Arginine 454 and lysine 370 are essential for the anion specificity of theorganic anion transporter, rOAT3

Organic anion transporters (OATs) and organic cation transporters (OCTs) me diate the flux of xenobiotics across the plasma membranes of epithelia. Sub strates of OATs generally carry negative charge(s) whereas substrates of OC Ts are cations. The goal of this study was to determine the domains and ami no acid residues essential for recognition and transport of organic anions by the rat organic anion transporter, rOAT3. An rOAT3/rOCT1 chimera contain ing transmembrane domains 1-5 of rOAT3 and 6-12 of rOCT 1 retained the spec ificity of rOCT1, suggesting that residues involved in substrate recognitio n reside within the carboxyl-terminal half of these transporters. Mutagenes is of a conserved basic amino acid residue, arginine 454 to aspartic acid ( R454D), revealed that this amino acid is required for organic anion transpo rt. The uptakes of p-aminohippurate (PAH), estrone sulfate, and ochratoxin A were similar to 10-, similar to 48-, and similar to 32-fold enhanced in o ocytes expressing rOAT3 and were only similar to2-, similar to6-, and simil ar to5-fold enhanced for R454D. Similarly, mutagenesis of the conserved lys ine 370 to alanine (K370A) suggested that K370 is important for organic ani on transport. Interestingly, the charge specificity of the double mutant, R 454DK370A, was reversed in comparison to rOAT3-R454DK370A preferentially tr ansported the organic cation, MPP+, in comparison to PAH (MPP+ uptake/PAH u ptake = 3.21 for the double mutant vs 0.037 for rOAT3). These data indicate that arginine 454 and lysine 370 are essential for the anion specificity o f rOAT3. The studies provide the first insights into the molecular determin ants that are critical for recognition and translocation of organic anions by a member of the organic anion transporter family.