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.
Highlights
Polyspecific organic cation transporters in the renal proximal tubule mediate the secretion of many endogenous compounds as well as various classes of clinically used drugs, including b-adrenergic blocking agents, antiarrhythmics, antihistamines, opiates, and various sedatives [1,2,3,4,5,6]
In 1994, Grundemann et al [17], using expression cloning in Xenopus laevis oocytes, cloned a polyspecific organic cation transport protein, rOCT1, from a cDNA library derived from rat kidney. rOCT1 is encoded by a 1.8-kb cDNA and has the properties of a basolateral membrane organic cation transporter, i.e. it is sensitive to membrane potential and insensitive to pH
Detection of a Novel Isoform of the mRNA Transcript of rOCT1 in the Rat Kidney—Using first strand cDNA from mRNA isolated from several rat kidneys and primers 1 and 2 (Table I, Fig. 1A) derived from the published cDNA sequence of rOCT1 [17], we obtained two PCR products of approximately 1.5 and 1.6 kb in size (Fig. 1B, lane 2)
Summary
CDNA Cloning—-Total RNA was isolated from male Harlan Sprague Dawley rat kidneys and other tissues using TriZOLR reagent (Life Technologies, Inc.). The synthesized cDNA and primers (10 mM) (see Table I and Fig. 1) specific for the rat kidney organic cation transporter (rOCT1) cDNA [17] were used in the subsequent PCR under the following conditions: 94 °C for 0.5 min, 55 °C for 1.5 min, 72 °C for 2 min, 35 cycles. Two primers (sense and antisense to each other) flanking the mutagenesis site (primers 9 and 10, see Table I) were used in PCR with plasmid DNA containing the rOCT1A inserts as the template and Pfu DNA polymerase (Stratagene). The PCR product was digested with DpnI restriction enzyme (Life Technologies, Inc.) followed by transformation and subcloning as described above. RNase Protection Assay—A 370-nucleotide EcoRI-XbaI fragment of rat rOCT1 cDNA was amplified with primers 11 and 12 (see Table I and Fig. 6A) by PCR. Primers were synthesized by the Biochemical Resource Center at the University of California, San Francisco
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