EXPRESSION CLONING OF A RAT-LIVER NA-INDEPENDENT ORGANIC ANION TRANSPORTER()

Using expression cloning in Xenopus laevis oocytes, we have isolated a cDNA encoding a rat liver organic anion-transporting polypeptide (oat p). The cloned oatp mediated Na+-independent uptake of sulfobromophtha lein (BSP) which was Cl--dependent in the presence of bovine serum alb umin (BSA) at low BSP concentrations (e.g., 2 muM). Addition of increa sing amounts of BSA had no effects on the maximal velocity of initial BSP uptake, but it increased the K(m) value from 1.5 muM (no BSA) to 2 4 muM (BSA/BSP molar ratio, 3.7) and 35 muM (BSA/BSP ratio, 18.4). In addition to BSP, the cloned oatp also mediated Na+-independent uptake of conjugated (taurocholate) and unconjugated (cholate) bile acids. Se quence analysis of the cDNA revealed an open reading frame of 2010 nuc leotides coding for a protein of 670 amino acids (calculated molecular mass, 74 kDa) with four possible N-linked glycosylation sites and 10 putative transmembrane domains. Translation experiments in vitro indic ated that the transporter was indeed glycosylated and that its polypep tide backbone had an apparent molecular mass of 59 kDa. Northern blot analysis with the cloned probe revealed crossreactivity with several m RNA species from rat liver, kidney, brain, lung, skeletal muscle, and proximal colon as well as from liver tissues of mouse and rabbit, but not of skate (Raja erinacea) and human.