L-Carnitine is an essential component of mitochondrial fatty acid ␤-oxidation and plays a pivotal role in the maturation of spermatozoa within the male reproductive tract. Epididymal plasma contains the highest levels of L-carnitine found in the human body, and initiation of sperm motility occurs in parallel to L-carnitine increase in the epididymal lumen. Using a specific carrier, epididymal epithelium secretes L-carnitine into the lumen by an active transport mechanism; however, the structure-activity relationship comprising the carnitine-permeation pathway is poorly understood. We discovered a novel carnitine transporter (CT2) specifically located in human testis. Analyzing the primary structure of CT2 revealed that it is phylogenetically located between the organic cation transporter (OCT/OCTN) and anion transporter (OAT) families. Hence, CT2 represents a novel transporter family. When expressed in Xenopus oocytes, CT2 mediates the high affinity transport of L-carnitine but does not accept mainstream OCT/ OCTN cationic or OAT anionic substrates. We synthesized and tested various carnitine-related compounds and investigated the physicochemical properties of substrate recognition by semi-empirical computational chemistry. The data suggest that the quaternary ammonium cation bulkiness and relative hydrophobicity be the most important factors that trigger CT2-substrate interactions. Immunohistochemistry showed that the CT2 protein is located in the luminal membrane of epididymal epithelium and within the Sertoli cells of the testis. The identification of CT2 represents an interesting evolutionary link between OCT/OCTNs and OATs, as well as provides us with an important insight into the maturation of human spermatozoa.

The tubular secretion of diuretics in the proximal tubule has been shown to be critical for the action of drugs. To elucidate the molecular mechanisms for the tubular excretion of diuretics, we have elucidated the interactions of human organic anion transporters (hOATs) with diuretics using cells stably expressing hOATs. Diuretics tested were thiazides, including chlorothiazide, cyclothiazide, hydrochlorothiazide, and trichlormethiazide; loop diuretics, including bumetanide, ethacrynic acid, and furosemide; and carbonic anhydrase inhibitors, including acetazolamide and methazolamide. These diuretics inhibited organic anion uptake mediated by hOAT1, hOAT2, hOAT3, and hOAT4 in a competitive manner. hOAT1 exhibited the highest affinity interactions for thiazides, whereas hOAT3 did those for loop diuretics. hOAT1, hOAT3, and hOAT4 but not hOAT2, mediated the uptake of bumetanide. hOAT3 and hOAT4, but not hOAT1 mediated the efflux of bumetanide. hOAT1 and hOAT3, but not hOAT2 and hOAT4 mediated the uptake of furosemide. In conclusion, it was suggested that hOAT1 may play an important role in the basolateral uptake of thiazides, and hOAT3 in the uptake of loop diuretics. In addition, it was also suggested that bumetanide taken up by hOAT3 and/or hOAT1 is excreted into the urine by hOAT4.

The liver plays an important role in the elimination of endogenous and exogenous lipophilic organic compounds from the body, which is mediated by various carrier proteins that differ in substrate specificity and kinetic properties. Here, we have characterized a novel member of the organic anion transporter family (SLC22) isolated from human liver. The transporter named organic anion transporter 7 (OAT7/ SLC22A9) showed 35% to 46% identities to those of other organic anion transporters of SLC22 family. When expressed in Xenopus oocytes, OAT7 mediated Na ؉ -independent, highaffinity transport of sulfate-conjugated steroids, estrone sulfate (ES; K m ‫؍‬ 8.7 M), and dehydroepiandrosterone sulfate (K m ‫؍‬ 2.2 M). In addition, OAT7 interacted with negatively charged sulfobromophthalein, indocyanine green, and several sulfate-conjugated xenobiotics. In contrast, glucuronide and glutathione conjugates exhibited no inhibitory effects on OAT7-mediated

Human organic anion transporter 4 (OAT4) is an apical organic anion/dicarboxylate exchanger in the renal proximal tubules and mediates high-affinity transport of steroid sulfates such as estrone-3-sulfate (E 1 S) and dehydroepiandrosterone sulfate. Here, two multivalent PDZ (PSD-95/Discs Large/ZO-1) proteins PDZK1 and NHERF1 were examined as interactors of OAT4 by a yeast two-hybrid assay. These interactions require the extreme C-terminal region of OAT4 and the first and fourth PDZ domains of PDZK1 and the first PDZ domain of NHERF1. These interactions were confirmed by surface plasmon resonance assays (K D : 36 nM, 1.2 M, and 41.7 M, respectively). In vitro binding assays and co-immunoprecipitation studies revealed that the OAT4 wild-type but not a mutant lacking the PDZ motif interacted directly with both PDZK1 and NHERF1. OAT4, PDZK1, and NHERF1 proteins were shown to be localized at the apical membrane of renal proximal tubules. The association with PDZK1 or NHERF1 enhanced OAT4-mediated E 1 S transport activities in HEK293 cells T he human organic anion transporter OAT4 (encoded by SLC22A11) is expressed in the kidney and the placenta and mediates the high-affinity transport of steroid sulfates such as estrone-3-sulfate (E 1 S) and dehydroepiandrosterone sulfate (DHEAS) (1). Because of its apical membrane localization in the renal proximal tubules (2), OAT4 had been presumed to be the apical exit pathway of organic anions that are taken up by the basolateral entrance pathway such as OAT1 and OAT3 (3). Recently, Ugele et al. (4) found the OAT4 protein expression at the fetal side of the syncytiotrophoblasts in the placenta and proposed a role for OAT4 in the placental uptake of fetal-derived steroid sulfates. On the basis of our finding that OAT4 is an apical organic anion/dicarboxylate exchanger (5), we suggested that OAT4 mainly functions as an apical entrance pathway for some organic anions in renal proximal tubules driven by an outwardly directed dicarboxylate gradient created by Na ϩ /dicarboxylate co-transporters (6). One possible role of OAT4 is as an apical backflux pathway (7) for some organic anions such as steroid sulfates, functionally coupled to the apical efflux transporters for organic anions, such as MRP2, MRP4, and NPT1 (putative human homologue of OAT V 1) (8 -13).At the extreme C-terminal (CT) end, OAT4 has a specific protein-protein interaction peptide sequence named the PDZ (PSD-95/Discs Large/ZO-1) motif (S-T-S-L) (14). PDZ domains have been identified in various proteins and are known to be modular protein-protein recognition domains that play roles in protein targeting and protein complex assembly (15-17). These multidomain molecules not only target and provide scaffolds for protein-protein interactions but also modulate the function of receptors and ion channels, by which they associate. Recently, we reported that the urate/anion exchanger URAT1 (18), which has a similar PDZ motif (S-T-Q-F) at its C-terminus, interacts with multivalent PDZ protein PDZK1 (19,20). In the same study, w...

ABSTRACT:Genetic variants of three human organic cation transporter genes (hOCTs) were extensively explored in a Korean population. The functional changes of hOCT2 variants were evaluated in vitro, and those genetic polymorphisms of hOCTs were compared among different ethnic populations. From direct DNA sequencing, 7 of 13 coding variants were nonsynonymous single-nucleotide polymorphisms (SNPs), including four variants from hOCT1 (F160L, P283L, P341L, and M408V) and three from hOCT2 (T199I, T201M, and A270S), whereas 6 were synonymous SNPs. The linkage disequilibrium analysis presented for three independent LD blocks for each hOCT gene showed no significant linkage among all three hOCT genes. The transporter activities of MDCK cells that overexpress the hOCT2-T199I, -T201M, and -A270S variants showed significantly decreased uptake of showed a 2-to 5-fold increase in K m values and a 10-to 20-fold decrease in V max values. The allele frequencies of the five functional variants hOCT1-P283L, -P341L, and hOCT2-T199I, -T201M, and -A270S were 1.3, 17, 0.7, 0.7, and 11%, respectively, in a Korean population; the frequency distributions of these variants were not significantly different from those of Chinese and Vietnamese populations. These findings suggest that genetic variants of hOCTs are not linked among three genes in a Korean population, and several of the hOCT genetic variants cause decreased transport activity in vitro compared with the wild type, although the clinical relevance of these variants remains to be evaluated.The human organic cation transporters hOCT1, hOCT2, and hOCT3 mediate electrogenic transport of small organic cations with different molecular structures, independent of sodium gradient (Koepsell, 1999). These organic cation substrates include clinically important therapeutics (e.g., metformin, procainamide, and cimetidine), endogenous compounds (e.g., dopamine and norepinephrine), as well as toxic substances [e.g., tetraethylammonium bromide (TEA), HPP ϩ , and methyl-4-phenylpyridinium acetate (MPP ϩ )] (Gorboulev et al., 1997;Zhang et al., 1997;Kang et al., 2006). Although these transporters show extensive overlaps in their substrate specificities, they exhibit distinct differences in tissue distribution; hOCT1 is primarily found in the sinusoidal membrane of hepatocytes and, to a lesser extent, in intestinal epithelial cells, whereas hOCT2 is mainly expressed in the basolateral membrane of kidney proximal tubules, and hOCT3 shows a widespread tissue distribution that includes the brain, heart, and liver. Based on their properties and tissue distributions, hOCT1, hOCT2, and hOCT3 are thought to play important roles in the excretion and distribution of organic cations in the liver, kidney, and brain (Jonker and Schinkel, 2004).Knockout mouse models have been generated for the Oct1, Oct2, and Oct3 genes to elucidate the in vivo function of the OCT transporters. Oct1-, Oct2-, and Oct3-deficient mice are viable and display no obvious phenotypic abnormalities (Jonker et al., 2001Zwart et al., 2001...