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

Abstract

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...

Neutral amino acid transport mediated by ortholog of imino acid transporter SIT1/SLC6A20 in opossum kidney cells

“…1) and normalized to the uptake measured in control monolayers. The data represent 3 independent experiments (means Ϯ SE, n ϭ 9), **P Ͻ 0.01. exchangers b 0,ϩ AT-rBAT and LAT2-4F2hc (7). They were also shown earlier to display an apical Na ϩ -dependent, electrogenic transport of some neutral L-amino acids (L-Phe, L-Ala, L-Val, and L-Gln) (21).…”

CATs and HATs: the SLC7 family of amino acid transporters

“…Interestingly, it is a quite efficient exporter of l-cysteine, an amino acid that is imported into proximal kidney tubule cells mainly via the apical b 0,+ AT-rBAT transporter (defective in cystinuria) as l-cystine [39]. The role of LAT2-4F2hc in the basolateral efflux of l-cysteine has been substantiated recently in cell culture models [3,21].…”

Kidney amino acid transport

“…Furthermore, it apparently displays similarly high levels of mRNA and protein expression. As yet, no genetic condition with impaired LAT2 function has been identified, but knock down experiments of LAT2 performed in the proximal tubule OK cell line model have shown a reduction of transepithelial L-cysteine transport [22].…”