Amino acid transporters: molecular structure and physiological roles

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amino acid transporter associated with the cystinuria-related type. COS-7 cells with rBAT but not with 4F2hc, BAT1. II membrane glycoprotein. J Biol Chem 1999; ...

Nephrol Dial Transplant (2000) 15 [Suppl 6 ]: 9–10

Nephrology Dialysis Transplantation

Amino acid transporters: molecular structure and physiological roles Yoshikatsu Kanai1,2, Hiroko Segawa1, Arthit Chairoungdua1, Ju Young Kim1, Do Kyung Kim1, Hirotaka Matsuo1, Seok Ho Cha1 and Hitoshi Endou1 1Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, and 2PRESTO, Japan Science and Technology Corporation, Japan

Amino acid reabsorption at the renal proximal tubules is mediated by the specialized amino acid transport systems situated on the apical membrane and basolateral membrane of the epithelial cells ( Figure 1). Acidic amino acids are reabsorbed by the apical membrane, Na+-dependent transport system X− glutamate A,G transporter. Neutral amino acids enter the epithelial cells via Na+-dependent transport system B0 and leave the epithelial cells via Na+-independent system L. Cystine and basic amino acids are reabsorbed via the apical membrane amino acid exchanger system b0,+. Basic amino acids leave the cells via basolateral membrane exchanger system y+L. It was demonstrated that glutamate transporter EAAC1, identified in 1992, corresponds to the apical membrane acidic amino acid transport system X− [1]. A,G The discovery of a family of amino acid transporters which are associated with single membrane-spanning type II membrane glycoproteins has brought about a breakthrough in the molecular cloning of amino acid transporters. 4F2 antigen (CD98), which originally was identified as a lymphocyte activation antigen, is composed of two subunits, a heavy chain type II membrane glycoprotein and a non-glycosylated light

chain, which are linked with each other via a disulfide bond. Now it is clear that the 4F2 light chain is an amino acid transporter [2]. Six transporters have been identified as 4F2 light chains [3]. In addition, a transporter has been identified which is structurally related to 4F2 heavy chain (4F2hc)-associated transporters and couples to the other type II membrane glycoprotein rBAT (related to b0,+ amino acid transporter), thereby establishing a family of amino acid transporters associated with type II membrane glycoproteins (LAT family) [4]. In renal proximal tubules, 4F2hc is localized on the basolateral membrane whereas rBAT exists on the apical membrane. It has been proposed that 4F2hc and rBAT carry the associated transporters to the basolateral membrane and the apical membrane, respectively. We performed expression cloning by co-expression of 4F2hc and a C6 glioma cell cDNA library and isolated a cDNA which encodes a Na+-independent neutral amino acid transporter, designated LAT1 (L-type amino acid transporter 1) [2]. For functional expression in Xenopus laevis oocytes, LAT1 required 4F2hc. LAT1 transported neutral amino acids with branched or aromatic side chains such as -Leu, -Ile,

Fig. 1. Transepithelial transport of amino acids in the renal proximal tubules. Correspondence and offprint requests to: Dr Yoshikatsu Kanai, Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan.

-Phe, -Met, -Tyr, -His, -Trp and -Val with K m values ~20 mM, and did not accept basic amino acids or acidic amino acids. The transport via LAT1 was Na+-independent and inhibited by a system L-specific

© 2000 European Renal Association–European Dialysis and Transplant Association

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inhibitor 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH ). These functional properties corresponded to those of the classically characterized amino acid transport system L. In in vitro translation, LAT1 was shown to be a non-glycosylated membrane protein consistent with the property of 4F2 light chain. In western blots, LAT1 was shown to be associated with 4F2hc via disulfide bonds. The expression of LAT1 was, however, detected less in kidney. Because 4F2hc is ubiquitously expressed, it is proposed that the structurally related transporters are associated with 4F2hc in kidney. We searched EST (expressed sequence tag) databases to identify several novel sequences related to LAT1. By cloning fulllength cDNAs and expressing them in Xenopus oocytes, we demonstrated that one of them encodes a Na+independent broad-scope neutral amino acid transporter designated as LAT2 (L-type amino acid transporter 2) [5]. LAT2 required 4F2hc for its functional expression. LAT2-mediated transport was not dependent on Na+ or Cl− and was inhibited by a system L-specific inhibitor BCH, indicating that LAT2 is a second system L transporter. Compared with LAT1 which prefers large neutral amino acids with branched or aromatic side chains, LAT2 exhibited remarkably broad substrate selectivity. It transported all the isomers of neutral a-amino acids with K values m between 30 and 300 mM. Because of these functional properties and the high level of expression of LAT2 in kidney, it is suggested that the heterodimeric complex of LAT2 and 4F2hc plays a major role in the transcellular transport of neutral amino acids at proximal tubules. In fact, LAT2 immunoreactivity was shown to be localized in the basolateral membrane of proximal tubules in kidney (Figure 1). One of the LAT1-related sequences found in EST databases was proved to be an amino acid transporter associated with rBAT, not with 4F2hc [4]. The cDNA which we isolated from rat kidney encoded a protein designated as BAT1 (b0,+-type amino acid transporter 1) which is structurally related to the system L amino acid transporters LAT1 and LAT2 (43% identity). In the non-reducing condition, a 125 kDa band was detected in rat kidney by anti-BAT1 antibody which seems to correspond to the heterodimeric complex of BAT1 and rBAT. The band was shifted to 41 kDa in the reducing condition, suggesting that BAT1 and rBAT are linked via a disulfide bond to form a heterodimeric complex. The BAT1 and rBAT proteins were shown to be co-localized on the apical membrane of renal proximal tubules where massive cystine transport has been proposed (Figure 1). When expressed in COS-7 cells with rBAT but not with 4F2hc, BAT1 exhibited the Na+-independent transport of cystine, basic and neutral amino acids with the properties of system b0,+. The affinity for cystine and basic amino acids was 200–500 mM, corresponding to the highaffinity cystine transport system in renal proximal tubules. The finding of BAT1 together with the 4F2hcassociated transporters established a family of amino

Y. Kanai et al.

acid transporters (LAT family) which associate with type II membrane glycoproteins. Although the LAT family is basically a family of Na+-independent transporters, it contains system y+L transporters which exhibit peculiar Na+ dependence [6 ]. When expressed with 4F2hc in Xenopus oocytes, y+LAT1 is Na+ independent for basic amino acids, whereas it is partially dependent on Na+ for neutral amino acids. This is because y+LAT1 requires a positive charge (a positive charge of basic amino acid side chains, or Na+, H+ or Li+ for neutral amino acids) for substrate binding [6 ]. This Na+ dependence is quite beneficial for y+LAT1, which mediates an obligatory exchange of basic and neutral amino acids, to be an exit path for basic amino acids at the basolateral membrane of epithelia ( Figure 1). Because of low Na+ concentration inside the cells, neutral amino acids are not accepted efficiently by the intracellular substratebinding site of y+LAT1, so that basic amino acids are accepted preferentially to be transported effectively out of the cells to the blood stream by the obligatory exchange [6 ]. This is a mechanism through which basic amino acids are transported against an electrical gradient through the plasma membrane. The finding of the LAT family has thus rapidly advanced the understanding of the molecular basis of epithelial transport of amino acids in proximal tubules. The amino acid transport systems L, y+L and b0,+ which play major roles in amino acid reabsorption at proximal tubules have turned out to belong to this family. In this family, two factors determine whether transporters are carried to the apical or the basolateral membrane of proximal tubules: (i) specific association between transporters and type membrane glycoproteins; and (ii) the mechanisms which determine the intracellular fate of the type II membrane glycoproteins. It will be a critical issue to identify their molecular basis.

References 1. Shayakul C, Kanai Y, Lee W-S, Brown D, Hediger MA. Localization of the high affinity glutamate transporter EAAC1 in rat kidney. Am J Physiol 1997; 273: F1023–F1029 2. Kanai Y, Segawa H, Miyamoto K, Uchino H, Takeda E, Endou H. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J Biol Chem 1998; 273: 23629–23632 3. Fukasawa Y, Segawa H, Kim JY et al. Identification and characterization of a Na+-independent neutral amino acid transporter that associates with the 4F2 heavy chain and exhibits substrate selectivity for small - and -amino acids. J Biol Chem 2000; 275: 9690–9698 4. Chairoungdua A, Segawa H, Kim JY et al. Identification of an amino acid transporter associated with the cystinuria-related type II membrane glycoprotein. J Biol Chem 1999; 274: 28845–28848 5. Segawa H, Fukasawa Y, Miyamoto K, Takeda E, Endou H, Kanai Y. Identification and functional characterization of a Na+independent neutral amino acid transporter with broad substrate selectivity. J Biol Chem 1999; 274: 19745–19751 6. Kanai Y, Fukasawa Y, Cha SH et al. Transport properties of a system y+L neutral and basic amino acid transporter: Insights into the mechanisms of substrate recognition. J Biol Chem 2000; 275: 20787–20793

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