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Manuel Palacín, Virginia Nunes, Mariona Font-Llitjós, Maite Jiménez-Vidal, Joana Fort, Emma Gasol, Marta Pineda, Lidia Feliubadaló, Josep Chillarón and Antonio Zorzano Physiology 20:112-124, 2005. doi:10.1152/physiol.00051.2004 You might find this additional information useful... This article cites 103 articles, 37 of which you can access free at: http://physiologyonline.physiology.org/cgi/content/full/20/2/112#BIBL This article has been cited by 5 other HighWire hosted articles: Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter C. A. Bippes, A. Zeltina, F. Casagrande, M. Ratera, M. Palacin, D. J. Muller and D. Fotiadis J. Biol. Chem., July 10, 2009; 284 (28): 18651-18663. [Abstract] [Full Text] [PDF] Distinct classes of trafficking rBAT mutants cause the type I cystinuria phenotype P. Bartoccioni, M. Rius, A. Zorzano, M. Palacin and J. Chillaron Hum. Mol. Genet., June 15, 2008; 17 (12): 1845-1854. [Abstract] [Full Text] [PDF] Amino Acid Transport Across Mammalian Intestinal and Renal Epithelia S. Broer Physiol Rev, January 1, 2008; 88 (1): 249-286. [Abstract] [Full Text] [PDF] Amino Acid Transport in Schistosomes: CHARACTERIZATION OF THE PERMEASEHEAVY CHAIN SPRM1hc G. Krautz-Peterson, S. Camargo, K. Huggel, F. Verrey, C. B. Shoemaker and P. J. Skelly J. Biol. Chem., July 27, 2007; 282 (30): 21767-21775. [Abstract] [Full Text] [PDF]

Medline items on this article's topics can be found at http://highwire.stanford.edu/lists/artbytopic.dtl on the following topics: Oncology .. Transgenic/Knockout Mice Veterinary Science .. Mammalia Developmental Biology .. Renal Aminoaciduria Medicine .. Cystinuria Physiology .. Mice Updated information and services including high-resolution figures, can be found at: http://physiologyonline.physiology.org/cgi/content/full/20/2/112 Additional material and information about Physiology can be found at: http://www.the-aps.org/publications/physiol

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Physiology (formerly published as News in Physiological Science) publishes brief review articles on major physiological developments. It is published bimonthly in February, April, June, August, October, and December by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by the American Physiological Society. ISSN: 1548-9213, ESSN: 1548-9221. Visit our website at http://www.the-aps.org/.

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System B0,+ amino acid transport regulates the penetration stage of blastocyst implantation with possible long-term developmental consequences through adulthood L. J. Van Winkle, J. K. Tesch, A. Shah and A. L. Campione Hum. Reprod. Update, March 1, 2006; 12 (2): 145-157. [Abstract] [Full Text] [PDF]


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PHYSIOLOGY 20: 112–124, 2005; 10.1152/physiol.00051.2004

The Genetics of Heteromeric Amino Acid Transporters

Manuel Palacín,1 Virginia Nunes,2 Mariona Font-Llitjós,2 Maite Jiménez-Vidal,1,2 Joana Fort,1 Emma Gasol,1 Marta Pineda,1 Lidia Feliubadaló,2 Josep Chillarón,1 and Antonio Zorzano1

Heteromeric amino acid transporters (HATs) are composed of a heavy (SLC3 family) and a light (SLC7 family) subunit. Mutations in system b0,+ (rBAT-b0,+AT) and in sys-

1Department

of Biochemistry and Molecular Biology, Faculty of Biology and Institut de Recerca Biomedica de Barcelona, Barcelona Science Park, University of Barcelona, and 2Centre de Genètica Mèdica i Molecular, Institut de Recerca Oncològica, Barcelona, Spain. mpalacin@pcb.ub.edu

tem y+L (4F2hc-y+LAT1) cause the primary inherited aminoacidurias (PIAs) cystinuria and lysinuric protein intolerance, respectively. Recent developments [including the identification of the first Hartnup disorder gene (B0AT1; SLC6A19)] and knockout mouse models have begun to reveal the basis of renal and intestinal reabsorption of amino acids in mammals.

acid transporters; CATs) and related proteins, which on average show <25% amino acid identity to the light subunits of HATs. The general features of HATs are as follows (reviewed in Refs. 15, 21, 61, and 100–102; only very recent references are cited):

Heteromeric amino acid transporters (HATs) are composed of a heavy subunit and a light subunit (Table 1) (15, 21, 100–102). Two homologous heavy subunits (HSHATs) from the SLC3 family have been cloned and are called rBAT (i.e., related to b0,+ amino acid transport) and 4F2hc [i.e., heavy chain of the surface antigen 4F2hc, also named CD98 or fusion regulatory protein 1 (FRP1)]. Nine light subunits (LSHATs; SLC7 family members from SLC7A5 to SLC7A13) have been identified. Six of them are partners of 4F2hc (LAT1, LAT2, y+LAT1, y+LAT2, asc1, and xCT); one forms a heterodimer with rBAT (b0,+AT); and two (asc2 and AGT-1) seem to interact with as yet unknown heavy subunits (100). Members SLC7A1–SLC7A4 of family SLC7 correspond to system y+ isoforms (i.e., cationic amino

• The heavy subunits (molecular mass of ~90 and ~80 kDa for rBAT and 4F2hc, respectively) are type II membrane N-glycoproteins with a single transmembrane domain, an intracellular NH2 terminus, and an extracellular COOH terminus significantly homologous to insect and bacterial glucosidases (FIGURE 1). Recently, X-ray diffraction of the extracellular domain of human 4F2hc revealed a three-dimensional structure similar to that of bacterial glucosidases {a triose phosphate isomerase (TIM) barrel

Table 1. Heteromeric amino acid transporters Heavy Chain (HSHAT)

Light Chain (LSHAT)

Gene

y+LAT1

SLC7A7

y+L

14q11.2

y+

SLC7A6

+

y L

16q22.1

LAT1

SLC7A5

L

16q24.3

LAT2

SLC7A8

L

14q11.2

asc1

SLC7A10

asc

19q12-13

xCT

SLC7A11

xc–

4q28-q32 2p16.3

cystinuria

b0,+AT

SLC7A9

b0,+

19q12-13

cystinuria

AGT1

SLC7A13

new

8q21.3

asc2

SCL7A12

asc

not present

4F2hc

Amino Acid Transport

SLC3A2 LAT2

rBAT

Human Chromosome

Inherited Aminoaciduria

11q13

SLC3A1

LPI

?

Heterometric amino acid transporters (HATs) are composed of a heavy and a light chain. Heavy subunits belong to solute carrier family SLC3, and light subunits belong to SLC7. AGT and asc2 heterodimerize with unknown heavy subunits. A functional asc2 gene is not present in the human genome. LPI, lysinuric protein intolerance.

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Heteromeric Amino Acid Transporters


REVIEWS Extracellular

Disulfide bridge C158 H110

COOH C109

NH2

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NH2

COOH

Cytosolic

FIGURE 1. A heteromeric amino acid transporter The heavy subunit (pink) and the light subunit (blue) are linked by a disulfide bridge (yellow) with conserved cysteine residues (cysteine 158 for the human xCT and cysteine 109 for human 4F2hc). The heavy subunits (4F2hc or rBAT) are type II membrane glycoproteins with an intracellular NH2 terminus, a single transmembrane domain, and a bulky COOH terminal. This part of the protein shows homology with bacterial glycosidases. The membrane topology of the light subunit xCT, as model of the other light subunit, shows 12 transmembrane domains, with the NH2 and COOH terminals located intracellularly and with a reentrant loop-like structure in the intracellular loop IL2-3 (His110 corresponds to the apex of the loop) (31). Residues with external (black) or internal (red) accessibility are shown.

[(␣␤)8] and eight antiparallel ␤-strands; FIGURE 1} (unpublished observations).

membrane unless it interacts with the heavy subunit.

• The light subunits (~50 kDa) are highly hydrophobic and not glycosylated. This results in anomalously high mobility in SDSPAGE (35–40 kDa). Recent cysteine-scanning mutagenesis studies support a 12-transmembrane-domain topology, with the NH2 and COOH terminals located inside the cell and with a reentrant-like structure in the intracellular loop IL2-3 for xCT, as a model for the light subunits of HATs (FIGURE 1) (31, 38).

• The light subunit confers specific amino acid transport activity to the heteromeric complex (LAT1 and LAT2 for system L isoforms, y+LAT1 and y+LAT2 for system y+L isoforms, asc1 and asc2 for system asc isoforms, xCT for system xc– isoforms, b0,+AT for system b0,+ isoforms, and AGT-1 for a system serving aspartate and glutamate transport (Table 1). Moreover, reconstitution in liposomes shows that the light subunit b0,+AT is fully functional in the absence of the heavy subunit rBAT (71).

• The light and the corresponding heavy subunit are linked by a disulfide bridge (FIGURE 1). For this reason, HATs are also named glycoprotein-associated amino acid transporters (21, 102). The intervening cysteine residues are located in the putative extracellular loop EL3-4 of the light subunit and a few residues away from the transmembrane domain of the heavy subunit (FIGURE 1). • The light subunit cannot reach the plasma

• HAT transport activities are, with the exception of system asc isoforms, tightly coupled amino acid antiporters (67).

Primary Inherited Aminoacidurias Primary inherited aminoacidurias (PIAs) are caused by defective amino acid transport activities, which

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REVIEWS Table 2. Primary inherited aminoacidurias Prevalence Cystinuria* Type I

Type of Inheritance

Gene

Chromosome

Mutations

AR

SLC3A1 SLC7A9 SLC7A9 SLC3A1

2p16.3 19q13.1

103 12 66 1

Transport System

1/7,000

Non-type I

ADIP

b0,+

very rare

AR?

?

?

?

?

LPI

>100 cases

AR

SLC7A7

14q11

31

y+L

Hyperdibasic aminoaciduria type 1

very rare

AD

?

?

?

?

Isolated lysinuria

very rare

AR?

?

?

?

?

Hartnup disorder

1/30,000

AR

SLC6A19

5p15

10

B0

Renal familial iminoglycinuria

1/15,000

AR

?

?

?

Imino(?)†

Dicarboxylic aminoaciduria

very rare

AR?

SLC1A1 ?

9p24 ?

KO null‡

– XAG

AR, autosomal recessive; ADIP, autosomal dominant with incomplete penetrance; AD, autosomal dominant; AR?, familial studies in the very few cases described for these diseases suggest an autosomal recessive mode of inheritance. *Among the 103 SLC3A1 and 66 SLC7A9 mutations described in cystinuria (which preferentially associated with phenotypes I and non-I, respectively), 12 SLC7A9 mutations associated to phenotype I in some heterozygotes, and 1 SLC3A1 mutation (dupE5–E9) associated in some heterozygotes with phenotype I (28). †The amino acids hyperexcreted in renal familial iminoglycinuria (glycine and proline) suggest that the defective transport system corresponds to system Imino, but no experimental evidence or candidate gene is available. ‡Slc1a1-null knockout mice present dicarboxylic aminoaciduria (62), suggesting this gene as a candidate for the human disease.

affect renal reabsorption of amino acids and may also affect intestinal absorption of amino acids and transport function in other organs. Several PIA have been described (Table 2). Reabsorption of dibasic amino acids is defective in lysinuric protein intolerance [LPI, also named hyperdibasic aminoaciduria type 2 or familial protein intolerance (MIM 222700) and first described by Perheentupa et al. in 1965 (63); reviewed in Ref. 86], autosomal dominant hyperdibasic aminoaciduria type I (MIM 222690) (106), and isolated lysinuria (56). Reabsorption of cystine and dibasic amino acids is defective in cystinuria (reviewed in Ref. 60). Hyperexcretion of cystine occurs only in isolated cystinuria (MIM 238200) (8). Reabsorption of zwitterionic amino acids (i.e., neutral amino acids at physiological pH) is defective in Hartnup disorder (MIM 234500) (3) and iminoglycinuria (MIM 242600) (72). Finally, reabsorption of glutamate and aspartate is defective in dicarboxylic aminoaciduria (MIM 222730) (53, 93). HATs are involved in PIA, because mutations in rBAT (SLC3A1) and b0,+AT (SLC7A9) cause cystinuria and mutations in y+LAT1 cause LPI. The relationship between LPI, autosomal dominant hyperdibasic aminoaciduria type I, and isolated lysinuria at the molecular level, if any, is unknown. Similarly, the gene responsible for isolated cystinuria 114

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remains to be identified.

Cystinuria Cystinuria is an autosomal inherited disorder, characterized by impaired transport of cystine and dibasic amino acids in the proximal renal tubule and the gastrointestinal tract [first described by Sir Archivald Garrod in 1908 (30) and reviewed in Refs. 60 and 80]. The overall prevalence of the disease is 1/7,000 neonates, ranging from 1/2,500 neonates in Libyan Jews to 1/100,000 among Swedes (60). Patients present normal to low-normal levels in blood, hyperexcretion in urine, and intestinal malabsorption of these amino acids. High cystine concentration in the urinary tract most often causes the formation of recurring cystine stones (i.e., urolithiasis) due to the low solubility of this amino acid. This is the only symptom associated with the disease. Therefore, treatment attempts to increase cystine solubility in urine (high hydratation, urine alkalinization, and formation of soluble cystine adducts with thiol drugs) (60). Cystinuria is not accompanied by malnutrition, suggesting that intestinal malabsorption is not severe. Absorption of di- and tripeptides via PepT1 may prevent malnutrition in cystinuria (17). Traditionally, three types of cystinuria have been recognized in humans: type I, type II, and type III

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Isolated cystinuria


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LPI LPI is a PIA with an autosomal recessive mode of inheritance (reviewed in Ref. 86). LPI is predominantly reported in Finland, with a prevalence of 1/60,000. LPI also occurs in Southern Italy and Japan (prevalence of 1/50,000 in the northern part of Iwate) (see Ref. 46 and reviewed in Ref. 58). In LPI there is massive excretion of dibasic amino acids, especially lysine, and the intestinal absorption of these amino acids is poor (reviewed in Ref.

57). As a result, plasma levels of dibasic amino acids are low. Arginine and ornithine are intermediates of the urea cycle that provide the carbon skeleton. Their reduced availability is thought to produce a functional deficiency of the urea cycle. Protein malnutrition and lysine deficiency contribute to the patient’s failure to thrive. Several organs are affected in LPI (reviewed in Ref. 86). Patients with LPI are usually asymptomatic while being breast fed, and symptoms (e.g., vomiting, diarrhea, and hyperammonemic coma when force-fed high-protein food) appear only after weaning. After infancy, patients with LPI reject a high-protein diet and show delay in bone growth, osteoporosis, hepatosplenomegaly, muscle hypotonia, and sparse hair. Most patients show normal mental development, but some may have moderate retardation. About two-thirds of the patients have interstitial changes in chest radiographs, sometimes with acute or chronic respiratory insufficiency that can lead to fatal pulmonary alveolar proteinosis and to multiple-organ dysfunction syndrome. Further symptoms, such as glomerulonephritis and erythroblastophagia, suggest that the immune system is affected in some patients. Low-protein diet and citrulline (i.e., a urea cycle intermediate that is not a substrate of system y+L) are used to correct the functional deficiency of the urea cycle (reviewed in Ref. 57). The final height in treated patients is slightly subnormal. This treatment neither corrects hepatosplenomegaly nor delays bone age or osteoporosis, probably due to lysine deficiency. The transport characteristics of two of the LSHATassociated transport systems are relevant to the inherited aminoacidurias cystinuria and LPI (Table 1): system b0,+ [due to the rBAT (SLC3A1) and b0,+AT (SLC7A9) heterodimer] is a tertiary active mechanism of renal reabsorption and intestinal absorption

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(80). This classification correlates poorly with molecular findings, and it has recently been revised to type I (MIM 220100) and non-type I (MIM 600918) cystinuria (with the latter corresponding to old types II and III). These two are distinguished on the basis of the cystine and dibasic aminoaciduria of the obligate heterozygotes (60): type I heterozygotes are silent, whereas non-type I heterozygotes present a variable degree of urinary hyperexcretion of cystine and dibasic amino acids that is higher in type II than in type III. This indicates that type I cystinuria is transmitted as an autosomal recessive trait, whereas non-type I is transmitted dominantly, with incomplete penetrance (27). Not surprisingly, urolithiasis has been described in a minority of non-type I heterozygotes (Table 3). Patients with a mixed type, inheriting type I and non-type I alleles from either parent, have also been described (33). Data on the relative proportion of the two types in specific populations are scarce. In 97 wellcharacterized families of the International Cystinuria Consortium (ICC) cohort of patients, mainly from Italy, Spain, and Israel, 38, 47, and 14% transmit type I, non-type I, and mixed cystinuria, respectively (Table 3) (28). This cohort is not a registry, and therefore it might not represent the whole population within those countries (20).

Table 3. Genotype and phenotype classification of cystinurial probands Phenotype Genotype

I

AA AA(B) BB B+ BB(A)

29 1

Non-I

Non-I Carriers

34

Mixed

Total Probands

%ab

2 1 7

31 1 42 3 1

39.7 1.3 53.8 3.8 1.3

3 1

Total Probands 30 34 3 11 78 100 % 38.5 43.6 3.8 14.1 100 Among the 164 probands studied by the International Cystinuria Consortium, 78 are fully genotyped and phenotyped (28). Thirty-eight probands are not fully genotyped [1 mutated SLC3A1 allele (A?), 1 mutated SLC7A9 allele (B?), and no mutated allele (??) have been identified in 11, 22, and 5 probands, respectively). Sixty-seven probands (25 AA, 23 BB, 5 A?, 11 B?, and 3 ??) have not been phenotyped due to lack or incoherent information on the amino acid excretion in urine of the obligate heterozygotes. AA, 2 mutated SLC3A1 alleles; BB, 2 mutated SLC7A9 alleles; B+, mutated SLC7A9 carrier; AA(B), 2 mutated SLC3A1 alleles and 1 mutated SLC7A9 allele; BB(A), 2 mutated SLC7A9 alleles and 1 mutated SLC3A1 allele.

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REVIEWS aa+, CssC

aa–

aa

Lumen

Na+

Apical

bo,+AT

rBAT

B0AT1

Na+

ASCT2

EAAT3

(di, tri)-aa H+

PepT

+ – K+

aa+, CssC CSH

aa–

aa0

Single aas

K+

y+LAT1

4F2 hc

ATPase

(di, tri)-aa

LAT2

4F2 hc

? TAT1

T

aa–

aa0 Na+

Basolateral

aa+

Na+ aa

Blood

FIGURE 2. Transporters involved in the renal and intestinal reabsorption of amino acids Transporters with a probed role in renal reabsorption or intestinal absorption of amino acids are colored. Transporters present in the apical or basolateral plasma membrane of the epithelial cells of the proximal convoluted tubule or of the small intestine, but with no direct experimental evidence supporting their role in reabsorption, are shown uncolored. Fluxes of amino acids in the reabsorption direction are in red. PepT (H+-dependent peptide transporter PepT1 in small intestine and Pept2 in kidney) cotransports di- and tripeptides [(di,tri)-aa] with protons. Then, intracellular hydrolysis renders single amino acids. The high intracellular concentration of neutral amino acids (aa0) due to the transport activity of apical (B0AT1) and basolateral (y+LAT1-4F2hc, T) transporters drives the active reabsorption of dibasic amino acids (aa+) and cystine (CssC), together with the membrane potential and reduction of CssC to cysteine (CSH). Basolateral efflux transporters for dicarboxylic amino acids are unknown (Taa–). T represents basolateral Na+dependent transporters with undefined roles in reabsoption (systems A and ASC).

of dibasic amino acid and cystine in the apical plasma membrane. It mediates the electrogenic exchange of dibasic amino acids (influx) for neutral amino acids (efflux) (reviewed in Ref. 59) (FIGURE 2). This exchange is favored by the membrane potential (negative inside the cell) and the high intracellular concentration of neutral amino acids, which is the result of the activity of sodium-dependent transport systems for neutral amino acids in the apical and basolateral domains of the epithelial cells. The influx of cystine through system b0,+ is favored by the intracellular reduction of cystine to cysteine. System y+L (due to a 4F2hc-y+LAT1 heterodimer) mediates the electroneutral exchange of dibasic amino acids (efflux) for neutral amino acids plus sodium (influx) (10, 13, 39). It is assumed that this transport system allows the efflux of dibasic amino acids against the membrane potential at the basolateral domain of epithelial cells (FIGURE 2). The 4F2hc-y+LAT2 heterodimer mediates system y+L in many other cell types (39, 65, 96).

The Molecular Basis of Cystinuria The above-mentioned characteristics of rBAT point116

PHYSIOLOGY • Volume 20 • April 2005 • www.physiologyonline.org

ed to this gene as a candidate for cystinuria. In 1994 it was demonstrated that mutations in SLC3A1 cause type I cystinuria (11). Since then 103 distinct rBAT mutations have been described, including nonsense, missense, splice-site, and frame-shift mutations, as well as large deletions and chromosome rearrangements (see Ref. 28 and references therein). Cystinuria resembling type I due to mutations in canine SLC3A1 has been reported in Newfoundland dogs (37). Similarly, Pebbels mice (homozygous for the rBAT mutation D140G) develop type I cystinuria with urolithiasis (64). Functional analysis of the most common SLC3A1 mutation, M467T, revealed a trafficking defect, with the protein reaching the plasma membrane inefficiently (14). A trafficking defect, also suggested for other SLC3A1 mutations (14, 74), is consistent with the proposed role of rBAT as an ancillary subunit of b0,+AT. SLC3A1 mutations may also affect transport properties of the holotransporter b0,+: the cystinuria-specific mutation R365W, in addition to temperature-sensitive protein stability and trafficking defect, shows a defect in the efflux of arginine but not in its influx (68). This suggests two transport pathways for the light subunit b0,+AT, one for influx

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? Taa–


REVIEWS

Explained alleles in cystinuria Very recently, the ICC performed an exhaustive mutational analysis on 164 probands (28): 86.8% of the independent alleles were identified. The coverage of identified alleles was similar in all cystinuria types (type I, 90.5%; non-type I, 87.6%; mixed, 89.3%; untyped patients, 83.6%). The unidentified alleles (13.2%) may be due to mutations in intronic or promoter regions (e.g., two cystinuria-specific sequence variants in the promoter region of SLC3A1 have been reported in Ref. 7), to SLC3A1 or SLC7A9 polymorphisms in combination with cystinuria-specific mutations in the other allele [as suggested by Schmidt et al. (76)],

or to unidentified genes. These three possibilities have not been confirmed or ruled out. Functional studies of promoter sequence variants have not been reported, and cystinuria association studies and functional analysis of candidate polymorphisms are required to demonstrate their role in the disease. Of particular interest is the possibility of a third cystinuria gene. In this sense, Goodyer’s group proposed SLC7A10 as a candidate (50). SLC7A10 is in the vicinity of the cystinuria gene SLC7A9 on chromosome 19q13.1 and codes for the renal light subunit asc1, which, with 4F2hc, forms the holotransporter asc with substrate specificity for cysteine and other small neutral amino acids. Moreover, these authors found the missense mutation E112D associated with cystinuria. In contrast, recent studies have ruled out this hypothesis: 1) cystinuria-specific mutations are not found in patients with alleles not explained by mutations in the two cystinuria genes (67, 78), 2) the conservative mutation E112D does not affect transport of 4F2hc-asc1 (67), and 3) asc1 mRNA is expressed in the distal tubule where renal reabsorption of amino acids is not relevant but where asc1 may have a role in the regulation of osmosis (67). Another candidate is a missing light subunit of rBAT. Coimmunoprecipitation studies and the SLC7A9 knockout model demonstrated that the protein products of SLC3A1 (rBAT) and SLC7A9 (b0,+AT) heterodimerize in mouse kidney brush border membranes, but a significant part of rBAT heterodimerizes with an unknown light subunit (X) (23, 25). Indeed, a heterodimer of rBAT is present in kidney brush-border membranes of the Slc7a9knockout mice (23). The protein X may also be present in human kidney brush borders, but a final demonstration is needed (25). Therefore, the gene coding for X could be a candidate for cystinuria. However, the similar hyperexcretion of amino acids detected in urine of patients with mutations in SLC3A1 or in SLC7A9 does not support this hypothesis (28). If X were coded by a cystinuria gene, one would expect higher aminoaciduria in patients with mutations in SLC3A1 than in patients with mutations in SLC7A9: b0,+AT mutations would affect system b0,+ only, whereas rBAT mutations would affect system b0,+ and the transport activity of the rBAT/X heteromeric complex. In summary, the possibility of a third cystinuria gene cannot be discarded, but it would be relegated to a very small proportion of patients and seems improbable (Only 3% of the probands of the ICC show no mutation in either of the two cystinuria genes; Ref. 28).

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and the other for efflux. This scenario is consistent with two additional sets of results: 1) the chicken intestinal system b0,+ has a sequential mechanism of exchange, compatible with the formation of a ternary complex (i.e., the transporter bound to its intracellular and extracellular amino acid substrates) (95); and 2) the analog aminoisobutyrate (AIB) induces an unequal exchange with other substrates through the rBAT-induced system b0,+ in oocytes (i.e., using the endogenous b0,+AT subunit) (16). The oligomeric structure of system b0,+ (i.e., rBAT-b0,+AT heteromer) is unknown. Functional coordination of two rBAT-b0,+AT heterodimers in a heterotetrameric structure would explain these results. If this is not the case, the transport defect associated with mutation R365W would suggest that a single b0,+AT subunit contains two translocation pathways. The gene causing non-type I cystinuria was assigned to 19q12-13.1 by linkage analysis (5, 89, 103). In 1999 the non-type I cystinuria gene was identified as SLC7A9 (24). SLC7A9 was a positional candidate gene for non-type I cystinuria because it has the appropriate chromosomal location, rBATassociated amino acid transport activity (system b0,+), and tissue expression (mainly in kidney and small intestine). The protein product encoded by SLC7A9 was termed b0,+AT for b0,+ amino acid transporter. Sixty-six SLC7A9 mutations causing cystinuria have been described (see Ref. 28 and references therein). Mutation G105R is the most frequent SLC7A9 mutation in the ICC cohort of patients (27.4% of the identified SLC7A9 alleles). Similarly to the human disease, the Slc7a9-knockout mouse presents non-type I cystinuria with urolithiasis (23). Several cystinuria-specific SLC7A9 missense mutations have been reported to lead to a transport function defect (27). Among these mutations, reconstitution in proteoliposomes showed that A182T-mutated b0,+AT is active but with a trafficking defect to the plasma membrane, whereas mutation A354T renders the transporter inactive (71).

Genotype-phenotype correlations in cystinuria Initial data suggested a one-to-one correlation between the phenotype and the mutated gene

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118

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in SLC3A1 (i.e., individuals AA); 2) all non-type I patients (including type non-type I heterozygotes with urolithiasis) have mutations in SLC7A9 (i.e., individuals BB and B+); 3) patients with mixed cystinuria carry mutations in SLC3A1 (2 probands AA) or in SLC7A9 (7 probands BB); and 4) 2 out of 126 fully genotyped probands carry mutations in both genes [probands AA(B) and BB(A)], suggesting a role for digenic inheritance in cystinuria (see below). Urolithiais shows a clear gender and individual variability among cystinuria patients (20). In the ICC cohort, the age of onset (first stone) ranges from 2–40 yr with a median of 12 and 15 yr for males and females, respectively. The incidence of onset before the age of 3 is lower in females than males. Similarly, the number of total stone events (i.e., spontaneously emitted stones plus those surgically removed) is higher in males than females (0.42 and 0.21 events per year in males and females, respectively). Of the 224 patients studied, ten with aminoaciduria and full genetic confirmation of the disease did not develop renal stones, and two of them are over 40 years of age. In contrast, clinical symptoms (i.e., urolithiais and its consequences) are almost identically represented in the two cystinuria types when either the clinical or the genetic classification is considered. The differences in severity between the genders and marked differences between siblings sharing the same mutations (19, 20) suggest that other lithogenic factors, genetic or environmental, contribute to the urolithiasis phenotype. Moreover, only about half of the Slc7a9-knockout mice, which have a mixed genetic background, develop urolithiasis (23). Moreover, lithiasic and nonlithiasic Slc7a9-knockout mice hyperexcrete similar levels of cystine. Studies in Slc7a9-knockout mice within different genetic backgrounds may unravel the genetic factors contributing to urolithiasis besides mutations in Slc7a9 and the cystine levels in urine.

Digenic inheritance causing partial phenotype To our knowledge, only four patients with mutations in both cystinuria genes have been reported (28, 35). There is no report of the urine phenotype of the Swedish patient AA(B). In the ICC database, only two sisters AA(B) and one male BB(A) from 2 families out of 126 fully genotyped families have been identified. They are classified as mixed cystinuria patients (i.e., each of the two mutated alleles in the same gene is associated with phenotype I or non-phenotype I in the obligate heterozygotes). The aminoaciduria levels of these patients and their double-heterozygote (i.e., AB) relatives indicate that digenic inheritance in cystinuria has only a partial effect on the phenotype, restricted to a variable impact on the aminoaciduria. Indeed,

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(mutations in SLC3A1 resulted in type I, and mutations in SLC7A9 resulted in non-type I) (12, 32). In contrast to this simple view, recent data show a more complex scenario. On the one hand, all SLC3A1 mutations in well-characterized families cause type I cystinuria, with the exception of mutation dupE5–E9, which shows the non-type I phenotype in four out of six heterozygotes studied (28). This mutation consists of a gene rearrangement c.(891+1524_1618-1600)dup (initially published in Ref. 77), which results in the duplication of exons 5–9 and the corresponding in-frame duplication of amino acid residues E298–D539 of rBAT, as shown by RNA studies (28, 77). Functional studies are necessary to explain the dominant negative effect of dupE5–E9 mutation on the rBAT/b0,+AT heteromeric complex. On the other hand, most of the heterozygotes carrying a SLC7A9 mutation have a nontype I urine phenotype (i.e., hyperexcretion of dibasic amino acids and cystine), but heterozygotes carrying SLC7A9 mutations may also have a type I phenotype (i.e., silent heterozygotes). Approximately 14% of the SLC7A9 heterozygotes have phenotype I in the ICC cohort of patients and their relatives (20). SLC7A9 mutations that have been found associated with phenotype I in some families are I44T, G63R, G105R, T123M, A126T, V170M (the Libyan Jewish mutation), A182T, G195R, Y232C, P261L, W69X, and c.614dupA (28, 49). There is no clear explanation of why these mutations associate with phenotype I, since some proteins show residual transport activity when expressed in heterologous expression systems but others do not (27). A182T is the most frequent SLC7A9 mutation associated with phenotype I (i.e., 6 out of 11 A182T heterozygotes in the ICC cohort), and this mutation leads to a protein with 50% residual transport activity at the plasma membrane (27, 71). Moreover, mixed cystinuria patients with two mutations in SLC7A9 presented a level of aminoaciduria in the lower range of non-type I patients with two mutations in this gene (28). This suggests that, in addition to individual and population variability, mild SLC7A9 mutations may be more prone to associate with silent phenotype in heterozygotes. The lack of a direct relationship between the mutated cystinuria gene and the type of cystinuria led the ICC to propose two parallel classifications to describe cystinuria (20) that are 1) based on the urine phenotype of the obligate heterozygotes (type I, non-type I, and mixed, as described above) and 2) based on the genotype of the patients (type A due to mutations in SLC3A1, type B due to mutations in SLC7A9, and type AB to define a possible digenic cystinuria). Table 3 summarizes the double classification for 78 cystinuria probands by the ICC (28) as follows: 1) most type I patients have two mutations


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The Molecular Basis of LPI The gene responsible for LPI was localized to 14q11.2 in Finnish and non-Finnish populations (47, 48). The cloning of y+LAT1, encoded by SLC7A7 (96), revealed characteristics that made this gene an excellent candidate for LPI:

• y+LAT1 heterodimerizes with 4F2hc to express system y+L amino acid transport activity in the basolateral plasma membrane of the epithelial cells of the renal proximal tubule and the small intestine (see above). • SLC7A7 is expressed in tissues affected in LPI (kidney, small intestine, lung, and white blood cells). • SLC7A7 maps to the correct location for LPI. Torrents and co-workers (97) performed mutational analysis of SLC7A7 in 1 Spanish and 31 Finnish LPI patients. A single Finnish mutant allele (1181-2A>T) was found, in which the splice site acceptor of intron 6 is inactivated and a cryptic acceptor 10 bp downstream is activated, with the result that 10 bp of the open reading frame are deleted and the reading frame is shifted. This mutation has been found in all Finnish LPI patients (i.e., “the Finnish mutation”) (54, 97). The Spanish patient with LPI was a genetic compound of two SLC7A7 mutations (a missense mutation, L334R, and a 4-bp deletion, 1291delCTTT). Simultaneously and independently, Borsani and coworkers (6) identified the Finnish mutation in four Finnish patients and found two additional SLC7A7 mutations (1625insATAC and 242del543) in five Italian patients with LPI. These two studies established that mutations in SLC7A7 cause LPI. Additional studies showed the nonsense mutation W242X and the insertion 1625insATAC as the most prevalent mutations in the south of Italy (88) and the nonsense mutation R410X as the most prevalent in Japan (55). A total of 31 SLC7A7 mutations have been described in 113 patients with LPI (222 explained alleles from a total of 226 studied alleles, including consanguineous ones; reviewed in Ref. 58). Identified SLC7A7 mutations include missense, nonsense, and splicing mutations, insertions, deletions, and large genomic rearrangements. No LPIassociated mutation has been found in SLC3A2, which encodes the heavy subunit of y+LAT1 (4F2hc). This strongly suggests that SLC7A7 is the only gene involved in the primary cause of LPI. It is believed that mutations in 4F2hc would be deleterious, and indeed, the targeted disruption of Slc3a2 in mouse is lethal during embryonic life (98). 4F2hc serves as the heavy subunit of six other heteromeric amino acid transporters (see above). Therefore, a defect in 4F2hc would result in six defective amino acid transport activities expressed in many cell types and tissues. Moreover, 4F2hc is a multifunctional protein with a putative role in ␤1-integrin function and cellular fusion (reviewed in Ref. 15). Thus defective 4F2hc would be life threatening. Functional studies in oocytes and transfected

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none of the individuals AB presented urolithiasis. Given that the frequencies of type A and B alleles are similar in this cohort, if digenic inheritance was the rule in cystinuria, we would expect a quarter of patients to be AA, a quarter of patients to be BB, and half of patients to be AB. This indicates that digenic inheritance affecting phenotype is an exception in cystinuria. However, we cannot rule out the possibility that some combinations of mutations A and B might produce enough cystine hyperexcretion to cause urolithiasis. A working hypothesis on the biogenesis of the rBAT-b0,+AT heterodimer and the urine phenotypes in cystinuria may explain the apparent lack of full digenic inheritance in cystinuria: the rBAT protein is produced in excess in kidney, and therefore neither an rBAT mutation in heterozygosis [e.g., mouse cystinuria model with the SLC3A1 mutation D140G (64)] nor any of the human cystinuria rBAT mutations except dupE5–E9 (28) lead to hyperexcretion of amino acids (phenotype I). Also, b0,+AT controls the expression of the functional rBAT-b0,+AT heterodimeric complex: interaction with b0,+AT stabilizes rBAT, and the excess of rBAT is degraded, as shown in transfected cells (4, 71). As a result, a half dose of b0,+AT [heterozygotes of severe human SLC7A9 mutations or of the SLC7A9 knockout mice (23, 28)] results in a significant decrease in the expression of rBAT-b0,+AT heterodimer complex (system b0,+), which causes hyperexcretion of cystine and dibasic amino acids. In this scenario, the lack of a full phenotype because of digenic inheritance indicates that in double heterozygotes (AB), the mutated rBAT, with trafficking defects (14, 68, 74), does not interact and/or does not compromise the heterodimerization and trafficking to the plasma membrane of the half dose of wild-type b0,+AT with the half dose of wild-type rBAT. Thus individuals AB behave as heterozygotes B with a variable degree of aminoaciduria, which could be greater than that of single heterozygotes within the family, depending on the particular combination of mutations. Demonstration of this hypothesis requires a deep study of both the impact of cystinuria-specific rBAT and b0,+AT mutations (including the dominant negative rBAT mutation dupE5–E9) in the biogenesis and the functional and structural stoichiometry of subunits of the heteromeric complex rBAT/b0,+AT both in cell culture studies and in vivo with the help of Slc3a1 and Slc7a9 double-mutant mice.

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REVIEWS cells showed that frameshift mutations (e.g., 1291delCTTT, 1548delC, and the Finnish mutation) produce a severe defect in trafficking to the plasma membrane (54, 94). In contrast, the missense mutations G54V and L334R inactivate the transporter (e.g., the mutated proteins reach the plasma membrane when coexpressed with 4F2hc, but no transport activity is elicited) (54, 94). Recently, functional studies in oocytes showed that the LPIspecific SLC7A7 mutations M1L, M50K, T188I, W242X, S386R, Y457X, and c.1471delTTCT lead to loss of transport function. In this last study, no attempt was made to elucidate whether these mutations affect trafficking or inactivate the transporter (Sperandeo MP and Sebastio G, personal communication).

The renal reabsorption of amino acids occurs in the proximal convoluted tubule (85), and the absorption of amino acids occurs in the small intestine (52). Most of the transporters responsible for these functions are the same in kidney and intestine (FIGURE 2). The most striking exception to this rule is the proton-dependent peptide transporters. PepT1 and PepT2 are expressed in small intestine and in kidney, respectively. The physiological role of PepT2 in kidney is unknown, and the quantitative contribution of PepT1 to assimilation of amino acids has not yet been evaluated in mammals or humans (17). A deeper study of the phenotype of the PepT2-knockout mice (73) and generation and study of the PepT1 may answer these questions. The molecular basis of cystinuria and LPI teaches us about the molecular bases of cystine and dibasic amino acid reabsorption. Mutations of the apical exchanger b0,+ (heterodimer rBAT-b0,+AT) lead to hyperexcretion of cystine and dibasic amino acids, as shown in human (11, 24), mouse (23, 64), and canine (37) cystinuria. Mutations of the basolateral exchanger y+L (heterodimer 4F2hc-y+LAT1) produce hyperexcretion of dibasic amino acids (6, 97). Similarly, there is intestinal malabsorption of cystine and dibasic amino acids in cystinuria and of dibasic amino acids in LPI (reviewed in Refs. 80 and 86). Thus the sequential transport activities of systems b0,+ (apical) and y+L (basolateral) play a major role in renal and intestinal reabsorption of amino acids. Besides these two players, not much is known about other transporters with a role in dibasic amino acid reabsorption. Cystinuria patients may show almost null cystine reabsorption in kidney, whereas dibasic reabsorption in kidney remains significant (reviewed in Ref. 25). This suggests that system b0,+ is the main (if not the only) apical reabsorption system for cystine, but other apical trans120

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The Renal and Intestinal Reabsorption of Amino Acids

port systems in addition to system b0,+ contribute to the renal reabsorption of dibasic amino acids. The study of the dibasic amino acid transport activities in the Slc7a9-knockout mice should help to identify those transporters. Similar data are not available for LPI patients, but the experiments of the Finnish group (70) suggest that system y+L is the main basolateral absorption system for dibasic amino acids in the small intestine: an oral load with the dipeptide lysyl-glycine increased glycine plasma concentrations, but plasma lysine remained almost unchanged in patients with LPI, whereas both amino acids increased in plasma of control subjects and in patients with cystinuria. This demonstrated the basolateral defect in LPI and points to a role of PepT1 in amino acid assimilation. Indeed, as a consequence of the renal and intestinal defects in LPI, plasma levels of dibasic amino acids are 1/2 to 1/3 of normal levels (reviewed in Ref. 86), whereas in human and mouse cystinuria cystine and dibasic amino acid levels in plasma are only moderately (20–30%) reduced (reviewed in Ref. 57). In all, these results suggest that system y+L has a higher impact than system b0,+ on renal and intestinal reabsorption of dibasic amino acids. An understanding of the molecular basis of zwitterionic renal and intestinal reabsorption was helped by the identification of B0AT1 (SLC6A19) as the gene causing Hartnup disorder (45, 81). B0AT1 corresponds to system B0 [also named system neutral brush border (system NBB)] and catalyzes the Na+-dependent transport of most neutral amino acids (9). Patients with Hartnup disorder present hyperexcretion and malabsorption of neutral amino acids (reviewed in Ref. 51). Interestingly, they also show moderate hyperexcretion of cystine and dibasic amino acids, suggesting a metabolic link with system b0,+ (reviewed in Ref. 81) (FIGURE 2). The basolateral Na+-K+-ATPase generates the electrochemical gradient of Na+, which is used by apical (system B0; i.e., B0AT1) and basolateral (systems A and ASC) Na+ cotransporters to generate a high intraepithelial concentration of zwitterionic amino acids. A reduced influx of zwitterionic amino acids when B0AT1 is defective (i.e., Hartnup disorder) would reduce the substrates for exchange with cystine and dibasic amino acids via system b0,+. The above-mentioned transporters with a role in reabsorption of amino acids are highly expressed in the corresponding apical or basolateral plasma membrane of the epithelial cells of the proximal convoluted tubules (S1 and S2 segments) in kidney and of the enterocytes of the small intestine (18, 25, 29). The heterodimer 4F2hc-LAT2 has a similar pattern of expression in kidney and small intestine, where it is located in the basolateral plasma membrane (18, 66). 4F2hc-LAT2 is an exchanger with


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• Within the SLC6 family there are two B0AT1sequence-related orphan transporters, XT2 (SLC6A18) and XT3 (SLC6A20). These three genes belong to a branch of amino acid transporters within the Na+-Cl–dependent neurotransmitter family (9). SLC6A18 is located in the vicinity of SLC6A19 in 5p15, and therefore it might be excluded from linkage to Hartnup disorder (45). Functional and tissue-expression studies are needed to ascertain the role of XT2 and XT3 in reabsorption of amino acids. • The amino acid transporter ASCT2 (also named ATB0) (SLC1A5) is expressed in the apical plasma membrane of the renal proximal convoluted and small intestine epithelial cells (1). This transporter exchanges most of the zwitterionic amino acids in a Na+-dependent manner (40, 42, 99). The role of ASCT2 in amino acid reabsorption has not been evaluated. • The metabolic link between systems b0,+, B0, and y+L requires a basolateral transport system to mediate the efflux of neutral amino acids (FIGURE 2). A defective amino acid transport system for basolateral efflux of zwitterionic amino acids would increase the intracellular concentration of

these amino acids, resulting in their hyperexcretion in urine and intestinal malabsorption. Candidate transporters for this function may be found within families SLC16 and SLC43. Amino acid transporters in these families mediate facilitated diffusion and may therefore mediate the efflux of zwitterionic amino acids from the high intracellular concentration to the interstitial space. T-type amino acid transporter 1 (TAT1; SLC16A10) transports aromatic amino acids in a Na+- and H+-independent manner (43, 44). TAT1 is expressed in human kidney and small intestine with a basolateral location. The SLC16 family (also named MCT for monocarboxylate transporters) contains members transporting monocarboxylates and thyroid hormones as well. Eight transporters within this family are orphan transporters (MCT5–7, MCT9, and MCT11–14) (34). Knockout mouse models for TAT1, and their related orphan transporters expressed in kidney cortex and small intestine, may help to identify basolateral transporters involved in reabsorption of zwitterionic amino acids. LAT3 (2) and LAT4 (5a) within family SLC43 mediates facilitated diffusion of zwitterionic amino acids with characteristics of system L. Neither of these two transporters is expressed in epithelial cells of the renal proximal convoluted tubule or the small intestine, but the SLC43 family has a third member with no identified transport function (EEG1; Ref. 90). Functional and tissueexpression studies are needed to ascertain the role of EEG1 in reabsorption of amino acids. The molecular basis of the renal and intestinal reabsorption of acidic amino acids is less known. The bulk (>90%) of filtered acidic amino acids is reabsorbed within segment S1 (i.e., the first part of the proximal convoluted tubule) (83, 84). Two apical acidic transport systems have been described: one of high capacity and low affinity and the other of low capacity and high affinity (reviewed in 36). The Na+/K+-dependent acidic amino acid transporter EAAT3, which localized to chromosome 9p24 (87) (also named EAAC1; SLC1A1) (system XAG–) is expressed in the brush-border membranes of segments S2 and S3 of the nephron (82). The transport characteristics of SLC1A1 correspond to the high-affinity system (41). The Slc1a1-knockout mice develop dicarboxylic aminoaciduria (62), demonstrating the role of this transporter in renal reabsorption of acidic amino acids. In contrast, a direct demonstration of the role of SLC1A1 in human dicarboxylic aminoaciduria is lacking. The apical low-affinity transport system for acidic amino acids in kidney has been determined in brush-border membrane preparations (104), but its molecular entity remained elusive (36). At renal basolateral plasma membranes, a high-affinity Na+/K+-dependent transport system for acidic amino acids has been reported (75), but its molec-

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broad specificity for small and large zwitterionic amino acids with characteristics of system L (66). This suggests that 4F2hc-LAT2 may have a role in reabsorption of zwitterionic amino acids. Indeed, antisense experiments in the polarized opossum kidney cell line OK, derived from proximal convoluted epithelial cells, demonstrated a role of LAT2 (SLC7A8) in the transepithelial flux of cystine, and the basolateral efflux of cysteine and influx of alanine, serine, and threonine (intracellular concentration of cysteine increases, and that of alanine, serine, and threonine decreases in LAT2-antisense OK cells in polarized culture) (26). To our knowledge, no inherited human disease has yet been related to LAT2 mutations. A final demonstration of the role of LAT2 in reabsorption would only be possible after the generation of LAT2-knockout mouse models. There is evidence for phenotypic variants of Hartnup disorder, suggesting that 5p15.33 (SLC6A19) is not the only locus to affect or modify the phenotype (79, 92). Two of the families with Hartnup disorder apparently present no mutations for SLC6A19, and linkage to 5p15 has been excluded in one of them (45). This suggests that a defect in other amino acid transporters may result in Hartnup disorder. Several candidates are already available:

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REVIEWS ular structure has not been identified. Another member of the SLC1 family, GLT1 [i.e., the glial high-affinity glutamate transporter (69), also named EAAT2; SLC1A2] is expressed in rat kidney cortex and porcine small intestine (22, 105). To our knowledge, the expression of GLT1 protein has not been studied in kidney or intestine. Slc1a2-knockout mice show lethal spontaneous epileptic seizures (91). Reabsorption of acidic amino acids could be studied in these mice to ascertain the role of GLT1, although this is difficult because only 50% survive longer than 6 wk.  We thank Robin Rycroft for editorial help.

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23. Feliubadalo L, Arbones ML, Manas S, Chillaron J, Visa J, Rodes M, Rousaud F, Zorzano A, Palacin M, and Nunes V. Slc7a9-deficient mice develop cystinuria non-I and cystine urolithiasis. Hum Mol Genet 12: 2097–2108, 2003. 24. Feliubadalo L, Font M, Purroy J, Rousaud F, Estivill X, Nunes V, Golomb E, Centola M, Aksentijevich I, Kreiss Y, Goldman B, Pras M, Kastner DL, Pras E, Gasparini P, Bisceglia L, Beccia E, Gallucci M, de Sanctis L, Ponzone A, Rizzoni GF, Zelante L, Bassi MT, George AL Jr, and Palacin M. Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (b0,+AT) of rBAT. International Cystinuria Consortium. Nat Genet 23: 52–57, 1999. 25. Fernandez E, Carrascal M, Rousaud F, Abian J, Zorzano A, Palacin M, and Chillaron J. rBAT-b0,+AT heterodimer is the main apical reabsorption system for cystine in the kidney. Am J Physiol Renal Physiol 283: F540–F548, 2002. 26. Fernandez E, Torrents D, Chillaron J, Martin Del Rio R, Zorzano A, and Palacin M. Basolateral LAT-2 has a major role in the transepithelial flux of L-cystine in the renal proximal tubule cell line OK. J Am Soc Nephrol 14: 837–847, 2003.

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Studies in our laboratory referred to above were supported in part by the Spanish Ministry of Science and Technology (SAF2003-08940-01/02), the European Union (EUGINDAT; LSHM-CT-2003-502852), the Spanish Instituto de Salud Carlos III (networks G03/054, C03/07 and C03/08), the Generalitat de Catalunya (2001 SGR00399; 2001 SGR00118), and the Comissionat per a Universitats i Recerca (Generalitat de Catalunya). M. FontLlitjós was a recipient of a CIRIT fellowship. M. JiménezVidal was supported by BIOMED BMH4 C798-3154. L. Feliubadaló was supported by EUGINDAT.

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