner proteins”) (129, 140, 145, 204). Exogenous expression in an epithelial cell model (MDCK cells) results in the colocalization of rBAT and b0,+ AT in the apical cell membrane. In the absence of rBAT b0,+ AT remains intracellular, which is consistent with a requirement for rBAT coexpression for b0,+ AT surface expression (9, 155). Both subunits are primarily expressed in kidney and intestine on the brush border membranes of proximal tubules or small intestine enterocytes, respectively (29, 145). Given rBAT’s sole known association to date is with b0,+ AT, it is surprising that its expression along the proximal tubule increases from S1 to S3, whereas that of b0,+ AT is maximal in S1 and decreases toward S3 (Fig. 9A–C). b0,+ -rBAT functions as a high affinity, potentially electrogenic obligatory exchanger of large neutral (notably cystine) against dibasic (arginine, lysine, and ornithine) amino acids (44, 145). Mouse models generated by the ablation of the heavy or light chains excrete higher levels of cystine and dibasic amino acids in the urine (63, 67, 144). In the Slc7a9−/− mouse while the total expression of b0,+ AT protein was ablated nonetheless significant rBAT protein covalently linked to unknown “light” subunit(s) remained in kidney. Nearly half these b0,+ null mice were shown to develop urinary cystine calculi during the first month of life which continued growing during the animals’ life span (63, 67). The rBAT mutant mouse called “pebbles” (Slc3a1pbl ) was identified in an ENU mutagenesis screen to contain a D140G substitution in the rBAT protein that has no effect on Slc3a1 mRNA transcriptional efficiency or stability. However the mutation did result in a b0,+ -rBAT protein trafficking defect producing elevated urinary cystine and cystine stones in mutant mice. Interestingly, although urinary basic amino acids levels were equal to or higher in pbl/pbl females than in males, females had delayed onset and/or a reduced penetrance of stone formation relative to males. All pbl/pbl male mice developed urolithiasis by one year, while only 23% of females developed stones by the same age. This difference was suggested to be either due to anatomical or metabolic differences between the male and female urinary systems (144).

Anionic amino acids EAAT3/EAAC1 (SLC1A1) functions as a high affinity electrogenic symporter/antiporter that cotransports L-glutamate, or L- or D-aspartate with Na+ and H+ in exchange for K+ efflux. Stoichiometrically, one negatively charged amino acid is cotransported with 3 Na+ , 1 H+ ions, and the antiport of 1 K+ ion (96). It was further shown by two-electrode voltage clamp using the exogenous expression Xenopus laevis oocyte system that disruption of the ionic environment can reverse the direction of substrate flux (98, 186). Thus far EAAT3 is the only anionic amino acid transporter detected



in apical membranes of proximal tubule cells where it is highly expressed in the later segments (S2, S3 >S1). It is also expressed in the distal convoluted tubule, as well as in the brush border of ileal enterocytes, various subtypes of neurons, intestine, liver, heart, and placenta (95, 170). Under normal conditions EAAT3 protein is distributed between plasma membrane and intracellular stores. A number of interacting proteins have been found to modulate EAAT3 activity (see “Partner proteins”) (4). Modulation of transporter activity is mainly due to localization to the cell surface rather than de novo synthesis. For example, Protein kinase C, the serum- and glucocorticoid-inducible kinase (SGK1), and the phosphoinositide-dependent kinase (PDK1) increase EAAT3 surface expression. Whereas, the inhibition of phosphatidylinositol 3-kinase (PI3K), or a direct interaction with the glutamate transport associated protein (GTRAP3-18) decreases plasma membrane EAAT3 localization. GTRAP3-18, which is an allosteric negative modulator of EAAT3, interacts with the carboxy-terminal end of the transporter to retard exit from the endoplasmatic reticulum (32, 162). The ablation of EAAT3 (Slc1a1−/− ) in a mouse model did not adversely impact viability or fertility, or result in any morphological, histological, or neurological abnormality. Nor was the loss of EAAT3 compensated for by an upregulation of other glutamate transporter mRNAs. Knockout mice did display a strong dicarboxylic aminoaciduria with L-glutamate and aspartate urinary excretion elevated 1400- and 10-fold, respectively confirming the vital role of EAAT3 in renal recovery of anionic amino acids (142).

Oligopeptides PEPT1 (SLC15A1) and PEPT2 (SLC15A2) PEPtide Transporter family members use the H+ gradient as the driving force for the electrogenic symport into cells of small peptide products of protein degradation (50). PEPT1 and PEPT2 have broad substrate selectivity for any di- and tri-peptide composed of any of the 20 proteinogenic amino acids. PEPT1 is a low affinity (millimolar range) high capacity transporter expressed in small intestine, kidney, extra hepatic biliary duct, and brain (61). While in comparison, PEPT2, which is expressed in the kidneys, brain, lungs, eyes, and mammary glands, transports substrates with high affinity (micromolar range) and low capacity (115). On apical proximal tubule membranes, as described for SLC6 family members, the low affinity PEPT1 is highly expressed in early (S1) and the high affinity PEPT2 in later proximal tubule segments (S2 and S3) (172, 184). In addition to oligopeptides the PEPTs have been shown to transport many other compounds, including drugs such as β-lactamate antibiotics (cephalosporins and penicillins), ACE inhibitors, and antiviral nucleoside prodrugs (217). Knockout mouse models for both transporters have been generated. Both are viable and have a mild phenotype without any evidence for compensatory changes in levels and/or function of amino acid and/or other peptide

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transporters. Although for Pept2−/− mice blood and urine amino acid levels were normal, most plasma amino acid levels were increased in Pept1−/− mice suggesting PEPT1 knockout altered amino acid handling (89). Highlighting the complementary physiological roles of the two PEPTs, clearance of the non-metabolized peptide glycylsarcosine was unchanged in Pept1−/− compared with Pept1+/+ mice, while intestinal absorption of glycylsarcosine was drastically reduced. In contrast, Pept2−/− glycylsarcosine clearance was twofold higher than in wild type littermates resulting in an increased urinary loss. Taken together these data indicate the high affinity PEPT2 is responsible for the majority of the renal reabsorption of small peptides (89, 137, 171).

Basolateral transporters Neutral amino acids LAT2 (SLC7A8) with its accessory 4F2hc (SLC3A2) family protein forms a heterodimeric amino acid transporter (204) for the Na+ -independent, obligatory exchange of a broad range of neutral amino acids. 4F2 functions as chaperone that targets the localization of LAT2 plasma membrane expression (see “Partner proteins”). LAT2-4F2hc mediates a 1:1 exchange of amino acids with a strongly assymetrical apparent affinity; the extracellular affinity for any given substrate is in the micromolar range while intracellular affinities are ∼1000-fold lower (i.e., millimolar range) (121). In addition to localization on basolateral membranes of renal proximal tubules, LAT2 is also highly expressed in small intestine, brain, muscles, lungs, bladder, and pancreas (53, 135). In contrast, 4F2hc has a ubiquitous localization. The highest expression of LAT2 in proximal tubule epithelium is in the early segments (S1-S2). It is hypothesized to function cooperatively with LAT4 and/or TAT1 to mediate the transfer of neutral amino acids to the renal interstitium. Although highly expressed in wild-type placenta and brain, as well as kidney, LAT2 knockout mice are viable and display only a moderate neurological phenotype of impaired coordination of movement. The total SLC7A8 knockout mouse (Slc7a8−/− ) does show elevated urinary excretion of glycine, L-serine, L-threonine, L-glutamine, L-leucine, and L-valine that might be due to a large extent to their increased concentration in plasma. In addition to the listed amino acids lost in the urine, L-lysine is also elevated in plasma. However, consistent with the lack of disturbance in the reabsorption of aromatic amino acids, a modest induction of renally expressed Slc16a10 mRNA (coding for TAT1) was reported for Slc7a8−/− mice (17). No human disease has been mapped to mutations in the SLC7A8 gene although it has been suggested as a candidate gene for isolated cystinuria (OMIM 238200) (23). TAT1 (SLC16A10) is a T-type aromatic amino acid transporter which by homology is assigned to the SLC16 monocarboxylate transporter gene family. It is highly expressed in kidney, small intestine, colon, liver, stomach, heart muscle, and testis. It localizes to the basolateral membranes of epithelial cells in the small intestine and kidney, and like

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LAT2 is highest in the early S1 and S2 proximal tubule segments. TAT1 transporter is a low affinity facilitated diffusion pathway (symmetric uniporter) for aromatic amino acids, Ldopa, and iodothyronines (101, 151). For example, TAT1 was found to transport L-Phe influx and efflux at similar maximal velocities and Km values around 30 mmol/L (151). When coexpressed in Xenopus laevis oocytes, TAT1 has been shown to be capable of functionally cooperating with LAT2-4F2. It is hypothesized to function similarly in vivo for the basolateral efflux step of transepithelial amino acid reabsorption (see Fig. 8) (150). The mice with the total ablation of TAT1 (Tat1−/− ) show no abnormal phenotype in histology, growth, fertility or neurological defect. No compensatory changes in any tested AA transporter mRNA were detected. However, under both normal and high protein diet aromatic amino acid concentrations in plasma, muscle and kidney but not liver were increased for Tat1−/− mice. When Tat1 null mice were fed a high protein diet they developed an aromatic aminoaciduria and a small urinary loss of LAT2 neutral amino acid substrates. Therefore, TAT1 expression is needed to equilibrate cellular concentrations and promote the release of aromatic amino acids across basolateral membranes of proximal kidney tubule and small intestine epithelial cells, as well as for the efflux of neutral amino acids by exchangers such as LAT2 (119). LAT4 (SLC43A2) transports the branched chain amino acids, L-methionine and phenylalanine by a low affinity Na+ and Cl− -independent, facilitative diffusion pathway (uniporter) (12). Unlike other system, L-type transporters notably from the SLC7 family, LAT4 does not require association of a SLC3 member protein for expression or membrane targeting. However, like LAT1 and LAT2, the amino acid analog BCH but not the system A inhibitor, MeAIB, can competively inhibit LAT4 transport. In addition, LAT4 transport is sensitive to inactivation by NEM. When expressed in Xenopus laevis oocytes LAT4 reportedly displays a biphasic high and low apparent affinity for substrates. For example, the two reported values for L-Phe Km are 180 μmol/L and 5 mmol/L. The expression of LAT4 is highest in placenta, kidney, and peripheral blood leukocytes and weaker in spleen, skeletal muscle, and heart (13, 43). In situ hybridization data indicate the transporter is expressed in distal nephron segments, namely the thick ascending limb, distal tubules, and collecting duct but our preliminary data indicate that Lat4 mRNA is substantially expressed in proximal tubules as well (Mariotta, Camargo and Verrey, unpublished data). A LAT4 knockout mouse has been generated but no functional data have been published as yet (F. Verrey, personal communication). SNAT3 also known as SN1 (SLC38A3) is a Na+ - and H+ dependent glutamine transporter localizing to the basolateral membrane of later proximal tubules (S3) (117, 126). It is also expressed in brain, retina, liver, kidney, pancreas, and adipose tissue. Amino acid transport is coupled to Na+ cotransport and H+ antiport. Human SNAT3 expressed in Xenopus laevis oocytes displays a low apparent affinity for glutamine, histidine, and asparagine with Km values in the range of 0.5 to



1.5 mmol/L, with both apparent Km and transport rate (Vmax ) influenced by pH [Makrides, personal communication; (62)]. SNAT3 transport direction has been shown to be reversible in response to both glutamine and H+ concentrations (18). Although extracellular pH influences SNAT3 transport activity, the glutamine gradient primarily drives transport direction. Additionally, SNAT3 expression has been shown to be increased by metabolic acidosis. The contribution of renal SNAT3 in pH control is discussed in more detail in the section on amino acid metabolism. The total SNAT3 knockout in mice is lethal between postnatal day 10 to 20 [C.Wagner, personal communication; (160)]. The targeted conditional knockout of SNAT3 in specific cell types is being generated but no data are available as yet.

Cationic amino acids y+ LAT1 and 4F2hc (SLC7A7-SLC3A2) form a heterodimeric amino acid transporter (204) that under normal physiological conditions acts to efflux dibasic amino acids in exchange for the uptake of neutral amino acids and Na+ (146). The intracellular affinities have not been determined. However by experimentally inducing an inverted exchange a high extracellular affinity for cationic amino acids, similar to the affinity for neutral amino acids, was measured. y+ LAT1 is highly expressed in small intestine, kidney, lungs, and leukocytes (53, 146, 197), whereas its accessory protein, 4F2hc is ubiquitously expressed. In the kidney, y+ LAT1 localizes selectively to early segments of proximal tubules (S1>S2>>S3) providing an efflux pathway for cationic amino acids taken up apically by b0,+ -rBAT (8, 53). The function of 4F2 is analogous to rBAT, and as for LAT2, y+ LAT1 interaction with the extracellular 4F2 domain targets it to the basolateral plasma membrane (8, 102, 146). Total knockout of 4F2 is embryonic lethal (199), while Slc7a7 gene inactivation results in intrauterine growth retardation with a very low survival rate. The phenotype of survivors mimics human Lysinuric Protein Intolerance (44). Specifically, the authors reported 18 Slc7a7−/− pups were born from >200 breedings of Slc7a7+/− mice (i.e., birthrate of 9%), of these 16 died within 24 h and 2 (1 male and 1 female) survived to adulthood (11% survival). Although surviving Slc7a7−/− mice were maintained on a special dietary regimen (low-protein with citrulline supplementation) a massive urinary excretion of arginine, ornithine, and orotic acid characteristic of human LPI was nonetheless observed (189).

Anionic amino acids AGT1 (SLC7A13) is a heterodimeric amino acid transporter (204) with an unknown accessory protein partner, which furthermore operates by an unknown mechanism. AGT is expressed in the kidney but not in the intestine. In kidney AGT localizes to the basolateral membrane of the proximal straight tubule, the outer medulla, and the distal tubule (120). This distribution is comparable to that of the apical glutamate transporter EAAT3. As a result, AGT1 could constitute



a net efflux pathway for anionic amino acids in kidney. However it was shown that co-expression of AGT with rBAT or 4F2hc does not stimulate transport. The indication that AGT is an anionic amino acid transporter came from testing in Xenopus laevis oocytes using fusion proteins of AGT1 with either 4F2hc or rBAT. Under these conditions AGT mediates a high affinity (low micromolar range) Na+ - and Cl− independent L-glutamate and L-aspartate transport (44). AGT preferred L-aspartate over D aspartate and cysteine strongly inhibited glutamate uptake without being transported. It is not clear whether like most SLC7 light chains AGT1 functions as obligatory exchanger. In this case, to achieve a net efflux of anionic amino acid substrates, AGT1 would have to import a different substrate in exchange that would have to be recycled to the exterior by another cooperating transporter or be metabolized. AGT1 may transport a wider range of substrates since uptake was only tested at low amino acid concentrations (20 μmol/L). Furthermore, competition of AGT L-aspartate uptake with 2 mmol/L of some neutral amino acids, for example, L-cysteine, substantially inhibited transport. Another possibility, which has not been tested, is that AGT countertransports anions such as bicarbonate, sulfate, phosphate, and dicarboxylic acids (22). If AGT1 is an amino acid uniporter, then it alone could provide a basolateral efflux pathway for glutamate and aspartate. Additional experiments and Slc7a13 transgenic animal models are needed to answer these open questions about the mechanism and potential role of AGT1 in basolateral efflux of anionic amino acids.

Amino acid and oligopeptide transporters in more distal segments of the nephron Only low concentrations of amino acids escape reabsorption in the proximal tubule to reach the loop of Henle and the distal nephron segments. Of the amino acid transporters known to be involved in amino acid reabsorption from the primary urine, only the anionic amino acid transporters EAAT3 (luminal) and AGT1 (basolateral) have been detected in the distal nephron segments. Furthermore, with the exception of taurine uptake, micropuncture and microinjection failed to detect any significant amino acid reabsorption activity in the deeper nephron regions beyond the proximal tubule. It is likely that the basolateral transporters that are expressed in later kidney tubule segments function primarily to fulfill housekeeping functions. Some of these transporters for which localization studies have been performed, are listed below. Asc1 (SLC7A10) forms a heterodimeric Na+ independent high affinity (Km ∼10-30 μmol/L) antiporter for small neutral amino acids (Gly, Ala, Ser, Cys, and Thr) and, with a lower affinity (Km ∼100-200 μmol/L), for large neutral amino acids. As expected for an exchanger, the presence of extracellular amino acids strongly increases Asc1 efflux; however perhaps its transport is not exclusively as an antiporter, since a significant efflux was shown to occur in the absence of trans-stimulation (73, 130, 147). In the CNS, Asc1 was suggested to be the major transporter for D-serine.

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Asc1 mRNA is expressed in brain, lung, kidney, and intestine (147). In kidney, Asc1 localizes to the loop of Henle, the distal tubule, and the collecting duct suggesting Asc1 may be mainly important in providing amino acids for endogenous cellular metabolism. When exogenously expressed in Xenopus laevis oocytes or mammalian cells Asc1 functionally heterodimerizes with 4F2hc and to a lesser extent with rBAT. The heavy chain associating with Asc1 in kidney is possibly 4F2hc because of its basolateral localization and the apparent absence of rBAT beyond the proximal tubule. It has, however, to be mentioned that 4F2 expression in later tubule segments has as yet only been reported at the mRNA level (146, 147, 158). Knockouts of Asc1 expression (Asc1−/− ) have been generated; unfortunately, the renal function of knockout mice has not been assessed at any developmental stage. Briefly, at birth Asc1−/− pups appeared normal. However by postnatal day 14 for both male and female Asc1 null mice, body, brain, and other organ weights were ∼20% to 30% less than wild-type littermates. Furthermore, the Asc1−/− mice had a hunched posture, and exhibited tremors, seizures, impaired movement, and a decreased rate of survival up to postnatal day 30, while Asc1+/− mice appeared phenotypically, developmentally, and reproductively normal (163, 210). Asc2 (SLC7A12) is a heterodimeric Na+ - and Cl− independent exchanger, which like AGT1, interacts with an unknown accessory light chain that is not rBAT or 4F2 (SLC3A1 or SLC3A2). However, when Asc2 is expressed in Xenopus laevis oocytes as fusion protein with rBAT or 4F2hc, it transports a similar selectivity of substrates at comparable affinities as Asc1. The mRNA is detected in muscle, placenta, spleen, brain, and kidney cortical and outer- and inner-medullary collecting ducts. Renal subcellular localization is not clear (40), but the axial localization and functional characteristics of Asc2 suggest a housekeeping role. TAUT (SLC6A6) cotransports 2 Na+ and 1 Cl− ion with 1 nonproteinogenic amino acid-like substrate such as taurine (which lacks a carboxyl group and is, therefore, not an “amino acid”), and β-alanine (nonproteinogenic degradation product of carnosine) with high affinity (TAUT Km for taurine and β-alanine are 5 and 56 μmol/L, respectively) (114, 152, 153, 185, 200). TAUT is widely expressed throughout the body in kidney, intestine, retina, brain, liver, muscles, and placenta. TAUT is expressed in the outer and the inner medulla localized on apical membranes in the inner medullary collecting duct. TAUT responds to salt load by increasing expression leading to an increased concentration within medullary cells of the osmolyte taurine (11, 214). Disruption of the TAUT gene (Taut−/− ) results in the severe degeneration of retinas, and a significantly reduced taurine concentration in plasma, kidney, liver, and eyes in mice. Furthermore, the fertility of knockouts is impaired, and their total exercise capacity is also reduced. Additionally, Taut−/− mice show a significant urinary loss of taurine and subsequent hypotaurinemia. Although there is no defect in antidiuresis upon water deprivation, Taut−/− mice display an impaired ability to lower urine osmolality and to

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increase urinary water excretion. The persistent urinary concentration and increased activity of the vasopressin system following restoration of free access to water after a period of dehydration further indicate a role for TAUT1 in the inhibition of vasopressin release after normalization of plasma osmolality (87, 90).

Aminoacidurias The appearance of an abnormally high excretion of specific amino acids in the urine, that is, an “aminoaciduria” can be caused by metabolic or transport defects. In metabolic disorders, one or more enzymes involved in the synthesis or catabolism of amino acids is defective leading to excretion of one or more abnormal amino acids or metabolites in the urine. The transport disorders, which are caused by deficient renal amino acid transport, result in inefficient reabsorption of amino acids and abnormal excretion. Specific aminoacidurias are classified by the chemical properties of the amino acids abnormally excreted [i.e., neutral, basic (cationic), acidic (anionic), or iminoaciduria]. The disorders identified to date are compiled in Table 6. Aminoacidurias due to transport disorders are caused by mutations in genes encoding amino acid transporters. The defective reabsorption of specific amino acids may result in disease by causing a systemic deficiency in particular amino acid(s) (i.e., due to urinary loss), or the precipitation of certain amino acid(s) (e.g., cystine) in the urine of the patients. Since many of the renal transporters are also expressed in intestine decreased absorption may also influence the phenotype of the disorder (167). The association of defects in amino acid transport and disease was first suggested a century ago (78, 79). The development of sensitive chromatography methods to quantify amino acid levles in urine, newborn screening programs (55), and the molecular characterization of the amino acid transporters has contributed greatly to the understanding and diagnosis of these genetic disorders (22, 25, 206).

Basic aminoacidurias Cystinuria (OMIM #220100): Patients with cystinuria are usually diagnosed by episodes of kidney or bladder cystine stones (nephro- or urolithiasis) which are due to elevated urinary cystine. Additionally, they also excrete dibasic amino acids (lysine, arginine, and ornithine) with their urine. High cystine excretion is caused by reduced reabsorption due mutations in the genes SLC3A1-SLC7A9 encoding the heterodimeric amino acid transporter b0,+ -rBAT (33, 64). The average prevalence is 1 in 7000 births, and to date 133 mutations in SLC3A1 and 95 mutations in SLC7A9 have been identified. Reported mutations include nonsense, missense, splicing, frameshift, and large sequence rearrangements (45). Inheritance can be autosomal recessive or dominant depending on the gene mutated. Mutations in SLC3A1 (chromosomal locus 2p21) leads to an autosomal recessive trait with heterozygous parents unaffected. On the other hand, mutations



Table 6


Aminoacidurias genetically elucidated in human Hallmark (elevation of individual AA in urine)

Aminoaciduria

Gene

Protein

Chromosome

Cystinuria A

SLC3A1

rBAT

2p21

Cystinuria B

SLC7A9

b0,+ AT

19q13.11

Lysinuric protein intolerance

SLC7A7

y+ LAT1

14q11.2

Lysine Arginine Ornithine

Hartnup disorder

SLC6A19

B0 AT1

5p15.33

Neutral amino acids

Iminoglycinuria∗

SLC6A19 SLC36A2

SIT1 PAT2

3p21.3 5q33.1

Proline Hydroxyproline

Dicarboxylic aminoaciduria

SLC1A1

EAAT3/EAAC1

9p24

Cystine Lysine Arginine Ornithine Cystine Lysine Arginine Ornithine

Glycine Aspartate Glutamate According to HUGO (www.genenames.org) and NCBI build 36. ∗ Pending independent confirmation.

in SLC7A9 (chromosomal locus 19q13.11) lead to a mild to moderately abnormal urinary amino acid pattern in most obligate heterozygotes (e.g., parents) and thus can be seen as an autosomal dominant trait. Before the genes were molecularly identified this “mixed” pattern caused many problems in the diagnosis and classification of the disease. The initial nomenclature, based on the excretion status of obligate heterozygotes [cystinuria types I, II (non-I), III (non-I)], has been “replaced” by a more meaningful system linked to the genotype (54). Currently, cystinuria is defined as types A or B, depending on the gene affected. Cystinuria type A is caused by mutations in SLC3A1, type B by mutations in SLC7A9. Disease severity is apparently similar for cystinuria types A and B. Lysinuric protein intolerance (OMIM #222700): Lysinuric protein intolerance (44) manifests early in life with patients experiencing episodes of diarrhea and hyperammonemia coinciding with increased protein intake (hence the name). Patients develop a severe phenotype similar to some metabolic disorders, lysosomal storage diseases, celiac disease, or autoimmune disorders (188). They present elevated urinary dibasic amino acids, lysine, arginine, and ornithine, but not cystine. Plasma levels of lysine, arginine, and ornithine are also decreased. LPI is caused by mutations in SLC7A7, a heterodimeric amino acid transporter family member (204) interacting with 4F2 (SLC3A2). The mutations causing this autosomal recessive disorder have been exclusively identified in the SLC7A7 gene (chromosomal locus 14q11.2) encoding the y+ LAT1 transporter. The incidence of LPI is very low, but in some populations reaches 1:50,000 births. Currently,


a total of 50 different LPI causative mutations of SLC7A7 have been described (168). Most of the LPI causative mutations consist of missense, nonsense or small deletions, while larger deletions, insertions, or splice-site mutations are less frequent. The phenotype of LPI patients includes not only urinary loss of dibasic amino acids, but additionally reduced intestinal absorption, and some immunological features [for review, see (21, 168)].

Neutral aminoacidurias Hartnup disorder (OMIM #234500): Hartnup is a disorder with variable symptoms. Currently, the disorder is diagnosed during newborn screening or after urinary amino acids analysis. Abnormally high neutral amino acids excretion is present in all patients. Under protein-restricted diet and/or high-stress patients can present with pellagra-like symptoms including light-sensitive dermatitis, intermittent cerebellar ataxia, and psychosis-like symptoms. The variability in symptoms may be due to the high frequency of compound heterozygosity, and/or to the differential interactions of specific mutations with intestinal and/or renal accessory proteins, and/or dietary habits. Hartnup is caused exclusively by mutations in the SLC6A19 gene (chromosomal locus 5p15.33). To date 22 deletions, missense, nonsense, and splice site mutations causing the disorder have been identified (36, 42, 104, 169, 179, 216). Mutations in genes encoding the renal and intestinal B0 AT1 accessory proteins, TMEM27 (TMEM27) and ACE2 (ACE2) are not responsible for the disorder since both genes

393


are located on the X chromosome, and no patients with an X-linked neutral aminoaciduria have been observed. The disorder has autosomal-recessive heritablity with an incidence of 1 to 15,000 live births.

Iminoacidurias Iminoglycinuria (OMIM #242600): Patients with iminoglycinuria present with elevated urinary levels of glycine, proline, and hydroxyproline. Interestingly, obligate heterozygotes may also display glycinuria. Patients are usually diagnosed as a result of newborn screening programs or diagnostics linked to other diseases. Iminoglycinuria has been suggested to be caused by mutations in multiple genes. These mutations were identified by sequencing candidate genes, encoding glycine and imino acid transporters. A complex combination of mutations and polymorphisms in the SLC36A2 (PAT2), SLC6A20 (SIT1), SLC6A19 (B0 AT1), and SLC6A18 (B0 AT3) genes have been suggested to cause iminoglycinurias (24). Preliminary genetic studies found no linkage with the chromosomal region encoding SLC6A20 (locus 3p21.31) (105), but genome-wide association studies showed that single nucleotides polymorphisms in SLC6A20 can be associated with metabolite excretion in healthy subjects (191). Additionally, exogenously expressed SIT1 mutants (corresponding to those found in iminoglycinuria patients) displayed decreased transport activity. Mutations in genes encoding the glycine and L-proline transporters, PAT1 (SLC36A1) and PAT2, are additional candidates. The SLC36A2 gene is the best candidate while analysis of the SLC36A1 (PAT1) chromosome locus (5q33.1) failed to uncover any linkage with the disorder (105). Genetic defects on SLC6A18 and SLC6A19 could cause a general neutral aminoaciduria, not solely the abnormal excretion of proline and/or glycine (36, 52, 180), disqualifying these genes as good candidates. The incidence of iminoglycinuria is 1:10,000 births.

Acidic aminoaciduria Dicarboxylic aminoaciduria (OMIM 222730): Patients with dicarboxylic aminoaciduria present with high acidic amino acids urinary excretion (aspartate and glutamate) (122, 192). It is caused by mutations in the SLC1A1 gene (chromosomal locus 9p24) encoding the glutamate and aspartate transporter EAAC1/EAAT3 (6, 97, 183). In two pedigrees, missense and deletion mutations were identified in homozygotes. Dicarboxylic aminoaciduria is an autosomal recessive disorder with an incidence of 1:35,000 births, as estimated from a 25-year screening program in Quebec-Canada (5).

Transporters Involved in Renal Amino Acid Metabolism Transporters both act in the reabsorption pathway for amino acids and participate in supplying amino acids to epithelial cells for endogenous needs. Furthermore, the renal proximal

394


tubule contributes to the regulation of whole body homeostasis through the recovery of amino acids from the primary filtrate and through roles in amino acid metabolism and interorgan exchange (Fig. 1). Renal amino acid metabolism has both local effects and influences diverse systemic physiological processes including pH, vasodilation, and urea cycle regulation. In comparison to liver and skeletal muscle the kidney is a relatively minor player in the control of circulating amino acid levels. Nonetheless, the kidney is the major site of glutamine (Fig. 11A) and proline disposal from arterial blood, and is an important source for serine and arginine (Fig. 11B) de novo synthesis, as well as, for the release of other amino acids (76, 201). Daily proximal tubule production by a 70 kg human contributes ∼3 to 4 g serine, ∼2 g arginine, and ∼1 g tyrosine, as well as lesser amounts (80%) of renal mitochondrial GSH transport.

Conclusion The molecular identification of most amino acid transporters has opened new avenues for research in defining molecular structure and function relationships, as well as, the molecular mechanisms mediating and controlling amino acid fluxes between body compartments. Of particular importance is the elucidation of transporter roles in establishing and maintaining amino acid homeostasis in both intracellular and extracellular spaces. Progress will require the kinetic, regulatory and expression data to be integrated in an understanding of the physiological regulatory networks controlling kidney amino acid metabolism. Such an integrated analysis of the physiological networks is necessary for determining the contribution of kidney proximal tubule transport and metabolic functions to body amino acid homeostasis in health and disease. An integrated view will also result in a better understanding of the complex pathophysiology of genetic and acquired renal diseases.

Acknowledgements The laboratory of F. Verrey is supported by Swiss NSF grant 31-130471/1 and the National Centre of Competence in Research (NCCR) Kidney.CH. V. Makrides was supported by a research grant from Ajinomoto.



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Trypanosoma gambiense: membrane transport of amino acids.

Transport of amino acids and nucleosides in the placenta.

Effects of amino acids on zinc transport in rat erythrocytes.

Transport systems for branched-chain amino acids in Pseudomonas aeruginosa.

Kinetic analysis of blood-brain barrier transport of amino acids.

Detecting Electron Transport of Amino Acids by Using Conductance Measurement.

Dynamic covalent transport of amino acids across lipid bilayers.

Membrane transport in Schistosoma mansoni: transport of amino acids by adult males.

Rapid axoplasmic transport in the olfactory nerve of the pike: I. Basic transport parameters for proteins and amino acids.

Energy coupling in the active transport of amino acids by bacteriohodopsin-containing cells of Halobacterium holobium.

Tritium suicide selection of mammalian cell mutants defective in the transport of neutral amino acids.

Kinetics of multiple transport systems for amino acids in the brain.

Phorbol esters stimulate the transport of anionic amino acids in cultured human fibroblasts.

Asymmetrical transport of amino acids across the blood-brain barrier in humans.

Placental amino acid uptake. III. Transport systems for neutral amino acids.

The transport of thyroxine into mouse neuroblastoma cells, NB41A3: the effect of L-system amino acids.

Transport of the large-neutral amino acids by the gamma-glutamyl cycle: a proposal.

Synthetic amino acids and the nature of L-DOPA transport at the blood-brain barrier.

Metabolism in the hypothermically perfused dog kidney. Utilization and production of amino acids.

Mediators of the direct effects of amino acids on the rat kidney.

Transport of amino acids across the blood-brain barrier: implications for treatment of maternal phenylketonuria.

Characteristics of the active transport of peptides and amino acids by germinating barley embryos.

Effect of alkali ions on the active transport of neutral amino acids into Streptomyces hydrogenans.

Transport of cationic amino acids by the mouse ecotropic retrovirus receptor.