The rBAT gene is responsible for L-cystine uptake via the b0,(+)-like amino acid transport system in a "renal proximal tubular" cell line (OK cells)

Abstract

Several studies have shown that the cRNA of human, rabbit, or rat rBAT induces in Xenopus oocytes sodium-independent, high affinity uptake of L-cystine via a system b0,(+)-like amino acid exchanger. We have shown that mutations in rBAT cause type I cystinuria (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-425; Calonge, M. J., Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P., Nunes, V., and Palacín, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9667-9671). Apart from oocytes, no other expression system has been used for transfection of functional rBAT activity. Furthermore, the b0,(+)-like transport activity has not been clearly described in the kidney or intestine. Here, we report that a "proximal tubular-like" cell line derived from opossum kidney (OK cells) expresses an rBAT transcript. Poly(A)+ RNA from OK cells induced by system b0,(+)-like transport activity in oocytes. This was hybrid-depleted by human rBAT antisense oligonucleotides. A polymerase chain reaction-amplified cDNA fragment (approximately 700 base pairs) from OK cell RNA corresponds to an rBAT protein fragment 65-69% identical to those from human, rabbit and rat kidneys. We have also examined transport of l-cystine in OK cells and found characteristics very similar to the amino acid exchanger activity induced by rBAT cRNA in oocytes. Uptake of L-cystine was of high affinity, sodium-independent and shared with L-arginine and L-leucine. It was trans-stimulated by amino acids with the same specificity as rBAT-induced transport activity in oocytes. Furthermore, it was localized to the apical pole of confluent OK cells. To demonstrate that the rBAT protein is functionally related to this transport activity, we have transfected OK cells with human rBAT antisense and sense sequences. Transfection with rBAT antisense, but not with rBAT sense, resulted in the specific reduction of rBAT mRNA expression and b0,(+)-like transport activity. These results demonstrate that rBAT is functionally related to the L-cystine uptake via system b0,(+)-like in the apical pole of the renal OK cell line.