The rBAT Gene Is Responsible for L-Cystine Uptake via the bo,+-like Amino Acid Transport System in a “Renal Proximal Tubular” Cell Line (OK Cells)

Mora, Conchi; Chillarón, Josep; Calonge, Marı́a Julia; Forgo, Judith; Testar, Xavier; Nunes, Virginia; Murer, Heini; Zorzano, António; Palacı́n, Manuel

doi:10.1074/jbc.271.18.10569

Cited by 51 publications

(43 citation statements)

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“…Polarized OK cells express the complete set of amino acid transporters known to be involved in the vectorial trans-epithelial flux of the amino acids hyperexcreted in cystinuria (Fig 7). OK cells express the rBAT-related system b o,ϩ exchanger activity in the apical pole of the cell (18), and as shown here, the basolateral system L isoform LAT-2/4F2hc and the basolateral system y ϩ L. System y ϩ L most probably represents the y ϩ LAT-1/ 4F2hc transporter and not the y ϩ LAT-2/4F2hc isoform be- cause the former is expressed in OK cells (present study) and the latter is only weakly expressed in kidney (37). Moreover, among the heteromeric amino acid transporters that mediate exchange of neutral amino acids, only LAT-2 transport activity, but not LAT-1, asc-1 and asc-2, is expressed in the basolateral domain of OK cells.…”

Section: Discussionmentioning

confidence: 99%

“…Cells were grown in Dulbecco modified Eagle medium/ Nutrition Mix F-12 medium supplemented with 10% fetal calf serum, as described (18). Subcultures were prepared by trypsinization and reseeding at high density (approximately 1 ϫ 10 6 cells/ml).…”

Section: Cell Culturementioning

confidence: 99%

“…The stably transfected clones were selected as described (18). Selection of clones with a lower expression of the oLAT-2 transcript was performed by Northern blot analysis.…”

Section: Olat-2 Constructs and Stable Transfection Of Ok Cellsmentioning

confidence: 99%

“…Indeed, OK cells express rBAT-associated system b o,ϩ transport activity in the apical pole (18). In these cells, like it is believed to occur during renal reabsorption (1), taken-up cystine is reduced to cysteine for efflux (19,20).…”

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Basolateral LAT-2 Has a Major Role in the Transepithelial Flux of L-Cystine in the Renal Proximal Tubule Cell Line OK

Fernández¹,

Torrents²,

Chillarón³

et al. 2003

Journal of the American Society of Nephrology

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Abstract. During renal reabsorption, the amino acid transporters b o,ϩ and y ϩ L have a major role in the apical uptake of cystine and dibasic amino acids and in the basolateral efflux of dibasic amino acids, respectively. In contrast, the transporters responsible for the basolateral efflux of the apically transported cystine are unknown. This study shows the expression of system L and y ϩ L transport activities in the basolateral domain of the proximal tubule-derived cell line OK and the cloning of the corresponding LAT-2 and y ϩ LAT-1 cDNAs. Stable transfection with a LAT-2 antisense sequence demonstrated the specific role of LAT-2 in the basolateral system L amino acid exchange activity in OK cells. This partial reduction of LAT-2 expression decreased apical-to-basolateral trans-epithelial flux of cystine and resulted in a twofold to threefold increase in the intracellular content of cysteine. In contrast, the content of serine, threonine, and alanine showed a tendency to decrease, whereas other LAT-2 substrates were not affected. This demonstrates that LAT-2 plays a major specific role in the net basolateral efflux of cysteine and points to LAT-2 as a candidate gene to modulate cystine reabsorption.In the kidney, most glomerulus-filtered amino acids are reabsorbed in the early proximal tubule (1). The amino acids are taken up through the brush border membrane and exit the epithelial cells through the basolateral membrane to the interstitial space. Two heteromeric amino acid transport exchangers (systems b o,ϩ and y ϩ L) have a major role in the reabsorption of cystine and dibasic amino acids (2-4). First, the heterodimer formed by rBAT and b o,ϩ AT is the amino acid transporter b o,ϩ , which mediates high-affinity uptake of cystine and dibasic amino acids coupled with the efflux of neutral amino acids (5-8) at the apical membrane of epithelial cells of the proximal tubule (9). Indeed, mutations in the rBAT (SLC3A1) gene cause type I cystinuria, and mutations in the b o,ϩ AT (SLC7A9) gene cause mainly non-type I and also type I cystinuria (recessive inherited aminoacidurias of cystine and dibasic amino acids) (6,10,11). Second, y ϩ LAT-1 dimerizes with 4F2hc to form the amino acid transporter y ϩ L, which mediates the efflux of cationic amino acids coupled with the influx of neutral amino acids plus sodium (12-14). Mutations in y ϩ LAT-1 (SLC7A7) cause lysinuric protein intolerance (15,16), an inherited aminoaciduria due to defective dibasic amino acid efflux from the basolateral membrane of proximal tubule epithelial cells (17). Thus, systems b o,ϩ and y ϩ L explain the trans-epithelial transport of dibasic amino acids. In contrast, the amino acid transporters that play a major role in the basolateral efflux of the apically taken-up cystine remain to be identified.Polarized OK cells, a proximal tubule-derived cell line from the American opossum, has been extensively used as a model for transport studies in renal epithelial cells. Indeed, OK cells express rBAT-associated system b o,ϩ transport activity in the...

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Section: Discussionmentioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

“…The stably transfected clones were selected as described (18). Selection of clones with a lower expression of the oLAT-2 transcript was performed by Northern blot analysis.…”

Section: Olat-2 Constructs and Stable Transfection Of Ok Cellsmentioning

confidence: 99%

mentioning

confidence: 99%

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Basolateral LAT-2 Has a Major Role in the Transepithelial Flux of L-Cystine in the Renal Proximal Tubule Cell Line OK

Fernández¹,

Torrents²,

Chillarón³

et al. 2003

Journal of the American Society of Nephrology

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“…Mis-sense mutations of human BAT have been demonstrated in cystinuria chromosomes [21][22][23][24][25], and expression of the most common mutation found in cystinuria patients [Met-467 changed to threonine (Met467Thr)] decreases transport activity in Xenopus oocytes by 90 % [21]. Transfection of cultured kidney proximal-tubular (OK) cells with BAT antisense leads to depletion of system b o,+ transport activity [26].…”

Section: Introductionmentioning

confidence: 99%

Cloning and functional expression of a cDNA from rat jejunal epithelium encoding a protein (4F2hc) with system y+L amino acid transport activity

Yao

Muzyka

Elliott

et al. 1998

Biochemical Journal

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Two different protein families, designated CAT (cationic amino acid transporter) and BAT (broad-specificity amino acid transporter) mediate the plasma membrane transport of cationic amino acids in animal cells. CAT transporters have 12-14 transmembrane domains and are selective for cationic amino acids. BAT proteins, in contrast, have one to four transmembrane domains and induce the transport of both cationic and zwitterionic amino acids when expressed in Xenopus oocytes. Mutations in the human BAT gene cause type I cystinuria, a disease affecting the ability of intestinal and renal brush border membranes to transport cationic amino acids and cystine. We have used functional expression cloning in oocytes to isolate a BAT-related cDNA from rat jejunal epithelium. The cDNA encodes the rat 4F2 heavy chain (4F2hc) cell-surface antigen, a 527-residue (60 kDa) protein that is 26% identical in amino acid sequence with rat renal BAT (also known as NBAT/D2). Expression of rat jejunal 4F2hc in oocytes induced the lysine-inhibitable Na+-dependent influx of leucine and the leucine-inhibitable Na+-independent influx of lysine. Lysine efflux was stimulated by extracellular (Na+ plus leucine). These characteristics identify the expressed amino acid transport activity as system y+L, a transporter that has been implicated in basal membrane transport of cationic amino acids in intestine, kidney and placenta.

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Linkage of type II and type III cystinuria to 19q13.1: Codominant inheritance of two cystinuric alleles at 19q13.1 produces an extreme stone-forming phenotype

Stoller

Bruce

et al. 1999

Am. J. Med. Genet.

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Cystinuria, a renal tubule disease affecting urinary cystine excretion with or without kidney stone formation, previously was mapped to chromosome region 2p.21. Mutations in the gene SLC3A1 or NBAT, the reported candidate gene for cystinuria at 2p.21, have been demonstrated in individuals with the autosomal recessive Type I cystinuria phenotype. Recently, the Type III cystinuria phenotype was mapped to chromosome region 19q13.1. Here we report a kindred of 39 persons in two families of cystinurics, Types II and III, that support linkage to 19q13.1 and exclude 2p.21. Based on a dominant model of inheritance, two-point analysis of the entire pedigree produced a maximum lod score (Z(max)) of 3.82 at marker D19S425. Multipoint analysis yielded a lod score of 4.96 at this marker, and a resultant lod score of 5.90 using a codominant model of inheritance. Furthermore, a candidate gene interval of 8.9 cM, flanked by markers D19S225 and D19S223, was obtained using multipoint and haplotype analyses. Thus, this kindred demonstrates the linkage of Type II cystinuria to 19q13.1 and confirms the linkage of Type III cystinuria at 19q13.1 while excluding the marker D19S225 that was previously included in the critical interval.

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The rBAT Gene Is Responsible for L-Cystine Uptake via the bo,+-like Amino Acid Transport System in a “Renal Proximal Tubular” Cell Line (OK Cells)

Cited by 51 publications

References 44 publications

Basolateral LAT-2 Has a Major Role in the Transepithelial Flux of L-Cystine in the Renal Proximal Tubule Cell Line OK

Basolateral LAT-2 Has a Major Role in the Transepithelial Flux of L-Cystine in the Renal Proximal Tubule Cell Line OK

Cloning and functional expression of a cDNA from rat jejunal epithelium encoding a protein (4F2hc) with system y+L amino acid transport activity

Linkage of type II and type III cystinuria to 19q13.1: Codominant inheritance of two cystinuric alleles at 19q13.1 produces an extreme stone-forming phenotype

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