Redox imbalance in cystine/glutamate transporter-deficient mice.

Cystine/glutamate transporter, designated as system x(-)(c), mediates cystine entry in exchange for intracellular glutamate in mammalian cells. This transporter consists of two protein components, xCT and 4F2 heavy chain, and the former is predicted to mediate the transport activity. This transporter plays a pivotal role for maintaining the intracellular GSH levels and extracellular cystine/cysteine redox balance in cultured cells. To clarify the physiological roles of this transporter in vivo, we generated and characterized mice lacking xCT. The xCT(-/-) mice were healthy in appearance and fertile. However, cystine concentration in plasma was significantly higher in these mice, compared with that in the littermate xCT(-/-) mice, while there was no significant difference in plasma cysteine concentration. Plasma GSH level in xCT(-/-) mice was lower than that in the xCT(-/-) mice. The embryonic fibroblasts derived from xCT(-/-) mice failed to survive in routine culture medium, and 2-mercaptoethanol was required for survival and growth. When 2-mercaptoethanol was removed from the culture medium, cysteine and GSH in these cells dramatically decreased, and cells started to die within 24 h. N-Acetyl cysteine also rescued xCT(-/-)-derived cells and permitted growth. These results demonstrate that system x(-)(c) contributes to maintaining the plasma redox balance in vivo but is dispensable in mammalian development, although it is vitally important to cells in vitro.

Expression of the activity of cystine/glutamate exchange transporter, system x(c)(-), by xCT and rBAT.

The expression of the activity of cystine/glutamate exchange transporter, designated system x(c)(-), requires two components, xCT and 4F2 heavy chain (4F2hc) in Xenopus oocytes. rBAT (related to b(0,+) amino acid transporter) has a significant homology to 4F2hc and is known to be located in the apical membrane of epithelial cells. To determine whether xCT can associate with rBAT and express the activity of system x(c)(-), xCT, and rBAT were co-expressed in Xenopus oocytes and in mammalian cultured cells. In the oocytes injected with rBAT cRNA alone, the activities of cystine and arginine transport were induced, indicating that the system b(0,+)-like transporter was expressed by associating the exogenous rBAT with an endogenous b(0,+)AT-like factor as reported previously. In the oocytes injected with xCT and rBAT cRNAs, the activity of cystine transport was further induced. This induced activity of cystine transport was partially inhibited by glutamate or arginine and completely inhibited by adding both amino acids. In these oocytes, the activity of glutamate transport was also induced and it was strongly inhibited by cystine. In NIH3T3 cells transfected with xCT cDNA alone, the activity of cystine transport was significantly increased, and in the cells transfected with both xCT and rBAT cDNAs, the activity of cystine transport was further enhanced. The enhanced activity was Na(+)-independent and was inhibited by glutamate and homocysteate. These results indicate that rBAT can replace 4F2hc in the expression of the activity of system x(c)(-) and suggest that system x(c)(-) activity could be expressed in the apical membrane of epithelial cells.