EXPRESSION AND CHARACTERIZATION OF A FUNCTIONAL-RAT LIVER NA+ BILE-ACID COTRANSPORT SYSTEM IN COS-7 CELLS

Expression and characterization of a functional rat liver Na+ bile acid cotransport system in COS-7 cell. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G382-G387, 1994. - A cDNA for the rat liver sodium dependent bile acid cotransporter was expressed in COS-7 cells to study the functional properties of the translated protein in a mammalian cell line. A 1.2-kb insert was ligated into a pMAMneo vector and transiently transfected using electroporation. After optimal conditions were established, the transiently transfected COS cells were screened with fluorescent-conjugated labeled bile acids for evidence of expression of the cotransporter after 48 h. The uptake of [H-3]taurocholate ([H-3]TC) was then determined in cells transfected with or without the bile acid insert. Progressive uptake of [H-3]TC (0.45 mu M) was observed for 30 min in the presence of sodium. In contrast, no uptake of [H-3]TC was observed in the absence of sodium, in nontransfected COS cells, or in COS cells transfected with the empty plasmid. Kinetic studies revealed a Michaelis constant (K-m) of 29 mu M, essentially identical to the K-m of this cotransporter described in intact rat hepatocyes and membrane vesicles. Uptake of [H-3]TC (5.0 mu M) at 5 min (n = 3-6) was inhibited by 100 mu M taurochenodeoxycholic acid (81%), tauroursodeoxycholic acid (77%), cholic acid (55%), chenodeoxycholic acid (74%), and ursodeoxycholic acid (56%) but not by 100 mu M taurodehydrocholate, 1 mM probenecid, or 100 mu M bilirubin. In contrast, bumetanide (500 mu M) inhibited [H-3]TC uptake by 52%. These studies indicate that the isolated cDNA codes for a physiological bile acid transporter present in rat hepatocytes and that posttranslational factors present in mammalian cells may not be as important in defining properties of this cotransport system.