Mutation of amino acid 475 of rat organic cation transporter 2 (rOCT2) impairs organic cation transport

Protein sequence alignments revealed one amino acid position, where organic cation transporters (OCTs, aspartate (D) at position 475 of rOCT2) and organic anion transporters (OATs, arginine (R) at position 466 of rOAT1) are charged oppositely. To address the impact of this amino acid for protein function we cloned rat organic cation transporter 2 (rOCT2), the renal electrogenic cation transporter of the basolateral side of proximal tubule cells. Site-directed mutagenesis was used to generate rOCT2-D475R (rOCT2-mut). Heterologous expression of rOCT2 wild-type (rOCT2-wt) in A6 cells resulted in a significant uptake of the fluorescent organic cation 4-(4-dimethylaminostyryl)-Nmethylpyridinium (ASP(+)). Accordingly, rOCT2-wt-transfected COS 7 cells showed an almost fourfold uptake of 25 muM [C-14]-TEA, whereas rOCT2-mut did not exhibit any uptake of [C-14] -TEA. These data indicate that rOCT2 transports both ASP(+) and TEA and that aspartate at position 475 of rOCT2 plays a critical role in transport function. (C) 2003 Elsevier SAS. All rights reserved.