INVITED REVIEW
Structure of renal organic anion and cation transporters

Gerhard Burckhardt and Natascha A. Wolff

Zentrum Physiologie und Pathophysiologie, D-37073 Göttingen, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
OATs
OCTs
COMPARATIVE ASPECTS OF OATs...
REFERENCES

Here we review the structural and functional properties of organic anion transporters (OAT1, OAT2, OAT3) and organic cation transporters (OCTN1, OCTN2, OCT1, OCT2, OCT3), some of which are involved in renal proximal tubular organic anion and cation secretion. These transporters share a predicted 12-transmembrane domain (TMD) structure with a large extracellular loop between TMD1 and TMD2, carrying potential N-glycosylation sites. Conserved amino acid motifs revealed a relationship to the sugar transporter family within the major facilitator superfamily. Following heterologous expression, most OATs transported the model anion p-aminohippurate (PAH). OAT1, but not OAT2, exhibited PAH-alpha -ketoglutarate exchange. OCT1-3 transported the model cations tetraethylammonium (TEA), N1-methylnicotinamide, and 1-methyl-4-phenylpyridinium. OCTNs exhibited transport of TEA and/or preferably the zwitterionic carnitine. Substrate substitution as well as cis-inhibition experiments demonstrated polyspecificity of the OATs, OCTs, and OCTN1. On the basis of comparison of the structurally closely related OATs and OCTs, it may be possible to delineate the binding sites for organic anions and cations in future experiments.

tetraethylammonium; N1-methylnicotinamide; proximal tubule; secretion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
OATs
OCTs
COMPARATIVE ASPECTS OF OATs...
REFERENCES

THE KIDNEYS EFFICIENTLY EXCRETE amphiphilic organic anions and cations of diverse chemical structures. Among these anions and cations are endogenous and exogenous compounds, including a number of clinically used drugs. Renal disposal of these compounds includes glomerular filtration and proximal tubular secretion processes.

Cellular transport mechanisms involved in proximal tubular secretion of organic anions and cations have been studied in detail and are reviewed elsewhere (21, 22, 37, 38, 41, 42, 78). In brief, amphiphilic organic anions are taken up into proximal tubular cells across the basolateral membrane by at least three transport systems with overlapping substrate specificities (54, 55). The prototypical organic anion, p-aminohippurate (PAH), is exchanged for intracellular alpha -ketoglutarate and is accumulated in the cell against its blood < cell concentration gradient and against the inside negative membrane potential. Uphill PAH transport is driven by the downhill efflux of alpha -ketoglutarate. PAH leaves the cell across the apical brush-border membrane by a not yet completely understood mechanism(s) that may involve exchange of PAH against another anion or electrogenic PAH uniport. Prototypical organic cations such as tetraethylammonium (TEA), N1-methylnicotinamide (NMN) or 1-methyl-4-phenylpyridinium (MPP) are taken up across the basolateral membrane of proximal tubular cells by electrogenic uniport driven by the inside negative membrane potential, or by exchange with intracellular organic cations, e.g., choline. The exit of organic cations across the brush-border membrane is accomplished by an organic cation/H+ antiporter (exchanger), driven by the lumen > cell H+ gradient established by Na+/H+ exchange. Some organic anions and organic cations, e.g., urate and choline, are also reabsorbed from proximal tubules, resulting in bidirectional transport.

The transport systems involved in the uptake of the organic anions and cations across the basolateral membrane exhibit broad substrate specificities to accommodate a large variety of chemically unrelated compounds. Common structural requirements for substrates are a hydrophobic moiety, the ability to form hydrogen bonds, and the presence of ionic or partial electrical charges (54, 55). Substrates with one or two negative charges preferably interact with the "PAH transporter," and those with positive charges with the "NMN transporter." In addition, uncharged, zwitterionic and even positively charged molecules can interact with the PAH transporter, provided they are hydrophobic enough. Therefore, a considerable number of compounds interact with both the PAH and the NMN transporter (57, 62, 63), suggesting that the transporters for organic anions and organic cations share some structural properties.

In recent years, renal transporters for organic anions and for organic cations have been cloned that indeed share structural properties. Here, we briefly review present knowledge of the structure of the organic anion transporters (OATs) and organic cation transporters (OCTs) and their functional properties following heterologous expression.


    OATs
TOP
ABSTRACT
INTRODUCTION
OATs
OCTs
COMPARATIVE ASPECTS OF OATs...
REFERENCES

Structure of OATs

Figure 1 depicts an alignment of six transporters of the OAT family. A rat renal PAH transporter was cloned independently in two laboratories [OAT1 (46); ROAT1 (48)]. It shares 96% of similar amino acids with the mouse renal OAT, cloned as the first member of this family and originally named NKT (25), and 91% with the human homologue, hOAT1, which was cloned in four laboratories [hROAT1 (40), hOAT1 (19, 39), hPAHT (27)]. More distantly related is the flounder renal OAT fOAT1 [fROAT (75)], which shares 57-58% of similar amino acids with rat, mouse, and human OAT1. The hOAT3 shows 51-52%, and rat OAT2 [NLT (47)] 45-46%, similarities, respectively, with the OAT1 proteins. rOAT3, which was cloned only recently (23), is not included in the alignment.


View larger version (134K):
[in this window]
[in a new window]
 
Fig. 1.   Comparison of 6 cloned organic anion transporters (OATs). SPTrEMBL or PIR accession numbers of sequences used are as follows (when only nucleotide sequence was available, its accession number is given in parentheses): rOAT1, o35956; mOAT1, q61185; hOAT1, (af057039); fOAT1, o57379; hOAT3, (af097491); rOAT2, q63314. Alignment was performed with PileUp program of Genetics Computer Group software package, version 9.1. Amino acids identical in at least all OAT1 from different species are shaded in gray. Consensus sequences common to all members of sugar porter family are underlined (see text).

Despite the different nomenclature, we shall use "OAT" for these OATs throughout this review, particularly because the OCTs, the first of which have been cloned earlier than the OATs, are abbreviated as OCT.

The shaded areas in Fig. 1 indicate amino acids conserved at least among rat, mouse, human, and flounder OAT1. Conserved regions, e.g., positions 1-40, 154-192, 352-394, 435-480, 517-531 in Fig. 1, may belong to functionally important parts of the protein. Of the 247 amino acids shared between these renal transporters, 104 are common also to OAT2 and OAT3. Among the amino acids conserved between all OATs are 4 cysteines and 10 prolines, which may be of importance for the secondary protein structure. Conserved charged amino acids comprise 1 aspartate, 6 glutamates, 2 lysines, and 10 arginines. Some of the positively charged lysines and arginines may be involved in the binding of the organic anions.

Some of these common amino acids belong to pairs of motifs (underlined in Fig. 1) conserved throughout the sugar porter family [SP; transporter commission (TC) no. 2.1.1] within the major facilitator superfamily (MFS; TC number 2.1) (36). These motif pairs are 1) G-(X)3-D-R/K-X-G-R-R/K (positions 165-174 in Fig. 1) and D-R/K-X-G-R (402-406; in OATs only G-R in positions 405-406 is conserved); 2) E-(X)6-R (twice; 224-231 and 459-466); and 3) P-E-S-P-R-X-L (281-287; in OATs P/X-E-S-X-R-W-L/X) and P-E-T-K (518-521; P-E-T-K/L), respectively. The occurrence of motif pairs suggested that MFS proteins have evolved by gene duplication (36). On the basis of structural data, rOAT1 was assigned TC no. 2.1.19.4 within the family of OCTs (TC no. 2.1.19; see Web site http://www-biology.ucsd.edu/~msaier/transport/titlepage/html). Other members of the OAT family have not yet been classified.

Table 1 summarizes the properties of the cloned OATs. They are 535-568 amino acids long. From human kidney two cDNAs differing only in length have been isolated that code for a 550 (19, 27, 39, 40)- and a 563 (19)-amino acid protein. Depending on the algorithm used, 7-12 transmembrane domains (TMD) were predicted for ROAT1 (48). Most authors, however, assumed 12 TMDs. Between the first and second predicted TMDs, a long hydrophilic loop is found in all OATs (and in all OCTs; cf. Fig. 3). Because this loop carries two to six potential N-glycosylation sites (N-X-S/T), it is most probably localized at the extracellular side of the plasma membrane. Inhibition of glycosylation by tunicamycin in mOAT1-expressing COS-7 cells resulted in an intracellular accumulation of newly synthesized transporters, suggesting that glycosylation is required for insertion of OAT1 into the plasma membrane (24).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Properties of cloned organic anion transporters

In all OATs, two or more potential protein kinase C phosphorylation sites (S/T-X-R/K) have been described. PAH transport by rOAT1 (73) and hOAT1 (27) expressed in Xenopus laevis oocytes or in HeLa cells, respectively, was inhibited after treatment of cells with phorbol esters. Staurosporin prevented the phorbol ester-induced inhibition of PAH transport, suggesting an inhibitory role of protein kinase C on OAT1. Similarly, fluorescein transport in killifish tubules (31), 2,4-dichlorophenoxyacetate secretion in flounder renal tubules (15), and PAH uptake into opossum kidney cells (49) were inhibited by phorbol esters, and staurosporin antagonized the phorbol ester effect. In one study in isolated rabbit S2 proximal tubule segments, an increased PAH uptake was found after exposure to phorbol esters (16). The reason for this discrepancy is not clear. Most studies, however, agree on a downregulation of PAH transport in renal tubules or via the cloned OAT1 by phorbol esters, suggesting that one or more of the identified protein kinase C consensus sequences may actually be utilized for regulation. Putative phosphorylation sites for protein kinase A, casein kinase II, and tyrosine kinase have also been reported. Whether any of these sites is used for the regulation of the OATs is not yet known.

Tissue Distribution of OATs

Except for fOAT1, the organ distribution of the cloned OATs was tested by Northern blots. Rat, mouse, and human OAT1 and human OAT3 mRNAs were most strongly expressed in kidneys and faintly in brain (19, 24, 25, 27, 39, 46, 48). OAT2 and OAT3 from rat, in contrast, showed highest expression in the liver (23, 45, 47). In situ hybridization studies performed in rat (46) and mouse (24) kidneys revealed positive signals in medullary rays of cortex, i.e., in the S2 segments of proximal tubules, the site of maximal proximal tubular PAH secretion (for review see Ref. 38). Immunohistochemical localization also demonstrated that rat (53) and human (19) OAT1 proteins are restricted to the S2 segments of proximal tubules. Within the proximal tubule cells, the antibodies reacted exclusively with the basolateral membrane, proving that OAT1 is a basolateral transporter involved in the uptake of organic anions from the blood.

Monoclonal antibodies against NLT (alias OAT2) labeled the sinusoidal membrane of rat hepatocytes, whereas the bile canalicular membrane was negative, indicating a role of OAT2 in the uptake of organic anions from the blood into the hepatocytes (47).

Functional Characterization and Physiological Importance of OATs

The cloned OATs have been expressed in Xenopus oocytes (4, 19, 23, 39, 45, 46, 48, 73, 75), HeLa cells (27), or COS-7 cells (24). Except for hOAT3, uptake of radiolabeled PAH in cells expressing these carriers was increased compared with water-injected oocytes or mock-transfected cells, respectively. Where investigated, PAH uptake was saturable. The reported Km values range between 5 and 70 µM [5 and 9 µM for hOAT1 (19, 27); 37 µM for mOAT1 (24); 14 and 70 µM for rOAT1 (46, 48); 21 µM for fOAT1 (75); and 65 µM for rOAT3 (23)]. From these data, it seems that the hOAT1 has a higher affinity for PAH than OATs from other species. Uptake of PAH by rOAT1 (46, 48), hOAT1 (27), and fOAT1 (75) was trans-stimulated by intracellularly accumulated alpha -ketoglutarate or glutarate. Similarly, efflux of radiolabeled glutarate from OAT1-expressing oocytes was trans-stimulated by PAH in the incubation medium (1). These data prove the functioning of OAT1 as a PAH/dicarboxylate antiporter. In cells expressing rOAT2 (45), however, preloading with glutarate was without an effect on PAH uptake. Whether rOAT2 functions as a uniporter, as suggested (45), remains to be clarified.

Besides PAH, several radiolabeled organic anions and cations were tested as substrates of heterologously expressed rat OAT1. These include endogenous compounds like alpha -ketoglutarate, cAMP, cGMP (46), folate (73), prostaglandin E2, and urate (46), the nonsteroidal anti-inflammatory drugs acetylsalicylate (aspirin), salicylate, and indomethacin (1), and the cytostatic drug methotrexate (46, 73). The human OAT1 transported alpha -ketoglutarate, but not methotrexate, prostaglandin E2 (27), and urate (39), suggesting species differences (27). Urate was also not translocated by fOAT1 (75) and hOAT3 (39). Labeled estrone sulfate was transported by rOAT3 (23), but not by rOAT2 (45), indicating differences in substrate specificity between OAT2 and OAT3. The organic cation tetraethylammonium (TEA) was not transported by OAT1-3 (39, 45, 46, 48, 73). Rat OAT3, however, accepted the cationic drug cimetidine (23).

Recently, an electrophysiological approach was used to demonstrate translocation of organic anions by fOAT1 expressed in Xenopus oocytes (3). At a concentration of 0.1 mM, PAH and the diuretics bumetanide, ethacrynic acid, and tienilic acid evoked an inward current that was most likely due to the exchange of a monovalent extracellular PAH (or diuretic) against a divalent intracellular alpha -ketoglutarate. Probenecid and, surprisingly, the loop diuretic furosemide did not generate an inward current, suggesting that these two compounds are not measurably translocated by fOAT1. Whether rat or human OAT1 do transport furosemide is unknown at present.

A great number of compounds have been tested as putative inhibitors of heterologously expressed OAT1. Table 2 lists only those compounds that were tested as putative inhibitors of PAH uptake in both rOAT1-expressing oocytes in vitro and rat kidney proximal tubules in vivo. These compounds comprise the second messengers cAMP and cGMP, the local hormone PGE2, the bile salt taurocholate, the end product of purine metabolism, urate, a number of drugs, and a homologous series of dicarboxylates with increasing chain length. It is obvious that rOAT1 interacts with many chemically unrelated compounds, supporting the notion that this transporter is polyspecific. Of particular clinical interest is the interaction with some beta -lactam antibiotics, an antiepilepsy drug, diuretics, an immunosuppressive drug, and a number of nonsteroidal anti-inflammatory drugs, suggesting that OAT1 may play an important role in renal secretion of these compounds. Because so far the actual translocation of these drugs has only been demonstrated for radiolabeled acetylsalicylate, salicylate, and ibuprofen (1), and, in electrophysiological studies, for the diuretics bumetanide, ethacrynic acid, and tienilic acid (3), the contribution of OAT1 in renal clearance of the other drugs remains to be determined.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Cis-inhibition of rOAT1 in vitro and of PAH transport in rat kidney in vivo

The apparent inhibition constant (Ki) values determined in vitro increased for naproxen < ibuprofen < indomethacin ~ salicylurate salicylate < acetylsalicylate < phenacetin paracetamol, indicating the importance of a negative charge and of hydrophobicity for interaction with the OAT1 (1). In the intact kidney, the apparent Ki values increased in the order of salicylurate < indomethacin < naproxen < ibuprofen salicylate ~ phenacetin ~ acetylsalicylate paracetamol (Table 2). These sequences are not identical, and apparent Ki values determined in rOAT1-expressing oocytes are always smaller than those found in vivo. However, both test systems revealed substrates with high affinity (ibuprofen, indomethacin, naproxen, salicylurate), with intermediate (acetylsalicylate, phenacetin, salicylate), and with low affinities (paracetamol), suggesting that rOAT1 reflects the renal PAH transporter. This conclusion is supported by the fact that dicarboxylates interact with rOAT1 only if their chain length exceeds four carbon atoms, as was found earlier for PAH transport in the intact kidney (Table 2). A similar finding was obtained with the OAT1 from flounder kidney (4), highlighting the importance of a proper distance between negative charges in a substrate for OAT1. A few compounds remain that for unknown reasons showed disparate results in vitro and in vivo. These include taurocholate (inhibition only in vivo), urate (mixed results in vitro), and ampicillin (no inhibition in vitro).

Compared with OAT1, relatively few compounds were tested as putative inhibitors of rOAT2 and rOAT3. Among the tested drugs, the diuretic bumetanide inhibited both transporters (23, 45). Another loop diuretic, furosemide, inhibited rOAT3 (23) and was not tested on rOAT2. The antibiotic cefoperazine and the tuberculostatic rifampicin inhibited rOAT2 (45), and the uricosuric drug probenecid, rOAT3 (23). The predominant expression of rOAT2 and rOAT3 in liver (23, 47) suggests that these transporters may be involved in the hepatic clearance of amphiphilic anionic compounds. rOAT3 may also play a role in the absorption of antibiotics from the cerebrospinal fluid (23).


    OCTs
TOP
ABSTRACT
INTRODUCTION
OATs
OCTs
COMPARATIVE ASPECTS OF OATs...
REFERENCES

Structure of OCTs

Figure 2 shows an alignment of 15 cloned OCTs ordered according to the degree of their relatedness. Among different mammalian species, the closest relationship exists for OCTN2 from rat [originally named UST2 (43)], mouse (26, 33), and human (50, 77) with 89-98% amino acid similarities. OCTN1 and OCTN2 share 37-41% similar amino acids with OCT3, OCT2, and OCT1. Rat (20) and mouse (74) OCT3 show 59-60% similarities to rat (34), mouse (accession no. AJ006036), human (10), and porcine (11) OCT2, and 56-57% similarities to the OCT1 transporters, respectively. Among themselves, the cloned OCT2 transporters share 83-92% similar amino acids, and 72-76% with rat (13), mouse [named Lx1 (44)], rabbit (52), and human (10, 79) OCT1, respectively. Among the OCT1 proteins of all species, 82-96% of the amino acids are similar.



View larger version (12672K):
[in this window]
[in a new window]
 
Fig. 2.   Comparison of 15 cloned (putative) organic cation transporters (OCTs). SPTrEMBL or PIR accession numbers of sequences used are as follows (when only nucleotide sequence was available, its accession number is given in parentheses): rOCTN2, o70594; mOCTN2, (AF111425); hOCTN2, o76082; hOCTN1, o14546; mOCTN1, (AB016257); rOCT3, o88446; mOCT3, (AF078750); rOCT2, p70485; mOCT2, o70577; hOCT2, o15244; pOCT2, o02713; rOCT1, i58089; mOCT1, o08966; rbOCT1, 077504; hOCT1, o15245. Alignment was performed as in Fig. 1. Amino acids identical in at least OCT1-3 from different species are shaded in gray. Consensus sequences common to all members of sugar porter family are underlined (see text).

As shown in Fig. 2, 285 amino acids are common to OCT1 and OCT2. Among OCT1, OCT2, and OCT3, 188 amino acids are identical, and between all OCTs and OCTNs 92 amino acids are conserved. Among the amino acids shared by OCTs and OCTNs are 4 cysteines and 13 prolines, suggesting an importance of these residues for secondary structure of the proteins. Conserved charged amino acids include three aspartic acids, six glutamic acids, and seven arginines that may be involved in either maintaining secondary structure through salt bridges or binding of charged substrates. As described earlier for the OATs, shared amino acids partly belong to pairs of conserved motifs (underlined in Fig. 2): 1) G-(X)3-D-R/K-X-G-R-R/K (positions 173-182), D-R/K-X-G-R (407-411); 2) E-(X)6-R (233-240 and 464-471); 3) P-E-S-P-R-X-L (290-296) and P-E-T-K (523-527), respectively (22, 44, 79). On the basis of these motifs, the OCT family has been assigned the TC no. 2.1.19 (Web site see above).

Structural properties of these transporters are summarized in Table 3. Most of them are between 551 and 557 amino acids long, with exception of rOCT2, which has 593 amino acids. Hydrophobicity analysis predicted 11 (hOCTN1, rOCT1) or 12 (all other family members) transmembrane-spanning alpha -helical domains (TMD1-TMD12). All OCT proteins share a large hydrophilic loop between TMD1 and TMD2, which carries two to five potential N-glycosylation sites and is most probably located at the extracellular side of the cell membrane (cf. Fig. 3). Antibodies generated against the large loop bound to nonpermeabilized human embryonic kidney (HEK-293) cells expressing rat OCT1, proving the extracellular localization of this loop (30). Given an even number of TMDs, both the NH2 and COOH termini must then be located at the cytoplasmic side of the membrane. In support of such a model, antibodies against the COOH terminus of OCT1 and OCT2 reacted with the proteins only after permeabilization of the cells (30).

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Properties of cloned (putative) organic cation transporters



View larger version (62K):
[in this window]
[in a new window]
 
Fig. 3.   Models of rat OCT1 and OAT1. Models are based on an assumption of 12 transmembrane domains. Shaded area, plasma membrane; Y, potential N-linked glycosylation; open circle , an amino acid residue; odash , amino acids D or E; oplus , amino acids K or R; otimes , an H residue. Arrows, negatively charged (OCT1) or positively charged (OAT1) amino acids conserved within OCT or OAT family, respectively (see text).

The N-glycosylation sites at positions 71/72 and 96/97/99 are conserved among OCT1, OCT2 and OCT3; those at positions 56/57, 64, and 91 among the OCTN1 and OCTN2. All proteins possess three to six potential protein kinase C phosphorylation sites. Those at positions 280-289 are found in all OCTs and OCTNs, whereas the OCTNs share a site at position 164. Transport studies with isolated rabbit renal proximal tubules (17) and with cell lines [IHKE-1, LLC-PK1 (18)] indicated a modulation of organic cation transport by phorbol esters. It is, however, not known, which of the potential phosphorylation sites is actually involved in regulation of organic cation transport by protein kinase C. Potential phosphorylation sites for protein kinase A have been found for most OCTs. Occasionally, sites for casein kinase II and tyrosine kinases have been reported. Studies on a regulatory role of protein kinase A, casein kinase II, and tyrosine kinase have not yet been performed.

Tissue Distribution of OCTs

The tissue distribution of the cloned transporters has mostly been investigated by Northern blot techniques. With the exception of human OCT1, which was present only in human liver, mRNA for all other OCTs was found in the kidneys. Expression of mRNA for rabbit OCT1 and rat OCT3 was very weak in kidneys compared with other organs. Published immunohistochemical data are so far available only for the human OCT2 (10), which was detected in distal convoluted tubules. Antibodies against rat OCT1 and OCT2 localized these transporters to the basolateral membrane in proximal tubules (H. Koepsell, personal communication). In situ hybridization experiments on rat kidneys revealed OCT2 mRNA expression in proximal tubules mostly in the outer medulla (14). In rat liver, mRNA for OCT1 was distributed evenly among the lobules, whereas protein expression was restricted to perivenous cells (30), suggesting cell-specific regulation of protein expression beyond gene transcription.

Besides kidneys and liver, mRNA for rat and rabbit OCT1, human OCT2, and rat OCT3 was detected in small intestine, hOCT2, rOCT3, and hOCTN2 also in the brain. A wide tissue distribution was found for human OCTN1 and OCTN2.

Functional Characterization of OCTs

Radiolabeled TEA was taken up by human OCTN1 (51) and OCTN2 (77) expressed in HEK-293 and HeLa cells, respectively. hOCTN2-mediated uptake of [14C]TEA was inhibited by unlabeled choline, cimetidine, MPP, procainamide, and TEA (77), indicating that this transporter interacts with several organic cations. Unlike in HeLa cells, hOCTN2 expressed in HEK-293 cells was unable to transport [14C]TEA (50). The reason for this discrepancy is not clear.

The human OCTN2 transports the zwitterionic carnitine in a sodium-dependent fashion (50). Importantly, an L352R mutation located in a putative membrane-spanning domain is responsible for a carnitine-deficiency syndrome in mice, the juvenile visceral steatosis (26, 33). The human systemic carnitine deficiency is related to mutations leading to a loss in the first two TMDs or to truncated proteins (33). These data, as well as the expression of OCTN2 in all tissues, suggests a general role in carnitine uptake.

The cloned OCT1, OCT2, and OCT3 transporters were heterologously expressed in Xenopus oocytes (5-7, 10, 13, 14, 20, 22, 29, 32, 35, 52, 76, 79), HEK-293 cells (2, 5, 11, 12, 20, 22, 29), Madin-Darby canine kidney cells (72), HeLa cells (20, 80), and HRPE cells (76). Most experiments on substrate specificity have been performed with rat OCT1 as well as with rat and human OCT2. Although uptake of radiolabeled cimetidine (2, 12), quinidine, and quinine (32) could not be demonstrated, choline (7, 10), dopamine (2, 6, 7, 12), epinephrine (2, 12), 5-hydroxytryptamine (5-HT; 2, 6, 12), MPP (2, 6, 7, 12, 13, 29, 52, 79), NMN (7), norepinephrine (2, 12), TEA (2, 7, 12, 13, 72, 80), and tyramine (2, 12) did show an enhanced tracer uptake in oocytes or cells expressing OCT1, proving the broad substrate specificity of this transporter. As expected for an electrogenic transporter, the addition of potential substrates to OCT1-expressing oocytes resulted in an inward current in voltage-clamped oocytes (6, 7). The "inward current" produced by the more hydrophobic "type 2" organic cations, cyanine 386, quinidine, quinine, and tubocurarine, however, later turned out to result from a trans-inhibition of choline efflux from the oocytes (32). Hence, the type 2 organic cations inhibit OCT1, but are not translocated.

The apparent Km values, which have been determined with various organic cations by either tracer flux measurements or electrophysiologically, differed among investigators. For example, for TEA, apparent Km values of 35 µM [rOCT1 expressed in oocytes; electrical current (7)], 38 µM [rOCT1 expressed in Madin-Darby canine kidney cells; tracer uptake (72)], 95 µM [rOCT1 expressed in oocytes, tracer uptake (13)], and 229 µM [hOCT1, expressed in HeLa cells (80)], were found, suggesting both species differences between rat and human OCT1 and the influence of the method, i.e., tracer vs. electrophysiological technique. Similarly, for choline, apparent Km values of 240 µM [rOCT1; oocytes; electrical current (7)]; 620 µM [rOCT1, oocytes; tracer uptake (10)]; and 1,100 µM [rOCT1, oocytes, tracer uptake (7)] were reported, whereas for MPP the apparent Km values were 9.6 (7), 13 (29), 14.6 (79), and 23 µM (52), very close irrespective of whether rat (7, 29), rabbit (52) or human (79) OCT1 was tested.

Collectively, in these experiments MPP turned out as the cation with the lowest Km value. High affinities with Km values below 100 µM were also found for acetylcholine (6), dopamine (6, 7), histamine (6) and 5-hydroxytryptamine (5-HT) (6), and, in three studies TEA (7, 13, 72). NMN [340 µM (7)] and choline (see above) showed moderate affinities toward the expressed OCT1.

Similar to OCT1, expressed OCT2 transported a variety of radiolabeled organic cations. MPP (5, 10), amantadine (5), 5-HT (5), memantine (5), and TEA (10, 11, 35, 72) exhibited high affinities with Km values <100 µM; choline (10), NMN (10), and dopamine [in one study (5)] were moderate (Km < 1,000 µM); and epinephrine (14), histamine (5), 5-HT [in another study (14)], and norepinephrine (5, 14) showed low affinities (Km > 1,000 µM), respectively. For OCT3, relatively few data are available with respect to transport. Radiolabeled dopamine (76), guanidine (20), MPP (76), and TEA (20) were taken up into OCT3-expressing cells. MPP had a high-to-moderate affinity, and TEA, with Km values of 2,500 and 6,200, a low affinity for OCT3.

Many more putative substrates have been tested in cis-inhibition studies by adding them to the incubation medium containing labeled TEA, MPP, or guanidine. Because the kinetics of inhibition were not tested, whether all inhibitors competed with the radiolabeled cations for the substrate binding site or bound to a separate site not related to translocation remains open. It is also not clear whether the inhibitor itself is transported. Table 4 lists only those compounds that were tested on organic cation transport in the intact kidney in vivo and on either rOCT1 or rOCT2 or on both. It is obvious from these selected compounds that OCT1 and OCT2 interact with endogenous and exogenous organic cations of diverse chemical structures. Among the most potent inhibitors of OCT1 were clonidine, cyanine-863, and, in some studies, cimetidine, decynium-22, and MPP, with IC50 values of <10 µM. In other studies, decynium-22 and MPP showed higher IC50 values of up to 100 µM or more. For cimetidine (35), choline, guanidine, 5-HT, and NMN (22, 72) moderate (IC50 < 1,000 µM), and for NMN (35) and dopamine, moderate-to-low (IC50 > 1,000 µM) affinities were found. As far as they are available, the results for OCT2 are comparable.

                              
View this table:
[in this window]
[in a new window]
 
Table 4.   Cis-inhibition of rOCT1 and rOCT2 in vitro and of organic cation transport in rat kidney in vivo

The apparent Ki values determined with the rat kidney in vivo were all higher than those found with heterologously expressed rat OCT1 and OCT2. Several reasons may account for this discrepancy. First, lipophilic organic cations exhibiting low IC50 values in vitro, e.g., cyanine-863, decynium-22, and MPP, may in vivo bind to membranes and interstitial components, reducing their effective concentration at the transporter. Second, experiments on expressed OCT1 and OCT2 required incubation of cells with radiolabeled substrate and test cations for up to 1 h, whereas experiments in intact rat kidneys were done with incubation periods of a few seconds. During more extended incubation periods, lipophilic compounds may accumulate in the cell membrane, resulting in a higher local concentration at the transporter. Third, the lipid compositions of the basolateral cell membrane in vivo and of the membrane of cells used for heterologous expression may differ. Fourth, apparent affinities toward organic cations in the extracellular fluid may depend on organic cations in the trans (intracellular)- compartment and/or on the phosphorylation state of the transporter. Fifth, because depolarization increased the Km values for choline and TEA in rOCT1- and OCT2-expressing oocytes fourfold (7, 35), different membrane potentials may account for part of the discrepancies. Finally, it remains possible that the organic cation transporter investigated in vivo and rOCT1 or OCT2 are not identical. However, a linear relationship between the Ki values for dopamine, 5-HT, MPP, norepinephrine, and TEA observed in vivo and those in rOCT1-expressing HEK-293 cells suggested that OCT1 is responsible for organic cation transport in rat renal proximal tubules (2).

Physiological Functions of Cloned OCT1, OCT2, and OCT3

Electrogenic organic cation transport and broad substrate specificity suggest that renal OCT1 and OCT2 are involved in the first step of secretion, i.e., uptake of organic cations across the basolateral membrane into proximal tubule cells. A decrease in pH inhibited uptake of radiolabeled organic cations via heterologously expressed pOCT2 (11), rOCT3 (20), hOCTN1 (51), and OCTN2 (77), suggesting a competition between protons and organic cations. Therefore, pOCT2 and hOCTN1 were claimed to represent the organic cation/H+ antiporter of the brush-border membrane. However, this conclusion may be premature because organic cation-induced currents were independent of pH in OCT3-expressing oocytes despite the fact that uptake was decreased at acidic pH (20). At present, the molecular identity of the organic cation/H+ antiporter remains not known with certainty.

With regard to the liver, there is little doubt that OCT1 is involved in the uptake of "type 1" organic cations into hepatocytes: a linear relationship existed between Ki values determined in isolated rat hepatocytes and those found with HEK-293 cells expressing rOCT1 (29). Compared with rOCT1, the human OCT1 exhibited either comparable (e.g., clonidine, desipramine, MPP, TEA) or lower affinities (e.g., decynium-22, procainamide, NMN, quinine, vecuronium) for organic cations, suggesting the existence of species differences that may prove important for hepatic uptake and metabolism of cationic xenobiotics in humans (79, 80).

The capability of OCTs to transport monoamine neurotransmitters raised considerable interest in their putative role in the central nervous system. mRNA for rat and human OCT2 was detected in the central nervous system (5, 10), and more detailed analysis located the OCT2 mRNA in cells in various regions including hippocampus, thalamus, and substantia nigra (5, 11). In this context it is interesting that an anti-Parkinsonian drug, amantadine, interacted with OCT2 (5) and that another substrate of OCT2, MPP, is particularly toxic for dopaminergic neurons in the substantia nigra. Hence, OCT2 may play a role in dopamine transport in the brain and be a target for anti-Parkinsonian drugs.

The relatively low affinities to cocaine, corticosterone, desipramine, and reserpine ruled out that OCT1 and OCT2 represent the classical monoamine transporters located at synapses or in synaptic vesicles of the central nervous system (2, 5, 13, 28). On the basis of its high affinity for steroid hormones, rOCT3, originally cloned from placenta (20, 76), has been postulated to be identical to the extraneuronal "uptake 2" system, which handles monoamines. In situ hybridization showed strong signals in cerebral cortex, hippocampus, pontine nucleus, and cerebellum in rat brain (20).


    COMPARATIVE ASPECTS OF OATs AND OCTs
TOP
ABSTRACT
INTRODUCTION
OATs
OCTs
COMPARATIVE ASPECTS OF OATs...
REFERENCES

It has become obvious that transporters for organic anions and organic cations in the basolateral membrane of proximal tubule cells not only share a number of substrates but also have structural similarities. Figure 3 depicts a model for rat OCT1 and rat OAT1. Both have 12 putative TMDs, and large hydrophilic loops between TMD1 and TMD2 as well as between TMD6 and TMD7. The loop between TMD1 and TMD2 carries three (rOCT1) or four (rOAT1) potential glycosylation sites and is therefore most probably facing the extracellular side. NH2 and COOH termini are located inside the cell according to this model. Most charged amino acids are found in the large hydrophilic loops, some of them also in the short loops between TMDs. With regard to specificity for organic cations of rOCT1, it is tempting to search for negatively charged amino acid residues (D, E) which are conserved between OCTs and are not found at a similar position in the OATs. Eight amino acids fulfill these criteria: E in the NH2 terminus (position 13 in rOCT1; conserved in OCT1-3 and OCTN1,2); E and D in the large extracellular loop (positions 69 and 95; OCT1-3); D at the beginning of TMD2 (position 150; OCT1-3); E in the large intracellular loop (position 325; conserved as E in OCT1-2, and as D in OCT3); D at the beginning of TMD8 (position 379; OCT1-3); D at the end of TMD8 (position 399; conserved as D in OCT1-2, and as E in OCT3); and D in the middle of TMD11 (position 475, OCT1-3). On the other hand, the organic anion recognition site in the OATs should involve positively charged amino acids (K, R, and, after ionization, H) that are conserved in all OATs and are not present in the OCTs. There are three amino acids that meet these requirements: H in TMD1 (position 34 in rOAT1, conserved in OAT1-3); K at the end of TMD8 (position 382, OAT1-3); and R in the middle of TMD11 (position 466, OAT1-3). Given the uncertainty of secondary structure predictions, the charged amino acids at the borders of TMDs 2 and 8 in rOCT1, and at TMD8 in rOAT1, may be actually located inside TMDs and participate in the translocation of organic anions. The histidine residue in TMD1 of all OATs is of particular interest, because the histidine reagent diethylpyrocarbonate (DEPC) inhibited PAH transport by mOAT1(24). PAH afforded protection from inhibition by DEPC, suggesting that this histidine residue may be related to the organic anion binding site. A conspicuous difference between OCTs and OATs is the charge in TMD11: OCT1-3 possess a negatively charged aspartate, and OAT1-3, a positively charged arginine. In vitro mutagenesis experiments are underway to determine the role of these residues in organic ion transport.

Perspectives

Expression and homology cloning techniques have revealed a new family of renal and hepatic transporters for amphiphilic organic anions and cations. After heterologous expression, the OCTs have been amply characterized with respect to specificity and affinity for their substrates. For the OATs, the substrate specificity has been tested, but the affinities for most substrates are unknown. Future experiments should not only determine these affinities but also help to distinguish between substrate binding to OATs and OCTs without transport and actual translocation of the competitor. These data are needed to establish a substrate structure-transport relationship, which is important for the understanding of the renal and hepatic handling of drugs.

The high degree of relatedness between organic anion and organic cation transporters offers the unique chance to delineate the binding and transport sites. Particularly conserved regions may represent functionally important domains of the transporter molecules. Positively charged, conserved amino acid residues in OATs, and negatively charged, conserved amino acids in OCTs, may be considered as possible binding sites for organic anions and cations, respectively. The construction and functional characterization of chimera between OATs and OCTs and of transporters with site-specific mutations will help to identify those binding sites. These experiments may also reveal how OATs and OCTs accommodate so many different, chemically unrelated compounds, which could, for example, be rendered possible by a large binding site with several niches.

With respect to renal secretion of organic anions and cations, we are beginning to understand on the molecular level, how organic anions and cations are taken up across the basolateral membrane from the blood into proximal tubule cells. Unfortunately, such knowledge is lacking for the apical membrane of proximal tubule cells. It remains an important task to identify the transporters involved in the exit of organic anions and cations into the primary urine.


    ACKNOWLEDGEMENTS

The authors thank K. J. Ullrich and B. C. Burckhardt for valuable suggestions, and E. Thelen for the artwork.


    FOOTNOTES

The authors' research on flounder and human OAT1 has been supported by Deutsche Forschungsgemeinschaft Grants Bu 571/4 and GRK 335.

Address for reprint requests and other correspondence: G. Burckhardt, Zentrum Physiologie und Pathophysiologie, Humboldtallee 23, D-37073 Göttingen, Germany (E-mail: gburckhardt{at}veg-physiol.med.uni-goettingen.de).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
OATs
OCTs
COMPARATIVE ASPECTS OF OATs...
REFERENCES

1.   Apiwattanakul, N, Sekine T, Chairoungdua A, Kanai Y, Nakajima N, Sophasan S, and Endou H. Transport properties of nonsteroidal anti-inflammatory drugs by organic anion transporter 1 expressed in Xenopus laevis oocytes. Mol Pharmacol 55: 847-854, 1999[Abstract/Free Full Text].

2.   Breidert, T, Spitzenberger F, Gründemann D, and Schömig E. Catecholamine transport by the organic cation transporter type 1 (OCT1). Br J Pharmacol 125: 218-224, 1998[Abstract].

3.   Burckhardt, B-C, Wolff NA, and Burckhardt G. Electrophysiological characterization of an organic anion transporter cloned from winter flounder kidney (fROAT). J Am Soc Nephrol. 11: 9-17, 2000[Abstract/Free Full Text].

4.   Burckhardt, G, Porth J, and Wolff NA. Functional and molecular characterization of renal transporters for p-aminohippurate (PAH). Nova Acta Leopoldina 78: 35-40, 1998.

5.   Busch, AE, Karbach U, Miska D, Gorboulev V, Akhoundova A, Volk C, Arndt P, Ulzheimer JC, Sonders MS, Baumann C, Waldegger S, Lang F, and Koepsell H. Human neurons express the polyspecific cation transporter hOCT2, which translocates monoamine neutrotransmitters, amantadine, and memantine. Mol Pharmacol 54: 342-352, 1998[Abstract/Free Full Text].

6.   Busch, AE, Quester S, Ulzheimer JC, Gorboulev V, Akhoundova A, Waldegger S, Lang F, and Koepsell H. Monoamine neurotransmitter transport mediated by the polyspecific cation transporter rOCT1. FEBS Lett 395: 153-156, 1996[ISI][Medline].

7.   Busch, AE, Quester S, Ulzheimer JC, Waldegger S, Gorboulev V, Arndt P, Lang F, and Koepsell H. Electrogenic properties and substrate specificity of the polyspecific rat cation transporter rOCT1. J Biol Chem 271: 32599-32604, 1996[Abstract/Free Full Text].

8.   David, C, Rumrich G, and Ullrich KJ. Luminal transport system for H+/organic cations in the rat proximal tubule. Kinetics, dependence on pH; specificity compared with the contraluminal organic cation-transport system. Pflügers Arch 430: 477-492, 1995[ISI][Medline].

9.   Dekant, W, and Vamvakas S. Biotransformation and membrane transport in nephrotoxicity. Crit Rev Toxicol 26: 309-334, 1996[ISI][Medline].

10.   Gorboulev, V, Ulzheimer JC, Akhoundova A, Ulzheimer-Teuber I, Karbach U, Quester S, Baumann C, Lang F, Busch AE, and Koepsell H. Cloning and characterization of two human polyspecific organic cation transporters. DNA Cell Biol 16: 871-881, 1997[ISI][Medline].

11.   Gründemann, D, Babin-Ebell J, Martel F, Örding N, Schmidt A, and Schömig E. Primary structure and functional expression of the apical organic cation transporter from kidney epithelial LLC-PK1 cells. J Biol Chem 272: 10408-10413, 1997[Abstract/Free Full Text].

12.   Gründemann, D, Breidert T, Spitzenberger F, and Schömig E. Molecular structure of the carrier responsible for hepatic uptake of catecholamines. Adv Pharmacol 42: 346-349, 1998[Medline].

13.   Gründemann, D, Gorboulev V, Gambaryan S, Veyhl M, and Koepsell H. Drug excretion mediated by a new prototype of polyspecific transporter. Nature 372: 549-552, 1994[ISI][Medline].

14.   Gründemann, D, Köster S, Kiefer N, Breidert T, Engelhardt M, Spitzenberger F, Obermüller N, and Schömig E. Transport of monoamine transmitters by the organic cation transporter type 2, OCT2. J Biol Chem 273: 30915-30920, 1998[Abstract/Free Full Text].

15.   Halpin, PA, and Renfro JL. Renal organic anion secretion: evidence for dopaminergic and adrenergic regulation. Am J Physiol Regulatory Integrative Comp Physiol 271: R1372-R1379, 1996[Abstract/Free Full Text].

16.   Hohage, H, Löhr M, Querl U, and Greven J. The renal basolateral transport system for organic anions: properties of the regulation mechanism. J Pharmacol Exp Ther 269: 659-664, 1994[Abstract].

17.   Hohage, H, Mörth DM, Querl IU, and Greven J. Regulation by protein kinase C of the contraluminal transport system for organic cations in rabbit kidney S2 proximal tubules. J Pharmacol Exp Ther 268: 897-901, 1994[Abstract].

18.   Hohage, H, Stachon A, Feidt C, Hirsch JR, and Schlatter E. Regulation of organic cation transport in IHKE-1 and LLC-PK1 cells. Fluorometric studies with 4-(4-dimethylaminostyryl)-N-methylpyridinium. J Pharmacol Exp Ther 286: 305-310, 1998[Abstract/Free Full Text].

19.   Hosoyamada, M, Sekine T, Kanai Y, and Endou H. Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney. Am J Physiol Renal Physiol 276: F122-F128, 1999[Abstract/Free Full Text].

20.   Kekuda, R, Prasad PD, Wu X, Wang H, Fei Y-J, Leibach FH, and Ganapathy V. Cloning and functional characterization of a potential-sensitive, polyspecific organic cation transporter (OCT3) most abundantly expressed in placenta. J Biol Chem 273: 15971-15979, 1998[Abstract/Free Full Text].

21.   Koepsell, H. Organic cation transporters in intestine, kidney, liver, and brain. Annu Rev Physiol 60: 243-266, 1998[ISI][Medline].

22.   Koepsell, H, Gorboulev V, and Arndt P. Molecular pharmacology of organic cation transporters in kidney. J Membr Biol 167: 103-117, 1999[ISI][Medline].

23.   Kusuhara, H, Sekine T, Utsunomiya-Tate N, Tsuda M, Kojima R, Cha SH, Sugiyama Y, Kanai Y, and Endou H. Molecular cloning and characterization of a new multispecific organic anion transporter from rat brain. J Biol Chem 274: 13675-13680, 1999[Abstract/Free Full Text].

24.   Kuze, K, Graves P, Leahy A, Wilson P, Stuhlmann H, and You G. Heterologous expression and functional characterization of a mouse renal organic anion transporter in mammalian cells. J Biol Chem 274: 1519-1524, 1999[Abstract/Free Full Text].

25.   Lopez-Nieto, CE, You GF, Bush KT, Barros EJG, Beier DR, and Nigam SK. Molecular cloning and characterization of NKT, a gene product related to the organic cation transporter family that is almost exclusively expressed in the kidney. J Biol Chem 272: 6471-6478, 1997[Abstract/Free Full Text].

26.   Lu, K-M, Nishimori H, Nakamura Y, Shima K, and Kuwajima M. A missense mutation of mouse OCTN2, a sodium-dependent carnitine transporter, in the juvenile visceral steatosis mouse. Biochem Biophys Res Commun 252: 590-594, 1998[ISI][Medline].

27.   Lu, R, Chan BS, and Schuster VL. Cloning of the human kidney PAH transporter: narrow substrate specificity and regulation by protein kinase C. Am J Physiol Renal Physiol 276: F295-F303, 1999[Abstract/Free Full Text].

28.   Martel, F, Ribeiro L, Calhau C, and Azevedo I. Comparison between uptake2 and rOCT1: effects of catecholamines, metanephrines and corticosterone. Naunyn Schmiedebergs Arch Pharmacol 359: 303-309, 1999[ISI][Medline].

29.   Martel, F, Vetter T, Russ H, Gründemann D, Azevedo I, Koepsell H, and Schömig E. Transport of small organic cations in the rat liver. The role of the organic cation transporter OCT1. Naunyn Schmiedebergs Arch Pharmacol 354: 320-326, 1996[ISI][Medline].

30.   Meyer-Wentrup, F, Karbach U, Gorboulev V, Arndt P, and Koepsell H. Membrane localization of the electrogenic cation transporter rOCT1 in rat liver. Biochem Biophys Res Commun 248: 673-678, 1998[ISI][Medline].

31.   Miller, DS. Protein kinase C regulation of organic anion transport in renal proximal tubule. Am J Physiol Renal Physiol 274: F156-F164, 1998[Abstract/Free Full Text].

32.   Nagel, G, Volk C, Friedrich T, Ulzheimer JC, Bamberg E, and Koepsell H. A reevaluation of substrate specificity of the rat cation transporter rOCT1. J Biol Chem 272: 31953-31956, 1997[Abstract/Free Full Text].

33.   Nezu, J-I, Tamai I, Oku A, Ohashi R, Yabuuchi H, Hashimoto N, Nikaido H, Sai Y, Koizumi A, Shoji Y, Takada G, Matsuishi T, Yoshino M, Kato H, Ohura T, Tsujimoto G, Hayakawa J-I, Shimane M, and Tsuji A. Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter. Nat Genet 21: 91-94, 1999[ISI][Medline].

34.   Okuda, M, Saito H, Urakami Y, Takano M, and Inui K-I. cDNA cloning and functional expression of a novel rat kidney organic cation transporter, OCT2. Biochem Biophys Res Commun 224: 500-507, 1996[ISI][Medline].

35.   Okuda, M, Urakami Y, Saito H, and Inui K-I. Molecular mechanisms of organic cation transport in OCT2-expressing Xenopus oocytes. Biochim Biophys Acta 1417: 224-231, 1999[ISI][Medline].

36.   Pao, SS, Paulsen IT, and Saier MH, Jr. Major facilitator superfamily. Microbiol Mol Biol Rev 62: 1-34, 1998[Abstract/Free Full Text].

37.   Pritchard, JB, and Miller DS. Proximal tubular transport of organic anions and cations. In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW, and Giebisch G.. New York: Raven, 1992, p. 2921-2945.

38.   Pritchard, JB, and Miller DS. Mechanisms mediating renal secretion of organic anions and cations. Physiol Rev 73: 765-796, 1993[Free Full Text].

39.   Race, JE, Grassl SM, Williams WJ, and Holtzman EJ. Molecular cloning and characterization of two novel human renal organic anion transporters (hOAT1 and hOAT3). Biochem Biophys Res Commun 255: 508-514, 1999[ISI][Medline].

40.   Reid, G, Wolff NA, Dautzenberg FM, and Burckhardt G. Cloning of a human renal p-aminohippurate transporter, hROAT1. Kidney Blood Press Res 21: 233-237, 1998[ISI][Medline].

41.   Roch-Ramel, F, Besseghir K, and Murer H. Renal excretion and tubular transport of organic anions and cations. In: Handbook of Physiology. Renal Physiology. Bethesda, MD: Am. Physiol. Soc, 1992, sect. 8, vol. II, chapt. 48, p. 2189-2262.

42.   Roch-Ramel, F, and Diezi J. Renal transport of organic ions and uric acid. In: Diseases of the Kidney, edited by Schrier RW, and Gottschalk CW.. Boston, MA: Little, Brown, 1997, p. 231-249.

43.   Schömig, E, Spitzenberger F, Engelhardt M, Martel F, Örding N, and Gründemann D. Molecular cloning and characterization of two novel transport proteins from rat kidney. FEBS Lett 425: 79-86, 1998[ISI][Medline].

44.   Schweifer, N, and Barlow DP. The Lx1 gene maps to chromosome 17 and codes for a protein that is homologous to glucose and polyspecific transmembrane transporters. Mamm Genome 7: 735-740, 1996[ISI][Medline].

45.   Sekine, T, Cha SH, Tsuda M, Apiwattanakul N, Nakajima N, Kanai Y, and Endou H. Identification of multispecific organic anion transporter 2 expressed predominantly in the liver. FEBS Lett 429: 179-182, 1998[ISI][Medline].

46.   Sekine, T, Watanabe N, Hosoyamada M, Kanai Y, and Endou H. Expression cloning and characterization of a novel multispecific organic anion transporter. J Biol Chem 272: 18526-18529, 1997[Abstract/Free Full Text].

47.   Simonson, GD, Vincent AC, Roberg KJ, Huang Y, and Iwanij V. Molecular cloning and characterization of a novel liver-specific transport protein. J Cell Sci 107: 1065-1072, 1994[Abstract/Free Full Text].

48.   Sweet, DH, Wolff NA, and Pritchard JB. Expression cloning and characterization of ROAT1. J Biol Chem 272: 30088-30095, 1997[Abstract/Free Full Text].

49.   Takano, M, Nagai J, Yasuhara M, and Inui K-I. Regulation of p-aminohippurate transport by protein kinase C in OK kidney epithelial cells. Am J Physiol Renal Fluid Electrolyte Physiol 271: F469-F475, 1996[Abstract/Free Full Text].

50.   Tamai, I, Ohashi R, Nezu J-I, Yabuuchi H, Oku A, Shimane M, Sai Y, and Tsuji A. Molecular and functional identification of sodium-ion-dependent, high affinity human carnitine transporter OCTN2. J Biol Chem 273: 20378-20382, 1998[Abstract/Free Full Text].

51.   Tamai, I, Yabuuchi H, Nezu J-I, Sai Y, Oku A, Shimane M, and Tsuji A. Cloning and characterization of a novel human pH-dependent organic cation transporter, OCTN1. FEBS Lett 419: 107-111, 1997[ISI][Medline].

52.   Terashita, S, Dresser MJ, Zhang L, Gray AT, Yost SC, and Giacomini KM. Molecular cloning and functional expression of a rabbit renal organic cation transporter. Biochim Biophys Acta 1369: 1-6, 1998[ISI][Medline].

53.   Tojo, A, Sekine T, Nakajima N, Hosoyamada M, Kanai Y, Kimura K, and Endou H. Immunohistochemical localization of multispecific renal organic anion transporter 1 in rat kidney. J Am Soc Nephrol 10: 464-471, 1999[Abstract/Free Full Text].

54.   Ullrich, KJ. Specificity of transporters for "organic anions" and "organic cations" in the kidney. Biochim Biophys Acta 1197: 45-62, 1994[ISI][Medline].

55.   Ullrich, KJ. Renal transporters for organic anions and organic cations. Structural requirements for substrates. J Membr Biol 158: 95-107, 1997[ISI][Medline].

56.   Ullrich, KJ. Features of substrates for interaction with renal transporters of organic anions and cations. Nova Acta Leopoldina 78: 23-34, 1998.

57.   Ullrich, KJ, Fritzsch G, Rumrich G, and David C. Polysubstrates: substances that interact with renal contraluminal PAH, sulfate, and NMeN transport: sulfamoyl-, sulfonylurea-, thiazide- and benzenamino-carboxylate (nicotinate) compounds. J Pharmacol Exp Ther 269: 684-692, 1994[Abstract].

58.   Ullrich, KJ, Papavassiliou F, David C, Rumrich G, and Fritzsch G. Contraluminal transport of organic cations in the proximal tubule of the rat kidney. I. Kinetics of N1-methylnicotinamide and tetraethylammonium, influence of K+, HCO-3, pH; inhibition by aliphatic primary, secondary and tertiary amines, and mono- and bisquaternary compounds. Pflügers Arch 419: 84-92, 1991[ISI][Medline].

59.   Ullrich, KJ, and Rumrich G. Contraluminal transport systems in the proximal renal tubule involved in secretion of organic anions. Am J Physiol Renal Fluid Electrolyte Physiol 254: F453-F462, 1988[Abstract/Free Full Text].

60.   Ullrich, KJ, and Rumrich G. Contraluminal renal anion and cation transport systems: interaction with fatty acids, eicosanoids, Krebs cycle intermediates, amino acids and analogues, cyclic nucleotides and steroid hormones. In: The Frontiers of Nephrology, edited by Berliner RW, Honda N, and Ullrich KJ.. Amsterdam: Elsevier, 1990, p. 55-65.

61.   Ullrich, KJ, and Rumrich G. Luminal transport system for choline+ in relation to the other organic cation transport systems in the rat proximal tubule. Kinetics, specificity: alkyl/arylamines, alkylamines with OH, O, SH, NH2, ROCO, RSCO and H2PO4-groups, methylaminostyryl, rhodamine, acridine, phenanthrene and cyanine compounds. Pflügers Arch 432: 471-485, 1996[ISI][Medline].

62.   Ullrich, KJ, Rumrich G, David C, and Fritzsch G. Bisubstrates: substances that interact with both, renal contraluminal organic anion and organic cation transport systems. II. Zwitterionic substrates: dipeptides, cephalosporins, quinolone-carboxylate gyrase inhibitors and phosphamide thiazine carboxylates; Nonionizable substrates: steroid hormones and cyclophosphamides. Pflügers Arch 425: 300-312, 1993[ISI][Medline].

63.   Ullrich, KJ, Rumrich G, David C, and Fritzsch G. Bisubstrates: substances that interact with renal contraluminal organic anion and organic cation transport systems. I. Amines, piperidines, piperazines, azepines, pyridines, quinolines, imidazoles, thiazoles, guanidines and hydrazines. Pflügers Arch 425: 280-299, 1993[ISI][Medline].

64.   Ullrich, KJ, Rumrich G, David C, and Fritzsch G. Interaction of xenobiotics with organic anion and cation transport systems in renal proximal tubule cells. In: Renal Disposition and Nephrotoxicity of Xenobiotics, edited by Anders MW, Dekant W, Henschler D, Oberleithner H, and Silbernagl S.. San Diego, CA: Academic, 1993, p. 97-115.

65.   Ullrich, KJ, Rumrich G, Fritzsch G, and Klöss S. Contraluminal para-aminohippurate (PAH) transport in the proximal tubule of the rat kidney. II. Specificity: aliphatic dicarboxylic acids. Pflügers Arch 408: 38-45, 1987[Medline].

66.   Ullrich, KJ, Rumrich G, Gemborys M, and Dekant W. Transformation and transport: how does metabolic transformation change the affinity of substrates for the renal contraluminal anion and cation transporters? Toxicol Lett 53: 19-27, 1990[ISI][Medline].

67.   Ullrich, KJ, Rumrich G, and Klöss S. Contraluminal para-aminohippurate transport in the proximal tubule of the rat kidney. III. Specificity: monocarboxylic acids. Pflügers Arch 409: 547-554, 1987[ISI][Medline].

68.   Ullrich, KJ, Rumrich G, and Klöss S. Contraluminal organic anion and cation transport in the proximal renal tubule: V. Interaction with sulfamoyl- and phenoxy diuretics, and with beta -lactam antibiotics. Kidney Int 36: 78-88, 1989[ISI][Medline].

69.   Ullrich, KJ, Rumrich G, Neiteler K, and Fritzsch G. Contraluminal transport of organic cations in the proximal tubule of the rat kidney. II. Specificity: anilines, phenylalkylamines (catecholamines), heterocyclic compounds (pyridines, quinolines, acridines). Pflügers Arch 420: 29-38, 1992[ISI][Medline].

70.   Ullrich, KJ, Rumrich G, Papavassiliou F, Klöss S, and Fritzsch G. Contraluminal p-aminohippurate transport in the proximal tubule of the rat kidney. VII. Specificity: cyclic nucleotides, eicosanoids. Pflügers Arch 418: 360-370, 1991[ISI][Medline].

71.   Ullrich, KJ, Rumrich G, Wieland T, and Dekant W. Contraluminal para-aminohippurate (PAH) transport in the proximal tubule of the rat kidney. VI. Specificity: amino acids, their N-methyl-, N-acetyl- and N-benzoylderivatives; glutathione- and cysteine conjugates, di- and oligopeptides. Pflügers Arch 415: 342-350, 1989[ISI][Medline].

72.   Urakami, Y, Okuda M, Masuda S, Saito H, and Inui KI. Functional characteristics and membrane localization of rat multispecific organic cation transporters, OCT1 and OCT2, mediating tubular secretion of cationic drugs. J Pharmacol Exp Ther 287: 800-805, 1998[Abstract/Free Full Text].

73.   Uwai, Y, Okuda M, Takami K, Hashimoto Y, and Inui K-I. Functional characterization of the rat multispecific organic anion transporter OAT1 mediating basolateral uptake of anionic drugs in the kidney. FEBS Lett 438: 321-324, 1998[ISI][Medline].

74.   Verhaagh, S, Schweifer N, Barlow DP, and Zwart R. Cloning of the mouse and human solute carrier 22a3 (Slc22a3/SLC22A3) identifies a conserved cluster of three organic cation transporters on mouse chromosome 17 and human 6q26-q27. Genomics 55: 209-218, 1999[ISI][Medline].

75.   Wolff, NA, Werner A, Burkhardt S, and Burckhardt G. Expression cloning and characterization of a renal organic anion transporter from winter flounder. FEBS Lett 417: 287-291, 1997[ISI][Medline].

76.   Wu, X, Kekuda R, Huang W, Fei YJ, Leibach FH, Chen J, Conway SJ, and Ganapathy V. Identity of the organic cation transporter OCT3 as the extraneuronal monoamine transporter (uptake2) and evidence for the expression of the transporter in the brain. J Biol Chem 273: 32776-32786, 1998[Abstract/Free Full Text].

77.   Wu, X, Prasad PD, Leibach FH, and Ganapathy V. cDNA sequence, transport function, and genomic organization of human OCTN2, a new member of the organic cation transporter family. Biochem Biophys Res Commun 246: 589-595, 1998[ISI][Medline].

78.   Zhang, L, Brett CM, and Giacomini KM. Role of organic cation transporters in drug absorption and elimination. Annu Rev Pharmacol Toxicol 38: 431-460, 1998[ISI][Medline].

79.   Zhang, L, Dresser MJ, Gray AT, Yost SC, Terashita S, and Giacomini KM. Cloning and functional expression of a human liver organic cation transporter. Mol Pharmacol 51: 913-921, 1997[Abstract/Free Full Text].

80.   Zhang, L, Schaner ME, and Giacomini KM. Functional characterization of an organic cation transporter (hOCT1) in a transiently transfected human cell line (HeLa). J Pharmacol Exp Ther 286: 354-361, 1998[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 278(6):F853-F866
0363-6127/00 $5.00 Copyright © 2000 the American Physiological Society