INTRODUCTION

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 -adrenergic blocking agents, antiarrhythmics, antihistamines, opiates, and various sedatives (1-6). The presence of polyspecific organic cation transporters asymmetrically distributed to the brush border and basolateral membrane of the renal epithelium promotes the vectorial movement of molecules in the secretory direction. The mechanisms of organic cation transport across each of these membranes have been studied in isolated renal plasma membrane vesicles from a number of species using the model compound, tetraethylammonium (TEA)¹ (7-12). In general, the studies have shown that TEA is transported from the blood into the cell across the basolateral membrane via a facilitative, but passive, electrogenic carrier-mediated system (9, 12, 13). The driving force is the inside negative membrane potential. Subsequently, TEA is transported into the tubule lumen across the brush border membrane via a secondarily active organic cation proton antiporter (2, 8, 9, 11, 14, 15). The inwardly directed proton gradient, which serves as the major driving force, is generated mostly by the Na⁺-H⁺ exchanger on the brush border membrane (6, 16).

Until recently, little was known about the molecular characteristics of organic cation transporters at either the basolateral or brush border membrane of the renal proximal tubule. However, 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. Recently, a second organic cation transporter, rOCT2, from a rat kidney cDNA library was cloned using homology cloning with degenerate oligonucleotide probes to rOCT1 (18).

Isoforms of proteins may represent the translation products of mRNA transcripts from multiple genes or the products of alternatively spliced mRNA transcripts from a single gene. The process of alternative RNA splicing may result in increased genetic flexibility. For example, alternatively spliced mRNA transcripts may be translated into proteins with distinct functional characteristics. On the other hand, the protein products may have identical functional properties but differ in terms of regulation mechanisms that are critical in the tissue-specific role of the protein or in the course of cell differentiation. It is increasingly clear that alternative RNA splicing plays a critical role in increasing the diversity of membrane transporters such as the Ca²⁺ pump (19-24). To understand the biological role of organic cation transporters in renal and other epithelia, it is essential to identify relevant, functional organic cation transporter isoforms. Although isoforms of organic cation transporters encoded by different genes have been identified (17, 18, 25), it is not known whether there are isoforms of organic cation transporters resulting from alternative RNA splicing.

By RT-PCR, we identified and cloned a novel isoform of rOCT1, rOCT1A, from the rat kidney. Sequence analysis, molecular modeling, and studies with synthetic constructs suggest that a functional organic cation transporter(s) is encoded by the mRNA of rOCT1A after initiation of protein synthesis at an internal start codon. This is the first evidence of a functional, alternatively spliced variant of a polyspecific organic cation transporter.

EXPERIMENTAL PROCEDURES

Total RNA was isolated from male Harlan Sprague Dawley rat kidneys and other tissues using TriZOL^R reagent (Life Technologies, Inc.). Poly(A)⁺ RNA (mRNA) was selected by affinity chromatography using oligo(dT)-cellulose spin columns (5 Prime  3 Prime, Inc., Boulder, CO). Total RNA or mRNA was primed with oligo(dT) primer to synthesize the first strand cDNA using the SuperScript^TM preamplification system for first strand cDNA synthesis (Life Technologies, Inc.). The synthesized cDNA and primers (10 µM) (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. The PCR products were electrophoresed through 1% agarose gels, and size-selected DNA fragments were extracted and subcloned into the pCR^TMII vector (original TA Cloning^® kit, Invitrogen) or the pGEM-T vector (Promega) using T4 DNA ligase followed by transformation into INVF One Shot^TM (original TA Cloning^® kit, Invitrogen) or DH5 (Life Technologies, Inc.) competent cells. Plasmid DNA was isolated using the Wizard^TM minipreps DNA purification system (Promega) and was analyzed by restriction enzyme analysis and/or sequencing.

Table I. Sequences of primers and their positions in the rOCT1 cDNA Sequences added that are not related to the rOCT1 cDNA, including restriction enzyme sites, are underlined, and mutation sites are indicated in italics. Sequence Position bp Primer 1 (sense) 5-GCAGGCCTGGCTAAACTGGTGAG-3 1-23 Primer 2 (antisense) 5-TTGCGGCCGCTCAGGTACTTGAGGACTT-3 1691 -1708 Primer 3 (antisense) 5-AGTGCGGAACAGGTC-3 1046 -1060 Primer 4 (sense) 5-CCAGAATCCCCCCGGTGG-3 887 -904 Primer 5 (sense) 5-AGCGGTGTGGCTGGAGCCAGGCAGAG-3 216 -241 Primer 6 (sense) 5-ATTGGGCCCCTGCGAGCATGGCTG-3 391 -414 Primer 7 (antisense) 5-ACACCCAGCTGCCCTTGCTGACCAT-3 695 -671 Primer 8 (antisense) 5-AACATGGATGTATAGTCTGGG-3 648 -628 Primer 9 (sense) 5-ATCGTCACTGAGTTTGGCCGTAAGCTC-3 start at 440  Primer 10 (antisense) 5-GAGCTTACGGCCAAACTCAGTGACGAT-3 start at 671  Primer 11 (sense) 5-GCTCTAGAGCCTGAGTTTAACCTGGTGTGT-3 447 -466     XbaI site Primer 12 (antisense) 5-GGAATTCCTGGAATGGCATAGGCCA-3 801 -817     EcoRI site

To obtain the genomic DNA sequence flanking the spliced region (exon/intron junctions), we used a novel method for walking upstream or downstream in genomic DNA from the cDNA sequence (26) with the Rat GenomeWalker^TM kit (CLONTECH) according to manufacturer instructions. Primers were designed from the cDNA sequence upstream or downstream of the splice site (primers 5-8) (see Table I). PCR reactions were performed with the Advantage^TM genomic PCR kit (CLONTECH) and used cycle parameters suggested by the manufacturer. The PCR products were subcloned as described above.

To generate the in-frame deletion (105 bp) variant of rOCT1, the QuickChange^TM site-directed mutagenesis kit (Stratagene) was used according to manufacturer protocol. Briefly, 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 then digested with DpnI restriction enzyme (Life Technologies, Inc.) followed by transformation and subcloning as described above.

Subcloned cDNA inserts isolated from multiple reverse transcription and/or PCR reactions were sequenced using universal and gene-specific primers by the Biomedical Resource Center DNA Sequencing Facility at the University of California, San Francisco with an automated sequencer (Applied Biosystems, model 373A). Sequence alignments of rat rOCT1 and rOCT1A were produced using the Gap and Bestfit programs in the Genetics Computer Group (Wisconsin Package, version 8) software package. The Motifs program in the Genetics Computer Group package was used to determine potential protein kinase C phosphorylation sites and N-glycosylation sites. The transmembrane domains of rOCT1A were predicted based on hydropathy analysis using the Kyte-Doolittle algorithm (27) in the Genetics Computer Group Pepplot program as well as the hydropathy analysis program in DNA Strider 1.2, a C program for DNA and protein analysis designed and written by Dr. C. Marck (Service de Biochimie et de Genetique Moleculaire, Gif-Sur-Yvette, France).

Healthy stage V and VI oocytes were defolliculated and then injected with 50 nl of water, mRNA, or capped cRNA (1 µg/µl in water) transcribed in vitro using T7 or SP6 RNA polymerase (mCAP^TM RNA capping kit, Stratagene) from linearized plasmid DNA (28). The uptake of [¹⁴C]TEA (53 mCi/mmol, American Radiolabeled Chemicals, Inc.) was measured in oocytes 3 days after injection using the methods previously described (28). Briefly, groups of 9-10 oocytes were incubated in the reaction mixture (100 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM Tris/Hepes, pH 7.4) containing 500 µM [¹⁴C]TEA with or without 5 mM cimetidine for 60-120 min at 25 °C. For inhibition studies, 5 mM concentrations of various unlabeled compounds were also included in the reaction mixture. For the Michaelis-Menten kinetic study, 30-100 µM [¹⁴C]TEA plus various amounts of unlabeled TEA were included in the reaction mixture. After incubation, oocytes were washed, and each oocyte was lysed individually with 100 µl of 10% SDS. The radioactivity was determined by liquid scintillation counting.

The TnT^®-coupled transcription/translation reticulocyte lysate system (Promega) and L-[³⁵S]methionine (1000 Ci/mmol, 10 mCi/ml, DuPont NEN) were used for eukaryotic in vitro translations following manufacturer protocol. Five microliters of translation product was denatured in 20 µl of Laemmli sample buffer plus 5% (v/v) 2-mercaptoethanol at 75 °C for 10 min and then loaded onto 4-20% gradient gels (Tris/glycine ready gels, Bio-Rad). To increase the sensitivity of detection of ³⁵S-labeled protein, gels were soaked in Amplify^TM (Amersham Life Science, Inc.) after the fixing step. Gels were dried and exposed to Hyperfilm-MP film (Amersham Life Science, Inc.).

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. After double digestion with EcoRI and XbaI, the PCR product was ligated into the pGEM^®-11Zf() vector (Promega). A 416-nucleotide biotin-labeled antisense RNA probe was synthesized from the linearized plasmid (cut by XbaI) by in vitro transcription with T7 RNA polymerase (BrightStar^TM BIOTINscript^TM T7 kit, Ambion). The RNase protection assay (RPA) was performed using a HybSpeed^TM RPA kit (Ambion) according to manufacturer protocol. rOCT1 and rOCT1A cRNA (600 pg) were also included in the RPA as controls and size indicators. After RNase digestion, the protected RNA fragments were precipitated, separated on a 5% polyacrylamide, 8 M urea gel, blotted onto a positively charged nylon membrane (BrightStar^®-Plus^TM positively charged nylon membrane, Ambion), and then fixed by cross-linking. The protected fragments corresponding to rOCT1 and rOCT1A were detected using the BrightStar^TM nonisotopic RNA detection system (Ambion) followed by membrane exposure to film.

Uptake values are expressed as pmol/oocyte/h and presented as mean ± S.E. or as specified in the figure legends. A minimum of 6-9 oocytes was used in each experiment. Statistical analysis was carried out by the unpaired Student's t test.

All chemicals were purchased from Sigma or Fisher. Molecular biology supplies and radiolabeled compounds were purchased from the indicated manufacturers. Primers were synthesized by the Biochemical Resource Center at the University of California, San Francisco.

RESULTS AND DISCUSSION

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). The PCR product at 1.6 kb matched the predicted size of the rOCT1 cDNA, whereas the 1.5-kb PCR product was of unknown origin. These two products were also detected after PCR using plasmid DNA isolated from a rat kidney cDNA library and the same primers (Fig. 1B, lane 3). Furthermore, both bands were detected from RT-PCR starting with total RNA isolated from the kidney of a single rat (data not shown). To confirm that the 1.5-kb PCR product was not generated from the mRNA transcript of the 1.6-kb PCR product due to either a PCR error or an error in reverse transcription, cRNA transcribed from the subcloned 1.6-kb PCR product was used in RT-PCR. This reaction resulted in a single detectable band on the gel (data not shown). Collectively, these data suggest that there are two RNA species in the rat kidney that can be amplified by RT-PCR using rOCT1-specific primers derived from the beginning and the end of the open reading frame (ORF) of the rOCT1 cDNA sequence. Furthermore, the resulting PCR product of primers 1 and 3 produced double bands (Fig. 1B, lane 5), whereas the product resulting from primers 2 and 4 produced a single band on the gel (Fig. 1B, lane 7), suggesting that the difference in size is near the 5-end of the cDNA. Sequence analysis demonstrated that the sequence of the 1.6-kb cDNA was identical to the published rOCT1 cDNA sequence (data not shown) (17).

To determine whether the 1.5-kb cDNA encodes a functional organic cation transporter, recombinant plasmids containing the cDNA insert oriented for sense transcription under the control of a T7 or SP6 promoter were used as templates for cRNA synthesis before injection into Xenopus laevis oocytes. A significant increase in the cimetidine-inhibitable [¹⁴C]TEA uptake was observed in cRNA-injected oocytes. [¹⁴C]TEA uptake in the cRNA-injected oocytes was increased 16-fold over that in the water-injected oocytes and 11-fold over that in the rat kidney total mRNA-injected oocytes (Fig. 2A). However, the relative enhancement in [¹⁴C]TEA uptake varied depending on the batch of oocytes (range approximately 2-20-fold).

To determine the specificity of transport, we studied the effect of various compounds on [¹⁴C]TEA uptake in the cRNA-injected oocytes. Consistent with the characteristics of polyspecific renal organic cation transporters, the organic cations (5 mM) TEA, guanidine, cimetidine, choline, N¹-methylnicotinamide, and procainamide all significantly inhibited the uptake of [¹⁴C]TEA (Fig. 2B, p < 0.05). Similar functional characteristics of both rOCT1 and rOCT2 have been observed (17, 18). In addition, p-aminohippuric acid (5 mM), a model organic anion, weakly inhibited [¹⁴C]TEA uptake (Fig. 2B, p < 0.05). Organic anions at high concentrations (e.g. 5 mM) have been shown previously to inhibit renal organic cation transporters (1, 29, 30). These data suggest that the 1.5-kb cDNA encodes a functional organic cation transporter having similar characteristics as rOCT1. Quantitative studies are under way to determine whether rOCT1 and rOCT1A differ in the potency of interaction with various substrates.

To determine the kinetic characteristics of TEA transport in oocytes injected with the cRNA of the 1.5-kb PCR product, we measured TEA uptake over a range of concentrations (30-500 µM). Fig. 2C shows the data along with the computer-generated nonlinear regression fit curve for the Michaelis-Menten equation. The V_max and K_m of TEA in this experiment were 5.4 ± 0.35 pmol/oocyte/h and 42 ± 11 µM, respectively. The K_m of TEA uptake (42 µM, Fig. 2C) is slightly lower than the value of 95 µM obtained previously for rOCT1 (17). The V_max is considerably lower (5.4 versus 81 pmol/oocyte/h (17)), suggesting that there may be fewer functional rOCT1A transporters present; however, V_max values are difficult to compare due to differences in experimental conditions between laboratories.

After ascertaining the function of the transporter, we carried out sequence analysis to deduce the primary sequence of the functional protein from the cDNA sequence. Because PCR may result in fidelity errors in amplification of DNA, we performed sequence analyses of cDNAs isolated from multiple reverse transcription and PCR reactions. A consistent sequence was obtained (Fig. 3A). DNA sequence alignment between the 1.5- and 1.6-kb clone (rOCT1) demonstrates that the 1.5-kb cDNA sequence is identical to that of the 1.6-kb cDNA with a deletion between bp 451 and 556 of the rOCT1 cDNA. This is consistent with the PCR results (Fig. 1B). The deletion results in a stop codon at bp 460 in reading frame 2 (RF2, the same reading frame encoding the ORF of rOCT1), and a large ORF is present from the ATG at bp 312 to the stop codon at bp 1602 in RF3 (Fig. 3A). This large open reading frame in RF3 encodes a protein (rOCT1A) of 430 amino acids that, after sequence alignment, is 92% identical to that of rOCT1 (the reading frame of rOCT1 is restored in the RF3 of rOCT1A cDNA after the deletion junction, and the only different region lies at the amino terminus) and 57% identical to that of rOCT2. (Alternative ATG sites at bp 408 or 540 of RF3 are other possible initiation sites and would encode truncated versions of rOCT1A.) Based upon hydropathy analysis using the Kyte-Doolittle algorithm and application of the positive inside rule (27, 31), rOCT1A is predicted to have 10 transmembrane domains with both amino and carboxyl termini intracellular (Fig. 3B). In comparison, both rOCT1 and rOCT2 are predicted to have 12 transmembrane domains (17, 18). As a result of the deletion, rOCT1A lacks the first two transmembrane domains as well as the three potential glycosylation sites in the first extracellular loop of rOCT1. Nevertheless, rOCT1A exhibits similar functional characteristics to those of rOCT1, which implies that the first two transmembrane domains and these three putative glycosylation sites are not essential for the transport function. However, other properties of the transporter such as synthesis, targeting, and sorting may be different between the two isoforms. Five potential protein kinase C phosphorylation sites at serine residues 160, 166, and 202 and threonine residues 170 and 424 were identified in the intracellular loops of rOCT1A. (The positions correspond to the deduced amino acid sequence of rOCT1A beginning at the internal ATG at bp 312.) Additionally, one potential N-glycosylation site was identified in the extracellular loop between helices 7 and 8. These sites are conserved in both rOCT1 and rOCT1A (17).

To determine the genomic nature of rOCT1A, genomic DNA fragments of rOCT1 flanking the splice sites were cloned and sequenced. As shown in Fig. 4, there is an intron of at least 6 kb in length between bp 451 and 452 of the rOCT1 cDNA followed by a short exon of 104 bp in length, which matches the deleted sequence. There is another intron of 439 bp in length between bp 555 and 556 of the rOCT1 cDNA. Based on the genomic sequence of rOCT1 in this region and the fact that rOCT1 and rOCT1A are 100% identical at the cDNA level (except for the 104-bp deletion in rOCT1A), rOCT1A represents an alternatively spliced isoform from a common precursor mRNA transcript. In addition, a recent chromosomal localization study of Roct1 excludes the possibility of multiple Roct1 genes or pseudogenes (32).

The 104 bp (exon B) in rOCT1 include part of the loop between transmembrane domain 1 and 2 and the whole transmembrane domain 2. This deletion interrupts a codon (AGG) between 555 and 556 that results in a frameshift and leads to an earlier stop codon in RF2. Frameshift in splice variants of membrane proteins in higher organisms (i.e. humans and other mammals) has been reported (19, 33, 34). In addition, the large intron of at least 6 kb between exon A and B presents the possibility of further alternative splicing. It will be of interest to ascertain whether other subtypes or isoforms of rOCT1 exist and to determine their functional significance.

In vitro translation experiments in the rabbit reticulocyte lysate system were performed to synthesize proteins from the cRNAs of rOCT1A and rOCT1 (as a comparison). Translation products were analyzed by SDS-polyacrylamide gel electrophoresis. Fig. 5 shows that translation of the cRNA of rOCT1 produced a single band with an apparent molecular size of 47 kDa (Fig. 5, lane 2). In contrast, translation of the mRNA encoding rOCT1A resulted in several bands with one major band of about 16 kDa in size and a major band(s) of about 37 kDa (Fig. 5, lane 3). The multiple bands at 37 kDa may be a result of translation beginning at multiple internal ATG sites as discussed previously and suggested by the sequence. The 16-kDa band may be encoded by the short open reading frame in RF2 (bp 38-460, 141 amino acids). Alternatively, it can be a result of proteolysis. The protein products produced in the in vitro translation experiments have apparent molecular masses smaller than predicted based upon the deduced sequences. However, anomalous migration of membrane proteins is not unusual (35, 36).

To determine whether the smaller band of about 16 kDa was due to the early stop codon at 460 bp in RF2, a mutant of rOCT1A was generated. This mutant contained a 105-bp in-frame deletion in the rOCT1 cDNA, thus eliminating the early stop codon and restoring the RF2 as in rOCT1 of the spliced RNA. As shown in Fig. 5, in vitro translation of the cRNA of this mutant produced a single band (lane 4). These data provide the indirect evidence suggesting that the 16-kDa band observed after in vitro translation of the cRNA of rOCT1A may be encoded by the short ORF in RF2 of rOCT1A. In addition, after in vitro translation, there is a notable size difference in the larger band from the cRNA of the mutant in comparison with the larger band(s) from the cRNA of rOCT1A. Such a size difference would not be expected with the difference in the length of the cDNA's encoding rOCT1A and the mutant, i.e. 1 bp.

Many naturally occurring cellular and viral mRNAs are polycistronic, and multiple proteins are synthesized by re-initiation of translation at internal AUGs after meeting the terminator codon of the upstream ORF. Examples of polycistronic mRNA transcripts in eukaryotes are rare and have never been reported in mammalian mRNA (37-39). Further studies are needed to determine whether the multiple bands observed in in vitro translation studies are a result of a polycistronic mRNA transcript of rOCT1A or proteolysis. Future studies utilizing antibodies recognizing different ORFs will provide the definitive evidence to determine whether multiple proteins from different ORFs were encoded. Mutagenesis studies will be important in determining the internal initiation site(s) of translation for rOCT1A.

Based upon comparative sequence analysis with rOCT1 and the general structure of membrane transporters that normally have multiple membrane spanning regions (40), we hypothesized that the larger protein is the functional transporter. Accordingly, we constructed a synthetic cDNA that would encode for the 16-kDa product (Pro 1) and two synthetic cDNAs that would encode for two possible functional transport proteins (Pro 2 and Pro 3) (Fig. 6A). The function of the synthetic proteins was tested by injecting oocytes with cRNA transcribed from the cDNA encoding Pro 1, 2, and 3. [¹⁴C]TEA uptake was enhanced significantly in oocytes injected with the cRNA of Pro 2 or 3 in comparison to water-injected oocytes (Fig. 6B, p < 0.05). In contrast, [¹⁴C]TEA uptake was not enhanced in oocytes injected with the cRNA of Pro 1 (2.35 ± 0.13 pmol/oocyte/h in cRNA-injected oocytes versus 2.51 ± 0.31 pmol/oocyte/h in water-injected oocytes, mean ± S.E.). The reasons why Pro 3 had a higher expressed activity than either Pro 2, rOCT1, or rOCT1A are unknown. Differences in efficiency of the in vivo translation or protein processing may explain the data. Alternatively, Pro 3 may have a higher intrinsic activity. These data suggest that Pro 2 and 3, representing possible proteins encoded by the cRNA of rOCT1A, can mediate the transport of TEA, whereas Pro 1 cannot. In in vitro translation experiments, Pro 2 migrated at a similar rate as the 37-kDa translation product of the cRNA of rOCT1A (data not shown), suggesting that Pro 2 might be the functional organic cation transporter. These data have implications to the function of rOCT1, since rOCT1A is 92% identical in sequence to rOCT1.

To characterize the rOCT1A RNA expression in the kidney, RPAs were performed. A 416-nucleotide biotin-labeled antisense RNA probe was synthesized to specifically detect rOCT1 and rOCT1A RNA fragments differing by approximately 110 nucleotides (Fig. 7A). As shown in Fig. 7B, two RNase-protected fragments of expected size for rOCT1 and rOCT1A (370 and 263 nucleotides, respectively) were detected in the RNA of total kidney, kidney cortex, and kidney medulla. In addition, from the intensities of the protected fragments, it appears that the rOCT1 RNA transcript is present at a higher level than that of rOCT1A in the kidney. Additional RPAs using this probe detected rOCT1 and rOCT1A RNA transcripts in the intestine but not in the heart or spleen (data not shown). Tissue distribution of rOCT1A transcript was also determined by RT-PCR using primers 1 and 3 (Table I). Fig. 7C shows that, consistent with the RPA results, transcripts of rOCT1 and rOCT1A (double bands at about 1.0 kb) are present in the rat kidney (total), kidney cortex, kidney medulla, intestine, liver, and colon but not in the stomach, brain, spleen, lung, and heart. These data suggest that the tissue distribution of the mRNA transcripts of rOCT1A and rOCT1 is similar but differs from that of rOCT2, which is confined to the kidney (18). We also noticed that although the mRNA transcript of rOCT1A and rOCT1 coexpress in the same tissues, their relative levels appear different in results from both RPA and RT-PCR. Whether the expression level of the RNA of rOCT1A and rOCT1 reflects the protein expression level and relates to their physiological roles in different tissues or whether the alternative splicing represents a regulatory mechanism will be further investigated.

In summary, a polyspecific organic cation transporter, rOCT1A, from rat kidney has been cloned and expressed. rOCT1A is an alternatively spliced variant of rOCT1 and is similar in function to rOCT1 and rOCT2. RT-PCR indicates that rOCT1A mRNA transcripts are also present in the intestine and liver, and RNase protection assays indicate that the RNA transcript of rOCT1A is present in detectable quantities in the rat kidney. Future studies will be performed to determine the underlying structural requirements for the function of organic cation transporters, the physiologic role of rOCT1A in the intact tissue, and the mechanisms involved in the translation of the rOCT1A mRNA transcript.