Human LAT1, a Subunit of System L Amino Acid Transporter: Molecular Cloning and Transport Function

Prasad, Puttur D.; Wang, Haiping; Huang, Wei; Kekuda, Ramesh; Rajan, D. Prasanna; Leibach, Frederick H.; Ganapathy, Vadivel

doi:10.1006/bbrc.1999.0206

Cited by 223 publications

(168 citation statements)

References 18 publications

(21 reference statements)

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“…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. This is in full agreement with the expression of these transporters in the epithelial cells of the renal proximal tubule; LAT-1 is barely expressed in human and mouse kidney (38,39), asc-1 is not expressed in kidney (28), asc-2 is localized in collecting ducts in mouse kidney (29), and only LAT-2 is conspicuously expressed in the epithelial cells of the renal proximal tubule and the small intestine (21)(22)(23)40).…”

Section: Discussionsupporting

confidence: 83%

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: Discussionsupporting

confidence: 83%

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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“…7a), whereas LAT-1-mediated transport is reported not to be influenced by pH level (44). This stimulation of uptake is due to the increase in the affinity without changing the V max (Fig.…”

Section: Na ϩ -Independent Broad Scope Neutral Amino Acid Transportermentioning

confidence: 84%

Identification and Functional Characterization of a Na+-independent Neutral Amino Acid Transporter with Broad Substrate Selectivity

Segawa¹,

Fukasawa²,

Miyamoto³

et al. 1999

Journal of Biological Chemistry

455

435

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We have isolated a cDNA from rat small intestine that encodes a novel Na ؉ -independent neutral amino acid transporter with distinctive characteristics in substrate selectivity and transport property. The encoded protein, designated L-type amino acid transporter-2 (LAT-2), shows amino acid sequence similarity to the system L Na ؉ -independent neutral amino acid transporter LAT-1 (Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda 273, 32437-32445). LAT-2 is a nonglycosylated membrane protein. It requires 4F2 heavy chain, a type II membrane glycoprotein, for its functional expression in Xenopus oocytes. LAT-2-mediated transport is not dependent on Na؉ or Cl ؊ and is inhibited by a system L-specific inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), indicating that LAT-2 is a second isoform of the system L transporter. Compared with LAT-1, which prefers large neutral amino acids with branched or aromatic side chains, LAT-2 exhibits remarkably broad substrate selectivity. It transports all of the L-isomers of neutral ␣-amino acids. LAT-2 exhibits higher affinity (K m ‫؍‬ 30 -50 M) to Tyr, Phe, Trp, Thr, Asn, Ile, Cys, Ser, Leu, Val, and Gln and relatively lower affinity (K m ‫؍‬ 180 -300 M) to His, Ala, Met, and Gly. In addition, LAT-2 mediates facilitated diffusion of substrate amino acids, as distinct from LAT-1, which mediates amino acid exchange. LAT-2-mediated transport is increased by lowering the pH level, with peak activity at pH 6.25, because of the decrease in the K m value without changing the V max value. Because of these functional properties and a high level of expression of LAT-2 in the small intestine, kidney, placenta, and brain, it is suggested that the heterodimeric complex of LAT-2 and 4F2 heavy chain is involved in the trans-cellular transport of neutral amino acids in epithelia and blood-tissue barriers.

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“…It is ubiquitous and is essential in cellular nutrition and transcellular transport of neutral amino acids. Various subtypes with different substrate specificities and transport properties have been described for system L (30)(31)(32)(33)(34). For example, LAT1 prefers neutral amino acid with branched or aromatic side chains (31), whereas LAT2 has a broad substrate specificity toward all the L-isomers of neutral amino acids, including L-cysteine (32,33).…”

Section: Discussionmentioning

confidence: 99%

The mechanism of transmembraneS-nitrosothiol transport

Zhang

Hogg

2004

Proc. Natl. Acad. Sci. U.S.A.

164

191

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S-nitrosothiols have been suggested to play an important role in nitric oxide (NO)-mediated biological events. However, the mechanisms by which an S-nitrosothiol (or the S-nitroso functional group) is transferred across cell membrane are still poorly understood. We have demonstrated previously that the degradation of S-nitrosoglutathione (GSNO) by cells absolutely required the presence of cystine in the extracellular medium and proposed a mechanism that involved the reduction of cystine to cysteine, followed by the reaction of cysteine with GSNO to form S-nitrosocysteine (CysNO), mixed disulfides, and nitrosyl anion. In the present study we have assessed the effect of cystine on the transfer of the S-nitroso functional group from the extracellular to the intracellular space. Using RAW 264.7 cells, we found that the presence of L-cystine enhanced GSNO-dependent S-nitrosothiol uptake, increasing the intracellular S-nitrosothiol level from Ϸ60 pmol͞mg of protein to Ϸ3 nmol͞mg of protein. A lthough S-nitrosothiols have been thought to play an important role in many nitric oxide (NO)-mediated biological events, the processes by which S-nitrosothiol or their bound S-nitroso groups are transferred between the extracellular and intracellular spaces are still poorly understood. Transport mechanisms involving cell-surface protein disulfide isomerase (1, 2), ␥-glutamyl transpeptidase (3, 4), or anion exchanger (5) have been proposed.Previous studies have shown that some cellular effects of either S-nitrosoglutathione (GSNO) or S-nitroso-N-acetylpenicillamine (SNAP) depend on the presence of L-cysteine (6-9). It was suggested that cysteine can increase the efficiency of NO release from GSNO by the formation of unstable Snitrosocysteine (CysNO) through a transnitrosation reaction (9). Increases in cytosolic GSNO and cysteine levels were observed during the incubation of CysNO with human red blood cells (RBCs), implying the transport of CysNO into RBCs and the transnitrosation between CysNO and cytosolic glutathione (GSH) (10). Mallis and Thomas (8) indicated that a stereospecific transporter is not involved in the direct transfer of CysNO across the cell membrane, because the combination of GSNO with L-cysteine or D-cysteine gave a similar CysNO-like effect. However, other studies indicate that amino acid transporter system L (L-AT) is involved in the uptake of CysNO by cells (7,(11)(12)(13). 2-Aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), a specific L-AT inhibitor, and L-leucine, a substrate for L-AT, were shown to inhibit CysNO-stimulated cellular effects. L-leucine uptake in PC12 cells was inhibited by CysNO, not by SNAP, sodium nitroprusside, 3-morpholinosydnonimine (SIN-1)⅐HCl, (ϩ or Ϫ)-N-{(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl}-3-pyridinecarboximide (NOR-4), or sodium nitrite (12). Although these studies suggest the involvement of specific transport mechanisms for S-nitrosothiols, the change of intracellular S-nitrosothiol level was not examined. Consequently, the direct involvement of these pathways...

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Human LAT1, a Subunit of System L Amino Acid Transporter: Molecular Cloning and Transport Function

Cited by 223 publications

References 18 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

Identification and Functional Characterization of a Na+-independent Neutral Amino Acid Transporter with Broad Substrate Selectivity

The mechanism of transmembraneS-nitrosothiol transport

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