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

Abstract

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.

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

“…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).…”

“…The following criteria were used to detect LAT-2 activity: (1) L-alanine is transported via LAT-2, asc-1, and asc-2 (22,23,28,29), but it is not transported via LAT-1 (30,31); (2) transport of L-alanine via LAT-2, but not via asc-1 and asc-2, is inhibited by BCH and L-tyrosine (23) To check the amino acid exchanger activity of the LAT-2-related L-system (21,22,24), polarized cells were loaded on the basolateral and apical membranes with the radioactive substrate (L-[ 3 H] leucine or L-[ 3 H] alanine), and the efflux across the basolateral membrane was measured. The efflux of Lleucine or L-alanine trans-stimulated by LAT-2 substrates was mostly sodium independent.…”

“…LAT-2 is expressed in the basolateral plasma membrane of the renal epithelial cells of the upper part of the proximal tubule (S1 and S2 segments) (21), where most of amino acid reabsorption occurs (1). LAT-2/4F2hc co-expressed in Xenopus oocytes show exchange of neutral amino acids of any size (21)(22)(23)(24). These characteristics suggest that LAT-2 may play a role in renal reabsorption, but which amino acids use this transporter for net efflux or influx in the context of a renal epithelial cell is unknown.…”

Fetal growth restriction: a workshop report

“…If these isoforms have different properties, altered isoform ratios or distribution may have significant effects on activity in vivo. For example the system L amino acids transporter has two isoforms; the first, LAT-1, is an exchange transporter [17], while there are reports that the second, LAT-2, is a uniporter [18]. Changes in the isoform ratio might have significant effects on transport without apparent changes in overall transporter expression.…”

Regulation of the plasma amino acid profile by leucine via the system L amino acid transporter

“…[21][22][23][24] Although all of the isoforms are strongly inhibited by BCH, substrate specificity differs slightly among isoforms; all LATs have a high affinity for BCAAs, methionine, and phenylalanine compared to other amino acids, but only LAT1 and LAT2 show high affinity for tyrosine. 21,22) The Km values for these amino acids are lower in LAT1 than in LAT2, but the tissue distribution of expression is wider in LAT2 than in LAT1. In this study, leucine administration affected the plasma concentrations of BCAAs, methionine, phenylalanine, and tyrosine, suggesting that LAT1 and LAT2 may be the main modulators responsible for the effects of leucine.…”