Molecular cloning and functional expression of a rabbit renal organic cation transporter

Terashita, Shigeyuki; Dresser, Mark J.; Zhang, Lei; Gray, Andrew T.; Yost, Spencer; Giacomini, Kathleen M.

doi:10.1016/s0005-2736(97)00207-1

Cited by 56 publications

(45 citation statements)

References 13 publications

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“…Despite several kinetic studies characterizing these transporters in plasma membranes and lysosomes, OCT1 is the only hepatic member of this putative liver-kidney organic cation transporter family that has been cloned and functionally characterized. 14,15,27,28 Other members of the putative organic cation superfamily include OCT2 and rOCT1A, which have also been cloned and functionally characterized, but do not appear to be expressed in the liver. 14,[28][29][30][31] Similarly, homologous cDNAs that encode for a polyspecific renal anion transporter, OAT1 (also known as the paraaminohippurate transporter), have been recently identified, but also appear to be kidney-specific.…”

Section: Discussionmentioning

confidence: 99%

Cloning and Functional Expression of A Mouse Liver Organic Cation Transporter

Green

Sterritt

et al. 1999

Hepatology

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Hepatic uptake of organic cations is essential for the metabolism and secretion of numerous endobiotics and drugs. Several hepatic organic cation transporters have been kinetically defined, yet have not been isolated or cloned. We have isolated a complementary DNA (cDNA) from both murine liver and kidney cDNA libraries (mOct1/ Slc22a1), and have functionally expressed it in Xenopus laevis oocytes. Although mOct1/Slc22a1 is homologous to previously cloned rat and human organic cation transporters, organic cation transport kinetics differed markedly. The liver and kidneys have critical roles in the secretion of drugs and endobiotics into the bile and urine. Hepatic elimination of many organic cationic drugs involves uptake across the basolateral surface of the hepatocyte, hepatic biotransformation, and canalicular secretion. 1 Previous studies in the intact rat, isolated perfused rat liver, and isolated hepatocytes show that the hepatobiliary transport of organic cations is carrier-mediated. [2][3][4] In addition, the organic cation choline is an essential nutrient and precursor for the synthesis of phospholipids. Hepatic choline levels are approximately 100-fold greater than blood concentrations, and de novo choline synthesis is minimal, suggesting the existence of a transport mechanism for hepatic choline uptake. 5,6 Several organic cation transporters have been kinetically defined on the plasma membrane of hepatocytes and in lysosomes. Functional studies in rat liver plasma membrane vesicles have shown at least two distinct organic cation:H ϩ exchangers, and two electrogenic organic cation transporters. [7][8][9][10][11][12][13] The complementary DNA (cDNA) encoding for the rat electrogenic polyspecific transporter (OCT1) has been isolated using functional expression cloning in Xenopus laevis oocytes, and assaying n-methyl-nicotinamide (NMN)-inhibitable uptake of the model quaternary organic cation tetraethylammonium (TEA). 14 A human homologue of rat OCT1 was recently cloned that transports 1-methyl-4-phenylpyridinium (MPP ϩ ), and uptake is also inhibited by the organic cation NMN. 15 In this study, we have isolated a murine cDNA (mOct1/ Slc22a1) from both liver and kidney libraries, and have functionally expressed it in X. laevis oocytes. Although mOct1/Slc22a1 is homologous with OCT1, transport kinetics differed markedly. mOct1/Slc22a1 transports several organic cations, but uptake is increased by inside:outside proton gradients, is unaffected by membrane potential differences, and is not inhibited by NMN. These data indicate that this cloned cDNA is a member of the hepatic and renal organic cation transporter family.

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Section: Discussionmentioning

confidence: 99%

Cloning and Functional Expression of A Mouse Liver Organic Cation Transporter

Green

Sterritt

et al. 1999

Hepatology

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“…Tetraethylammonium uptake by rat (29) and human (34) OCT1 was inhibited by unlabeled cimetidine in the medium with apparent K i values of 5.7 and 166 M, respectively. Similarly, transport of 1-methyl-4-phenylpyridinium by rabbit OCT1 was also inhibited by cimetidine (26). Translocation of radiolabeled cimetidine, however, was slow or negligible in human and rat OCT1 (10,34).…”

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confidence: 92%

Transport of cimetidine by flounder and human renal organic anion transporter 1

Burckhardt

Brai

Wallis

et al. 2003

American Journal of Physiology-Renal Physiology

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Burckhardt. Transport of cimetidine by flounder and human renal organic anion transporter 1. Am J Physiol Renal Physiol 284: F503-F509, 2003. First published November 12, 2002 10.1152/ajprenal.00290.2002The H 2-receptor antagonist cimetidine is efficiently excreted by the kidneys. In vivo studies indicated an interaction of cimetidine not only with transporters for basolateral uptake of organic cations but also with those involved in excretion of organic anions. We therefore tested cimetidine as a possible substrate of the organic anion transporters cloned from winter flounder (fROAT) and from human kidney (hOAT1). Uptake of [ 3 H]cimetidine into fROAT-expressing Xenopus laevis oocytes exceeded uptake into control oocytes. At Ϫ60-mV clamp potential, 1 mM cimetidine induced an inward current, which was smaller than that elicited by 0.1 mM PAH. Cimetidine concentrations exceeding 0.1 mM decreased PAH-induced inward currents, indicating interaction with the same transporter. At pH 6.6, no current was seen with 0.1 mM cimetidine, whereas at pH 8.6 a current was readily detectable, suggesting preferential translocation of uncharged cimetidine by fROAT. Oocytes expressing hOAT1 also showed [ 3 H]cimetidine uptake. These data reveal cimetidine as a substrate for fROAT/hOAT1 and suggest that organic anion transporters contribute to cimetidine excretion in proximal tubules.winter flounder renal organic anion transporter; organic anion transport; organic cation transport; kidney AS A SPECIFIC ANTAGONIST OF histamine H 2 receptors, cimetidine acts on gastric parietal cells to inhibit HCl secretion stimulated by histamine, pentagastrin, and acetylcholine. Between 50 and 80% of the dose administered intravenously was recovered in urine as unchanged cimetidine, and elimination half-life was ϳ2 h in healthy volunteers with normal kidney and hepatic function (24). The mean steady-state plasma concentration on a standard 1,000-mg daily dose was 1 g/ml (range 0.64-1.64 g/ml). A renal clearance of 600 ml/ min (24), exceeding the glomerular filtration rate by approximately fivefold, indicated an efficient tubular secretion of cimetidine. Therefore, cimetidine must interact with renal transporters, most probably with those located in the proximal tubules.Because at physiological pH part of the cimetidine is positively charged, the organic cation transport systems of the proximal tubule cell are expected to be involved in cimetidine secretion. The two-step model of transcellular organic cation secretion consists of electrogenic uptake at the basolateral side, moving organic cations down their electrochemical gradient from blood into proximal tubular cells, and organic cation/proton exchange mediating uphill exit into the lumen. The cloned organic cation transporters, OCT1, OCT2, and OCT3, are electrogenic and likely contribute to organic cation uptake across the basolateral membrane (for reviews, see Refs. 6 and 18). In accordance with their function, rat OCT1 and OCT2 were localized to the basolateral membrane of proximal tubule ce...

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“…1-methyl-4-phenylpyridinium (MPP ϩ )), evidently plays a key role in all species in governing the entry of OCs from the blood into the renal tubule, thereby controlling the first step in renal secretion of OCs. The orthologs of OCT1 and OCT2 have been cloned from human (13,14), rat (15,16), mouse (17,18), and rabbit (19,20), and in each species, these homologs share ϳ70% amino acid identity and Ͼ90% sequence similarity. This high degree of shared structure is consistent with the shared functional characteristics of these homologous transporters.…”

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confidence: 99%

A Conserved Glutamate Residue in Transmembrane Helix 10 Influences Substrate Specificity of Rabbit OCT2 (SLC22A2)

Zhang

Shirahatti

Mahadevan

et al. 2005

Journal of Biological Chemistry

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OCT1 and OCT2 are involved in renal secretion of cationic drugs. Although they have similar selectivity for some substrates (e.g. tetraethylammonium (TEA)), they have distinct selectivities for others (e.g. cimetidine). We postulated that "homolog-specific residues," i.e. the 24 residues that are conserved in OCT1 orthologs as one amino acid and in OCT2 as a different one, influence homologspecific selectivity and examined the influence on substrate binding of three of these conserved residues that are found in the C-terminal half of the rabbit orthologs of OCT1/2. The N353L and R403I substitutions (OCT2 to OCT1) did not significantly change the properties of OCT2. However, the E447Q replacement shifted substrate selectivity toward an OCT1-like phenotype. Substitution of glutamate with cationic amino acids (E447K and E447R) abolished transport activity, and the E447L mutant displayed markedly reduced transport of TEA and cimetidine while retaining transport of 1-methyl-4-phenylpyridinium. In a novel homology model of the three-dimensional structure of OCT2, Glu 447 was found in a putative docking region within a hydrophilic cleft of the protein. Renal excretion is the principal pathway for elimination of many clinically used drugs and is the exclusive pathway for eliminating many end products of drug-metabolizing enzymes (1-3). Transporters in the renal tubule epithelium mediate secretion and thus play a critical role in detoxification (4). In addition, transporters control the exposure of renal cells to nephrotoxic drugs and environmental toxins and thereby influence xenobiotic-induced nephrotoxicity. A large fraction of these agents fall into the chemical class commonly referred to as "organic cations" (OCs), 2 i.e. a diverse array of primary, secondary, tertiary, or quaternary amines that have a net positive charge on the amine nitrogen at physiological pH. The proximal tubule is the primary site of renal OC secretion (3,5), and the first step in the process is OC entry from the blood into proximal cells across the peritubular (i.e. basolateral) membrane. Three homologous transporters (OCT1, OCT2, and OCT3) have been cloned and subsequently shown to be expressed in the basolateral membrane of proximal cells (1,6). In all species examined, including the human (7), the kinetic and selectivity profile of OCT3 and the comparatively low levels of mRNA and protein expression of this homolog in the kidney suggest that renal secretion is dominated by some combination of OCT1 and OCT2 activity. Perhaps the most convincing evidence of the significance of OCT1 and OCT2 in renal OC secretion is the observation that secretion of the prototypic OC, tetraethylammonium (TEA), is completely eliminated in OCT1/2 Ϫ/Ϫ mice (8).In human kidney, OCT2 appears to be the predominant OC transporter. Expression of mRNA for OCT2 far exceeds that for OCT1, and immunocytochemistry clearly shows a basolateral expression for OCT2 and little or no presence of OCT1 (9). There are, however, clear species differences in this profile, with subst...

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Molecular cloning and functional expression of a rabbit renal organic cation transporter

Cited by 56 publications

References 13 publications

Cloning and Functional Expression of A Mouse Liver Organic Cation Transporter

Cloning and Functional Expression of A Mouse Liver Organic Cation Transporter

Transport of cimetidine by flounder and human renal organic anion transporter 1

A Conserved Glutamate Residue in Transmembrane Helix 10 Influences Substrate Specificity of Rabbit OCT2 (SLC22A2)

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