cDNA Cloning, Functional Characterization, and Tissue Distribution of an Alternatively Spliced Variant of Organic Cation Transporter hOCT2 Predominantly Expressed in the Human Kidney YUMIKO URAKAMI, MAIKO AKAZAWA, HIDEYUKI SAITO, MASAHIRO OKUDA, and KEN-ICHI INUI Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto, Japan. Abstract. A cDNA coding a novel organic cation transporter, hOCT2-A, was isolated from human kidney. The hOCT2-A cDNA is an alternatively spliced variant of hOCT2 with an insertion of 1169 bp. The open reading frame encodes a 483- amino acid protein that has 81% amino acid identity with hOCT2. From hydropathy analysis, hOCT2-A is predicted to have nine transmembrane domains. hOCT2-A mRNA is ex- pressed mainly in kidney and weakly in brain, liver, colon, skeletal muscle, bone marrow, spinal cord, testis, and placenta. When expressed in HEK293 cells, hOCT2-A stimulated the uptake of tetraethylammonium (TEA) in an electrogenic man- ner. The transport of TEA by hOCT2-A–transfected cells was saturable with the apparent Km value of 63 M. hOCT2-A stimulated the uptake of TEA, 1-methyl-4-phenylpyridinium, and cimetidine as well as did hOCT2. The uptake of guanidine and choline by hOCT2-transfected cells also increased mark- edly but not that by hOCT2-A–transfected cells. The uptake of TEA mediated by hOCT2-A but not by hOCT2 was inhibited significantly by organic cations such as procainamide, N- acetylprocainamide, and levofloxacin, indicating that hOCT2-A differs from hOCT2 in its affinity for several com- pounds. These findings suggested that hOCT2-A contributes to the renal clearance of endogenous and exogenous organic cations. Secretion is an important physiologic function for the mainte- nance of body fluid homeostasis and detoxification of drugs and xenobiotics. The proximal tubular cells play a principal role in limiting or preventing toxicity by actively secreting organic cations from the circulation into the urine (1– 4). Func- tional studies using isolated membrane vesicles (5–7) and cultured renal epithelial cells (8,9) suggested that renal tubular secretion of cationic substances is controlled by the concerted action of two distinct classes of organic cation transporters: one driven by the transmembrane potential difference in the baso- lateral membranes and the other driven by the transmembrane H + gradient in the brush border membranes. Several organic cation transporters of the OCT gene have been identified: OCT1 (10), OCT2 (11), OCT3 (12), OCTN1 (13), and OCTN2 (14,15). rOCT1A has been identified from rat kidney as a spliced variant of rat (r) OCT1 (16). We isolated the second member of the OCT family, rOCT2, in rat kidney, which has 67% amino acid identity to rOCT1 (11). rOCT1 is expressed abundantly in the liver and kidney (10), whereas rOCT2 is expressed predominantly in the kidney (11). Func- tional studies using Xenopus oocytes (10,11,17,18) and trans- fected mammalian cells (19 –21) suggested that rOCT1 and rOCT2 have multispecific (polyspecific) substrate specificity and translocate various structurally unrelated cationic com- pounds in an electrogenic manner. Immunohistochemical anal- ysis showed that both rOCT1 and rOCT2 proteins were local- ized in the basolateral membrane of renal tubular cells (22,23). Human (h) OCT1 and hOCT2 were isolated from the human liver and kidney, respectively (24). It is interesting that hOCT2 is expressed mainly in the kidney, whereas hOCT1 is tran- scribed mainly in the liver (24). A recent study by Pietig et al. (25) showed that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated by hOCT2, is electrogenetic. Recently, the spliced isoforms in some genes encoding renal transporters have been identified (26). In some cases, the spliced isoforms have been functional, resulting in a variety of physiologic consequences, including changes in the polariza- tion of isoforms, changes in pharmacologic or kinetic proper- ties, and changes in tissue expression or intrarenal localization (26). Because the renal organic cation transporters mediate secretion of various organic cations in the basolateral or brush border membranes, we hypothesized that several spliced iso- forms derived from the OCT genes would be expressed in the kidney. We report here the identification of a novel organic cation Received January 22, 2002. Accepted March 16, 2002. Correspondence to Dr. Ken-ichi Inui, Department of Pharmacy, Kyoto Uni- versity Hospital, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan; Phone: 81-75-751-3577; Fax: 81-75-751-4207; E-mail: inui@kuhp.kyoto-u.ac.jp 1046-6673/1307-1703 Journal of the American Society of Nephrology Copyright © 2002 by the American Society of Nephrology DOI: 10.1097/01.ASN.0000019413.78751.46 J Am Soc Nephrol 13: 1703–1710, 2002