News Physiol Sci 13: 11-16, 1998;
1548-9213/98 $5.00
News in Physiological Sciences, Vol. 13, No. 1, 11-16,
February 1998
© 1998 Int. Union Physiol. Sci./Am. Physiol. Soc.
Structure and Function of Renal Organic Cation Transporters
Hermann Koepsell,
Andreas Busch,
Valentin Gorboulev and
Petra Arndt
H. Koepsell, V. Gorboulev, and P. Arndt are at the Institute of Anatomy, University of Würzburg, Koellikerstr. 6, 97070 Würzburg, Germany; A. Busch at the Institute of Physiology, University of Tübingen, Gmelinstr. 5, 72076 Tübingen, Germany.
 |
Abstract
|
|---|
Polyspecific transport systems in the kidney mediate the excretion and reabsorption of organic cations. Electrogenic import systems and electroneutral export systems in the basolateral and luminal plasma membranes of proximal renal tubules are involved. Two subtypes of electrogenic import systems have been cloned from rats and humans and functionally characterized.
|
Introduction
|
|---|
Many ingested drugs like quinine and salicylate are absorbed in the small intestine and enter the liver where they are transported into hepatocytes. From the hepatocytes, the drugs may be excreted into the bile or reabsorbed into the blood. In the kidney, the drugs can be excreted by ultrafiltration in the glomeruli or by secretion in the proximal tubules. For the excretion of hydrophobic compounds, which are mainly bound to plasma proteins and do not pass the filtration barrier in the glomeruli, high-affinity transport systems in the proximal tubules are required. Ultrafiltrated drugs or endogeneous compounds like choline or monamine neurotransmitters may also be reabsorbed. In the luminal and basolateral membranes of renal proximal tubules, polyspecific transport systems for anions and cations have been described that are responsible for the reabsorption and secretion of drugs and xenobiotics in the kidney (11, 12).
|
Four polyspecific transport systems mediate reabsorption and excretion of organic cations in renal proximal tubules
|
|---|
Cation transport in the renal proximal tubule has been studied by microperfusion experiments and by uptake measurements with membrane vesicles (11–15). In microperfusion experiments, cation uptake across the basolateral and luminal membranes was determined. These experiments allowed the distinction of three different transport systems for cations that appear to be polyspecific and are sufficient to describe secretion and reabsorption of cations in the proximal tubule. Furthermore, P-glycoprotein, which is involved in multidrug resistance, has been localized to the luminal membrane of renal proximal tubules. This transporter also participates in the excretion of hydrophobic cations (8). Figure 1
summarizes the different cation transport systems in the proximal tubule.
View larger version (22K):
[in this window]
[in a new window]
|
FIGURE 1. Polyspecific transport systems for organic cations described in rat renal proximal tubules. A potential-dependent cation (Cat+) transporter (system 1) was localized in the basolateral membrane, and the electroneutral H+/cation antiporter (system 2) was localized in the brush-border membrane and in intracellular vesicles. Brush-border membrane contains another potential-dependent cation transporter (system 3) and P-glycoprotein (system 4). Na+/H+ antiporter in the brush-border membrane and H+-ATPase in intracellular vesicles are also indicated.
|
|
The first step in cation secretion in proximal tubules is the uptake from the interstitium. This step is mediated by a polyspecific uptake system in the basolateral membrane (Fig. 1
, system 1), which is accelerated by an inside negative membrane potential and is independent of Na+ and pH (12, 13). A similar transport system may be present in the sinusoidal membrane of hepatocytes (4). Experiments with perfused livers and with isolated hepatocytes suggest that two separate uptake systems exist in liver: type 1 transports small hydrophilic cations such as choline or the model substrate procainamide ethobromide, and type 2 transports large cations such as the cationic drugs quinine and d-tubocurarine. The second step in renal cation excretion, the cation transport out of tubular epithelial cells, is mediated by a potential-independent H+/cation antiporter (Fig. 1, system 2). This transport system is also polyspecific and appears to be electroneutral (11, 12). It is energized by the inwardly directed H+ gradient, which is generated by the luminal Na+/H+ antiporter (see Fig.1). An H+/cation antiporter has also been detected in intracellular vesicles (10). Here, the H+ gradient generated by the H+-adenosinetriphosphatase (ATPase) is the driving force for the accumulation of cations. It is not clear whether the vesicles serve as buffer compartments for cations and whether exocytosis of these vesicles contributes significantly to the transcellular cation movement. The functional role of P-glycoprotein in the brush-border membrane (Fig. 1, system 4) is not fully understood. It may be responsible for the secretion of hydrophobic cations that are not transported by the luminal H+/cation antiporter.
In the renal proximal tubule, organic cations may also be reabsorbed from the primary filtrate (11). For example, this has been demonstrated for the endogenous cation choline, which is reabsorbed at low and secreted at high plasma concentrations. An electrogenic cation transporter in the brush-border membrane(Fig. 1, system 3) is probably responsible for cation reabsorption. Because this transporter has a relatively high affinity for choline, it was primarily designated as a choline transporter (15). However, this transporter is also polyspecific (14, 15). The apparent Michaelis-Menten (Km) constant values of several cations for this transporter were distinctly different from the Km values that were estimated for the luminal H+/cation antiporter and the potential-dependent cation uptake system in the basolateral membrane.
The direction of transcellular cation movement in the proximal tubule is determined by the functional properties of the engaged transporters in the basolateral and luminal membranes and by the acting driving forces. The transporter properties include substrate specificity, transport mechanisms, and regulation of transporter function and expression. The driving forces are dependent on the intracellular and extracellular cation concentrations, the membrane potentials, and/or the transmembrane H+ gradients. Cloning and functional characterization of the individual transporters is required to understand the reabsorption and secretion of cations.
|
Identification of a new family of polyspecific cation transporters
|
|---|
Employing expression cloning with a cDNA library from rat kidney and screening for uptake of the model cation tetraethylammonium (TEA), we isolated a gene (rOCT1) that encodes an integral membrane protein with 556 amino acids (5). rOCT1 is a member of a new transporter family. It is localized on chromosome 1q11–12 (6) and is expressed in renal proximal tubules, hepatocytes, and small intestinal epithelial cells (5). Figure 2 shows a schematic representation of the transporter. Hydropathy plots predicted 11 transmembrane 
-helices. However, an additional transmembrane domain (Fig. 2, transmembrane 4) was assumed, since it was predicted in recently cloned homologous transporters. We localized the first hydrophilic loop to the extracellular membrane side because several glycosylation sites were predicted on this loop. In the topology proposed in Fig. 2, the second large hydrophilic loop is localized intracellularly. This loop contains several potential protein kinase C-dependent phosphorylation sites. They may be functional sites for transport regulation, since activation of protein kinase C alters TEA transport in proximal tubules from rabbit kidney (for literature, see Ref. 5) as well as in Xenopus oocytes expressing rOCT1. rOCT1 contains four short intracellular consensus sequences that have been described in nutrient transporters of different genetic families (for literature, see Ref. 5).
After expression of rOCT1 in Xenopus oocytes, highly active uptake of radioactively labeled TEA was induced that could be inhibited by organic cations with different molecular structures such as cyanine 863, quinine, procainamide, 1-methyl-4-phenylpyridinium (MPP), O-methylisoprenaline, dopamine, norepinephrine, serotonin, tetramethylammonium, and choline (1, 2, 5, 7). The apparent affinities of the different cationic inhibitors vary by four orders of magnitude and are independent of the employed expression system. To elucidate whether the different compounds are transported, some were tested by tracer uptake measurements in Xenopus oocytes. Saturable transport by rOCT1 was demonstrated for N1-methylnicotinamide (NMN), choline, dopamine, and MPP and could be inhibited by cyanine 863 and quinine (1, 2). Cation transport by rOCT1 was tested for symmetry and transstimulation by performing efflux measurements with rOCT1-expressing oocytes that were injected with radioactively labeled MPP (1). Expressed MPP efflux was observed when no cations were on the outside (trans-zero). Cation transport was transinhibited by cyanine 863 and transstimulated by TEA or MPP.
With rOCT1-injected oocytes, several electrical measurements were also performed (1, 2). When different cations were added to the bath, the membrane potential was decreased, suggesting electrogenic transport by rOCT1. Employing voltage-clamped oocytes, we investigated currents induced by the addition of cations to the bath. Whereas in noninjected oocytes no significant currents were induced by 1 mM of different cations, in rOCT1-injected oocytes inward currents between 20 and 100 nA were observed. The cation concentrations that induced half-maximal currents were decreased with increasing membrane potential (1, 2). At a clamped membrane potential of -50 mV, the half-maximal concentrations for TEA, choline, MPP, and dopamine were similar to the Km values determined from the tracer uptake measurements. Significant currents were also induced when the membrane potential was clamped to zero (2). These data indicate that the inward currents induced by TEA, NMN, choline, MPP, and dopamine represent electrogenic transport and suggest that cation transport by rOCT1 is driven by both the membrane potential and the concentration gradient. Electrogenic cation transport by rOCT1 is not dependent on pH or Na+, since the currents induced by TEA and choline were not altered by pH changes in the bath that were between 6.5 and 8.5 or by replacing extracellular NaCl with D-glucose (2). Interestingly, similar maximal transport rates were estimated for substrates with Km values differing by two orders of magnitude.
We employed inhibitors of TEA uptake for electrical measurement experiments to test whether they induced currents. In addition, we tested the polyamines spermine and spermidine, the neurotransmitter acetylcholine, and the divalent muscle relaxants pancuronium and d-tubocurarine, which have been classified as type 2 substrates in the liver (4). When added to the bath of rOCT1-expressing oocytes that were clamped at –50 mV, all these cations induced inwardly directed currents. Cation-induced inward currents can also be generated by transinhibition of the outflux of endogeneous cations through rOCT1; future tracer flux experiments are needed to elucidate which of these cations are actually transported by rOCT1. Preliminary data showed that quinine, which is a high-affinity inhibitor of TEA transport by rOCT1 and induces inward currents in voltage-clamped oocytes, is not transported by rOCT1.
The data indicate that rOCT1 is a new prototype of polyspecific transporter that can only be compared with P-glycoprotein in efficiency and polyspecificity. Whereas P-glycoprotein is an ATP-dependent export system for hydrophobic substrates that accumulate in the lipid membrane, rOCT1 is an import pump that transports cations with different molecular structures into cells. The cations are removed from the aqueous phase, and the driving force may be provided by the cation gradient and the membrane potential. In situ hybridization experiments and polymerase chain reactions with reversely transcribed mRNAs isolated from microdissected nephron segments showed that rOCT1 mRNA is transcribed in rat renal proximal tubules (unpublished observations). A functional comparison of electrogenic cation transport, expressed by rOCT1 with the potential-dependent cation uptake observed in the luminal and basolateral membrane of rat subcortical proximal tubules by microperfusion experiments (13, 14), suggests that rOCT1 is responsible for the basolateral uptake in these tubules. For example, the apparent Km values for uptake of TEA (0.1 mM) by rOCT1 were identical to the Km value determined for basolateral uptake in vivo. The order of magnitude was different from the in vivo Km value of TEA (16 mM ) estimated for the luminal uptake (14).
|
rOCT1 homologous genes from rats and humans with distinct functions have been isolated
|
|---|
By homology screening of renal cDNA libraries, one homologous transporter from rat named rOCT2 (2, 9) and two homologous transporters from humans termed hOCT1 and hOCT2 (3) were isolated. In addition, an OCT1-type cDNA from mouse (mOCT1) was cloned (GenBank accession no. U38652). Figure 3 shows a schematic representation of the five homologous genes. Twelve presumed membrane-spanning domains are indicated. The first hydrophilic loop is highly conserved and has three stretches of 8–14 amino acids that are identical in the OCT genes. This loop contains eight conserved acidic amino acid residues that may be engaged in cation transport. In the presumed large intracellular loop, one potential protein kinase C dependent phosphorylation site is conserved that may play a role in regulation (see above).
View larger version (19K):
[in this window]
[in a new window]
|
FIGURE 3. Schematic representation of open reading frames of rOCT1 (Ref. 5) and homologous genes from rat (rOCT2, Refs. 2, 9), mouse (mOCT1, GenBank accession no. U38652), and humans (hOCT1 and hOCT2, Ref. 3). Presumed membrane-spanning domains (1–12), potential glycosylation sites (Y-like symbol), and potential phosphorylation sites [protein kinase C dependent ( ), protein kinase A dependent ( ), tyrosine kinase dependent ( ) and casein kinase dependent ( )] are indicated.
|
|
For the distribution of the OCT1 and OCT2 transporters, distinct species differences were observed. In rat, large amounts of rOCT1 mRNA were detected in kidney, liver, and small intestine, whereas, in humans, large amounts of hOCT1 mRNA could be detected only in liver. The transcription of OCT2 appears to be more tissue specific because large mRNA amounts of rOCT2 and hOCT2 were detected only in kidney. In situ hybridization experiments with rat and human kidney suggested that rOCT2 and hOCT2 are transcribed in proximal and distal tubules. Immunohistochemical studies in human kidney revealed that hOCT2 is located at the luminal membrane (3).
rOCT2, hOCT1, and hOCT2 were functionally expressed in Xenopus oocytes and/or in a human embryonic kidney cell line. With rOCT2 and hOCT2, highly active cation transport could be expressed, whereas only small uptake rates were obtained with hOCT1. Similar to rOCT1 transport of TEA, choline, NMN, and MPP could also be expressed by rOCT2, hOCT1, and hOCT2. The uptake by these transporters was also independent of Na+ or pH and was also stimulated by an inside negative membrane potential. Similar to rOCT1, cation-induced inward currents could be demonstrated in voltage-clamped oocytes expressing rOCT2 and hOCT2. In Table 1, the substrate specificities for cation uptake by rOCT1 (2, 5), rOCT2 (unpublished observations), and hOCT2 (3) are compared. Apparent Km values were determined for uptake of TEA, choline, NMN, and MPP, and apparent inhibitor constant (Ki) values were estimated for various other cations. The data indicate differences between the identical OCT subtypes in humans and rat and between different OCT subtypes in rat. The affinity for transport by and/or for binding to the OCT-type transporters is dependent on cation stucture and the properties of the specific OCT-type transporters. Different structural features of the cations such as positive charge, charge distribution, hydrophobicity, and size of the cations are surely important; however, none of these features will be sufficient to predict the cation interaction with one of these transporters.
The data show that mammals have OCT-type cation transporters that are polyspecific and independent of Na+ and pH. These transporters probably function as cellular import systems, since they are driven by the membrane potential. In the kidney, OCT1-type transporters are probably localized in the basolateral membrane of subcortical renal proximal tubules and are engaged in cation excretion. The localization of the OCT2-type transporters has not been finally determined and the luminal electrogenic cation import system may an OCT-homologous transporter which has not been identified. Renal cation transport is especially important for the excretion of xenobiotics and cationic drugs, whereas cation reabsorption in the kidney plays an important role in the homeostasis of endogenous cations such as choline, NMN, polyamines, and monoamine neurotransmitters.
|
Concluding remarks
|
|---|
To understand renal reabsorption and secretion of endogeneous and exogeneous organic cations in humans, the cation transporters in the basolateral and luminal membranes of proximal and distal tubules must be identified, localized and their functional properties determined. Cloning of two different cation import systems from rat and humans, which belong to a new transporter family, is an important step in this direction, since the functional characterization of individual cation transport systems has now become possible. The characterization of expressed cation transporters performed so far reveals new insights into cation transport: 1) the potential-dependent cellular import systems for cations belong to the OCT family, whereas the cellular export system(s), the H+/cation antiporter(s), probably belongs to a different transporter family; 2) the potential-dependent organic cation transporters in the kidney are polyspecific as has been postulated from in vivo measurements; 3) during potential-dependent cation transport in the kidney, electrical charge is transferred over the plasma membrane; and 4) the potential-dependent cation transporters may operate in both directions. To obtain a more profound understanding of cation transport by OCT-type transporters, functional studies have to be performed in an oocyte preparation in which the buffer composition on both sides of the membrane can be controlled and in which rapid kinetics can be performed. Thereby, different steps in the transport cycle may be distinguished and the transporters may be investigated for symmetry. In addition, transported cations have to be distinguished from those that bind to the substrate binding site but are not transported. It is of high theoretical interest to identify and characterize the cation binding site(s) of the polyspecific OCT-type transporter to obtain a deeper understanding of polyspecific cation transport.
|
Acknowledgments
|
|---|
The authors' work was supported by grants from the Deutsche Forschungsgemeinschaft.
|
References
|
|---|
-
Busch, A. E., S. Quester, J. C. Ulzheimer, V. Gorboulev, A. Akhoundova, S. Waldegger, F. Lang, and H. Koepsell. Monoamine neurotransmitter transport mediated by the polyspecific cation transporter rOCT1. FEBS Lett. 395: 153–156, 1996.[Medline]
-
Busch, A. E., S. Quester, J. C. Ulzheimer, S. Waldegger, V. Gorboulev, P. Arndt, F. Lang, and H. Koepsell. Electrogenic properties and substrate specificity of the polyspecific rat cation transport rOCT1. J. Biol. Chem. 271: 32599–32604, 1996.[Abstract/Free Full Text]
-
Gorboulev, V., J. C. Ulzheimer, A. Akhoundova, I. Ulzheimer-Teuber, U. Karbach, S. Quester, C. Baumann, F. Lang, A. E. Busch, and H. Koepsell. Cloning and characterization of two polyspecific organic cation transporters from man. DNA Cell Biol. 16: 871–881, 1997.[Medline]
-
Groothuis, G. M. M., and D. K. F. Meijer. Drug traffic in the hepatobiliary system. J. Hepatol. 24: 3–28, 1996.
-
Gründemann, D., V. Gorboulev, S. Gambaryan, M. Veyhl, and H. Koepsell. Drug excretion mediated by a new prototype of polyspecific transporter. Nature 372: 549–552, 1994.[Medline]
-
Koehler, M. R., V. Gorboulev, H. Koepsell, C. Steinlein, and M. Schmid. Roct1, a rat polyspecific transporter gene for the excretion of cationic drugs maps to chromosome 1q11-12. Mamm. Genome 7: 247–248, 1996.[Medline]
-
Martel, F., T. Vetter, H. Russ, D. Gründemann, I. Azevedo, H. Koepsell, and E. Schömig. Transport of small organic cations in the rat liver: the role of the organic cation transporter OCT1. Naunyn Schmiedeberg's Arch. Pharmacol. 354: 320–326, 1996.[Medline]
-
Miller, D. S. Daunomycin secretion by killifish renal proximal tubules. Am. J. Physiol. 269 (Regulatory Integrative Comp. Physiol. 38): R370–R379, 1995.[Abstract/Free Full Text]
-
Okuda, M., H. Saito, Y. Urakami, M. Takano, and K.-I. Inui. cDNA cloning and functional expression of a novel rat kidney organic cation transporter, OCT2. Biochem. Biophys. Res. Commun. 224: 500–507, 1996.[Medline]
-
Pritchard, J. B., D. B. Sykes, R. Walden, and D. S. Miller. ATP-dependent transport of tetraethylammonium by endosomes isolated from rat renal cortex. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F966–F976, 1994.[Abstract/Free Full Text]
-
Roch-Ramel F., K. Besseghir, and H. Murer. 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.
-
Ullrich, K. J. Specificity of transporters for "organic anions" and "organic cations" in the kidney. Biochim. Biophys. Acta 1197: 45–62, 1994.[Medline]
-
Ullrich, K. J., F. Papavassiliou, C. David, G. Rumrich, and G. Fritzsch. 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 bisquarternary compounds. Pflügers Arch. 419: 84–92, 1991.[Medline]
-
Ullrich, K. J., and G. Rumrich. Luminal transport system for choline+ in relation to the other organic cation transport systems in the rat proximal tubule. Pflügers Arch. 424: 1–15, 1996.
-
Wright, S. H., T. M. Wunz, and T. P. Wunz. A choline transporter in renal brush-border membrane vesicles: energetics and structural specificity. J. Membr. Biol. 126: 51–65, 1992.[Medline]
This article has been cited by other articles:
|
|
|
|
 
B. M. Schmitt and H. Koepsell
Alkali Cation Binding and Permeation in the Rat Organic Cation Transporter rOCT2
J. Biol. Chem.,
July 1, 2005;
280(26):
24481 - 24490.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Cetinkaya, G. Ciarimboli, G. Yalcinkaya, T. Mehrens, A. Velic, J. R. Hirsch, V. Gorboulev, H. Koepsell, and E. Schlatter
Regulation of human organic cation transporter hOCT2 by PKA, PI3K, and calmodulin-dependent kinases
Am J Physiol Renal Physiol,
February 1, 2003;
284(2):
F293 - F302.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. H. Sweet and J. B. Pritchard
rOCT2 is a basolateral potential-driven carrier, not an organic cation/proton exchanger
Am J Physiol Renal Physiol,
December 1, 1999;
277(6):
F890 - F898.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Introduction
Four polyspecific transport...
