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Cloning and functional characterization of a rat renal organic cation transporter isoform (rOCT1A)
L Zhang1, M J Dresser, J K Chun
1Department of Biopharmaceutical Sciences, University of California, San Francisco, California 94143, USA.
Abstract:
Polyspecific organic cation transporters in the renal proximal tubule mediate the secretion of many clinically used drugs as well as endogenous metabolites. Recently, two organic cation transporters (rOCT1 and rOCT2) were cloned from rat kidney. In this study, we report the cloning and functional expression of an rOCT1 isoform, rOCT1A, from rat kidney. Genomic DNA cloning and sequencing demonstrated that rOCT1A is an alternatively spliced variant of rOCT1 with a deletion of 104 base pairs near the 5'-end. The uptake of [14C]tetraethylammonium (TEA) in oocytes injected with the cRNA-encoding rOCT1A was increased 16-fold over 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). [14C]TEA uptake in the cRNA-injected oocytes was saturable (Km = 42 +/- 11 microM) and was inhibited significantly by organic cations, including cimetidine and N1-methylnicotinamide. The amino acid sequence was deduced from the cDNA after examination of all three reading frames. Two overlapping open reading frames were found. Studies with synthetic constructs suggest that a functional organic cation transporter is encoded by the larger open reading frame. The larger open reading frame encodes a 430-amino acid protein (termed rOCT1A) that is 92% identical to rOCT1 and 57% identical to rOCT2. From hydropathy analysis, rOCT1A is predicted to have 10 transmembrane domains with both amino and carboxyl termini intracellular. RNase protection assays demonstrate the presence of rOCT1A mRNA transcripts in rat kidney cortex, medulla, and intestine. These studies demonstrate the presence of a functional, alternatively spliced organic cation transporter (rOCT1A) in rat kidney.
Insights
Researchers identified a new organic cation transporter, rOCT1A, in rat kidneys. This alternatively spliced variant of rOCT1 plays a role in transporting organic cations, impacting drug and metabolite secretion.
Area of Science:
- Pharmacology
- Molecular Biology
- Renal Physiology
Background:
- Polyspecific organic cation transporters are crucial for renal proximal tubule secretion of drugs and endogenous metabolites.
- Two organic cation transporters, rOCT1 and rOCT2, have been previously cloned from rat kidney.
Purpose of the Study:
- To clone and functionally characterize a novel isoform of the rat organic cation transporter 1 (rOCT1) from rat kidney.
- To investigate the molecular and functional properties of this rOCT1 isoform, designated rOCT1A.
Main Methods:
- Genomic DNA cloning and sequencing to identify alternatively spliced variants.
- Functional expression in Xenopus oocytes to study substrate uptake.
- Kinetic analysis and inhibition studies with known organic cations.
- Sequence analysis, hydropathy prediction, and mRNA expression analysis (RNase protection assays).
Main Results:
- An alternatively spliced variant, rOCT1A, was identified, differing from rOCT1 by a 104-base pair deletion.
- rOCT1A demonstrated significant [14C]tetraethylammonium ([14C]TEA) uptake in oocytes, 16-fold higher than controls.
- Uptake was saturable (Km = 42 microM) and inhibited by organic cations like cimetidine.
- rOCT1A encodes a 430-amino acid protein, 92% identical to rOCT1, predicted to have 10 transmembrane domains.
- rOCT1A mRNA was detected in rat kidney cortex, medulla, and intestine.
Conclusions:
- A functional, alternatively spliced organic cation transporter, rOCT1A, is present in the rat kidney.
- rOCT1A contributes to the polyspecific transport of organic cations in renal and intestinal tissues.
- This finding expands the understanding of organic cation transporter diversity and their role in drug and metabolite handling.