CLONING AND FUNCTIONAL-CHARACTERIZATION OF A RAT RENAL ORGANIC CATIONTRANSPORTER ISOFORM (ROCT1A)

Polyspecific organic cation transporters in the renal proximal tubule mediate the secretion of many clinically used drugs as well as endogen ous metabolites. Recently, two organic cation transporters (rOCT1 and rOCT2) were cloned from rat kidney. In this study, we report the cloni ng and functional expression of an rOCT1 isoform, rOCT1A, from rat kid ney. Genomic DNA cloning and sequencing demonstrated that rOCT1A is an alternatively spliced variant of rOCT1 with a deletion of 104 base pa irs near the 5'-end. The uptake of [C-14]tetraethylammonium (TEA) in o ocytes injected with the cRNA-encoding rOCT1A was increased 10-fold ov er that in water-injected oocytes (29 +/- 2.8 pmol/oocyte/h versus 1.8 +/- 0.13 pmol/oocyte/h, mean +/- S.E., p < 0.05). [C-14]TEA uptake in the cRNA-injected oocytes was saturable (K-m = 42 +/- 11 mu M) and wa s inhibited significantly by organic cations, including cimetidine and N-1-methylnicotinamide. The amino acid sequence was deduced from the cDNA after examination of all three reading frames. Two overlapping op en reading frames were found. Studies with synthetic constructs sugges t that a functional organic cation transporter is encoded by the large r open reading frame. The larger open reading frame encodes a 430-amin o acid protein (termed rOCT1A) that is 92% identical to rOCT1 and 57% identical to rOCT2. From hydropathy analysis, rOCT1A is predicted to h ave 10 transmembrane domains with both amino and carboxyl termini intr acellular. RNase protection assays demonstrate the presence of rOCT1A mRNA transcripts in rat kidney cortex, medulla, and intestine. These s tudies demonstrate the presence of a functional, alternatively spliced organic cation transporter (rOCT1A) in rat kidney.