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Christopher Volk*DOI:&10.1002/wmts.100
Wiley Interdisciplinary Reviews: Membrane Transport and Signaling pages 1&13, Author InformationDepartment of Natural Sciences, Bonn-Rhein-Sieg University of Applied Sciences, Rheinbach, Germany*Correspondence to: Publication HistoryIssue published online: 24 DEC 2013Article first published online: 18 DEC 2013Manuscript Accepted: 4 NOV 2013Manuscript Received: 24 OCT 2013Manuscript Revised: 24 OCT 2013
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The SLC22 protein family includes more than 30 different proteins that operate as transporters for organic cations (OCTs), organic cations and zwitterions (OCTNs) or organic anions (OATs). These transporters play a pivotal role in the secretion of organic ions in kidney and liver. Furthermore, they contribute to the homeostasis of organic ions in several other tissues such as brain, placenta, intestine, and lung. Substrates include not only endogenous compounds such as monoamine neurotransmitters, choline, carnitine, &-ketoglutarate, urate, or steroid hormones, but also a broad spectrum of therapeutic drugs. Therefore, they control the bioavailability of many drugs and are responsible for several side effects or drug&drug interactions. SLC22 proteins are polyspecific transporters as they are able to transport structurally different compounds. All SLC22 proteins share a common membrane topology with 12 &-helical transmembrane domains. Mutational analyses and homology modeling of the steric structure of the proteins led to the conclusion that they possess a large cleft that is accessible from the aqueous phase. Located within this cleft is an inner cavity containing different interaction sites for different substrates. During the transport cycle the transporter undergoes conformational changes including an outward-open conformation, a transient occluded state and an inward-open conformation. WIREs Membr Transp Signal &13. doi: 10.1002/wmts.100Conflict of interest: The author has declared no conflicts of interest for this article.For further resources related to this article, please visit the .A large number of endogenous metabolites as well as xenobiotic drugs are organic ions, and uptake, distribution, and excretion of these compounds are strongly depending on the expression of transport systems for organic ions. Members of the solute carrier (SLC) 22 gene family are transporters for organic cations, anions, and zwitterions, thus playing a major role in the homeostasis of organic ions. Additionally, members of the SLC0, SLC47, and ATP-binding cassette protein (ABC) families that are not subjects of this review are involved in the transport of organic ions.Since the cloning of the first SLC22 transporter, rOCT1 from rat kidney, many SLC22A proteins have been cloned, including 23 human proteins, of which 13 were functionally characterized. The SLC22 family includes subgroups of transporters for organic cations (OCTs), organic cations and zwitterions (OCTNs), and organic anions (OATs). The transporters within each subgroup overlap in substrate spectra and tissue distribution. For this reason, it is difficult to clarify the physiological role of each individual SLC22 protein in every tissue. The function of these transporters, however, is most significant in kidney and liver.In the kidney, they show the strongest expression in proximal tubule cells (Figure ). Here, secretion of organic ions in renal tubules is a crucial process for the elimination of a vast number of endogenous compounds as well as xenobiotics. This process involves two steps. First, the compounds are taken up across the basolateral membrane into the tubular cells and following, they are released over the luminal membrane into the primary urine. For many organic cations the basolateral uptake is mainly performed by OCTs. In humans, this is accomplished by OCT2, while in rodents OCT1 and OCT2 together fulfill this function. The luminal release of cations is primarily managed by other transporters such as the multidrug and toxin extrusion transporters of the SLC47 family (MATE1, MATE2K) or ABC proteins (P-glycoprotein). Furthermore, OCT1 might also contribute to tubular reabsorbtion of cations as it was also detected at the tubular luminal membrane.Figure&1. SLC22 transporters in a human renal proximal tubule cell. Abbreviations: DC, OA+, OC&, ZI, zwitterion. The difference in line thickness in case of the OCTs indicates that uptake of organic cations is the favored direction of transport.The first step in tubular secretion of organic anions is mediated by OAT1, OAT2, and OAT3 in humans and OAT1 and OAT3 in rodents, respectively. These transporters are expressed in the basolateral membrane of the tubules and take up organic anions in exchange for &-ketoglutarate, succinate, or fumarate. The counterions are supplied by sodium-coupled dicarboxylate transporters. In the luminal membrane different OATs are expressed in humans (OAT4, OAT10, URAT1) and rodents (OAT2, OAT5, OAT9, OAT10, URAT1). These transporters mainly manage tubular reabsorption of various organic anions. A contribution to the luminal secretion has also been discussed since most SLC22 transporters principally are able to operate bidirectionally, depending on the apparent driving forces such as concentration gradients or the membrane potential. Other transporters such as ABC transporters (MDR2, MDR4) and organic anion transporting polypeptides (OATPs) of the SLC0 family might play a more important role in organic anion secretion at the luminal membrane. Additionally, OCTN1 and OCTN2 are expressed in the tubular luminal membrane. OCTN1 contributes to the luminal secretion of organic cations and the reabsorption of zwitterions, while the main function of OCTN2 is the reabsorption of l-carnitine.In the liver, SLC22 transporters have only been detected at the sinusoidal membrane of hepatocytes. Here they mediate the uptake of organic cations and anions as the first step of biliary secretion. In humans, OCT1 and OCT3 are responsible for the uptake of cations into hepatocytes while in rodents only OCT1 fulfills this function. Organic anion uptake is performed by OAT2 and OAT7. The luminal secretion is mainly performed by MATE1 and ABC transporters as MDR1 for organic cations and by various ABC transporters (MDR2, BCRP, BSEP) for organic anions.The coexpression of transporters with partially overlapping ranges of substrates might enlarge the total range of transported compounds in a particular tissue. Furthermore, this could increase the concentration range for a substrate that can be sufficiently controlled by a tissue since related transporters often differ significantly in affinity to specific substrates. Guanidine, for instance, is transported with a 10-fold higher affinity by rOCT1 compared with rOCT2.The importance of SLC22 transporters is further highlighted by the finding that defects of these proteins are linked to several diseases such as systemic carnitine deficiency or Crohn's disease. Because a vast number of drugs are substrates of SLC22 transporters, they are also responsible for drug&drug interactions and severe side effects of drugs, e.g., the nephrotoxicity of platinum compounds during the treatment of cancer. Furthermore, several single nucleotide polymorphisms have been identified for many SLC22 proteins and some of them strongly alter activity or specificity of transport. This may be responsible for interindividual differences in the response to therapeutic drugs, as has been reported in case of metformin.As a common feature, the characterized SLC22A transporters are polyspecific, e.g., they are able to translocate molecules with different structures. The modes of transport, however, differ between the subgroups. They might work as facilitated diffusion systems (OCTs, OCTNs), antiporters (OCTNs, OATs), or cotransporters (OCTNs) and some of these transporters can change their transport mode according to the substrate.In addition to the transported substrates, numerous compounds have been identified that are not transported but inhibit the activity of SLC22 proteins. This group includes endogenous compounds such as steroid hormones as well as a large number of different drugs. Because this topic has been addressed extensively in previous reviews it is not discussed in detail in this review.In total, more than 30 different SLC22 transporters have been cloned with identified orthologs in a broad variety of species. Here, we focus on the transporters of humans and rodents that have been functionally characterized (Table ) (for a complete overview of the cloned SLC22 proteins in human, mouse and rat see Ref ). A particular emphasis is put on structural properties of these transporters and their polyspecific binding sites.Table&1.&Characterized SLC22 Proteins from Humans and RodentsProteinHuman Gene SymbolRodent Gene SymbolPredominant SubstratesTransport ModeOCT1SLC22A1Slc22a1Organic cationsFacilitated diffusionOCT2SLC22A2Slc22a2Organic cationsFacilitated diffusionOCT3SLC22A3Slc22a3Organic cationsFacilitated diffusionOCTN1SLC22A4Slc22a4Organic cations, zwitterionsFacilitated diffusionCotransport with Na+Exchange for H+OCTN2SLC22A5Slc22a5Zwitterions, organic cationsCotransport with Na+Facilitated diffusionOCTN3&Slc22a21CarnitineFacilitated diffusionOCT6SLC22A16&CarnitineCotransport with Na+OAT1SLC22A6Slc22a6Organic anionsExchange for dicarboxylatesOAT2SLC22A7Slc22a7Organic anionsExchange for dicarboxylates or glutamateOAT3SLC22A8Slc22a8Organic anionsExchange for dicarboxylatesOAT4SLC22A11&Organic anions Exchange for dicarboxylates (influx) or Cl& (efflux) OAT5SLC22A10&n.i.n.i.oat5&Slc22a19Estron-3-sulfateExchange for dicarboxylates?OAT6SLC22A20Slc22a20Organic anionsExchange for dicarboxylatesOAT7SLC22A9&Estron-3-sulfate, DHEASExchange for short chain fatty acidsOAT8&Slc22a24Organic anionsExchange for dicarboxylatesOAT9&Slc22a27Zwitterions, organic anionsn.i.OAT10SLC22A13Slc22a13Nicotinate, urateExchange for lactate or succinateURAT1SLC22A12Slc22a12UrateExchange for organic or inorganic anionsThis group includes three transporters (OCT1-3) that have been cloned and characterized in several species, including human, mouse, rat, and rabbit. All of them operate as uniporters, managing facilitated diffusion of organic cations driven by the electrochemical gradient. The negative membrane potential therefore strongly supports cellular uptake of organic cations and a significant efflux can only be expected in the presence of a strong outward-oriented concentration gradient or in case of depolarization of the membrane potential.OCT1 (SLC22A1)OCT 1 was first cloned from rat and following from humans. In rat, a functional active splice variant lacking trans-membrane domain 1 (TMD 1) was also identified. OCT1 is strongly expressed in the sinusoidal membrane of hepatocytes. Additionally, weaker expression levels of hOCT1 have been shown in a broad variety of tissues, including small intestine, lung, heart, kidney, skeletal muscle, brain, placenta, eye, adrenal gland, immune cells, and skin. In rodents, in addition to the liver, a strong expression of OCT1 was also observed in the kidney. Substrates of OCT1 include endogenous compounds such as choline, acetylcholine, and monoamine neurotransmitters such as serotonin, dopamine, and agmatine, the polyamine putrescine, model compounds such as tetraethyl-ammonium (TEA), 1-methyl-4-phenylpyridinium (MPP+), N-methylnicotinamide (NMN), and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP), and drugs such as the antidiabetic metformine, antiviral drugs such as lamivudine and acyclovir, and antineoplastics such as oxaliplatin, picoplatin, or sorafenib. In addition, toxins such as aflatoxin B1 or ethidiumbromide are also transported. (For a detailed overview on substrates and inhibitors of OCTs see Ref .) The major function of OCT1 most likely is mediating the uptake of organic cations in hepatocytes as the initial step of biliary secretion. Moreover, in humans it might contribute to the reabsorption of organic cations from the primary urine as OCT1 has been detected at the luminal membrane of proximal and distal tubules. In contrast, in rodents the transporter is located at the basolateral membrane of proximal tubules. Here it is, together with OCT2, mediating the initial step of tubular secretion of organic cations. Additionally to its hepatic and renal function, OCT1 may be involved in the absorption of organic cations in the small intestine and in the passage of substrates over the blood&brain barrier.OCT2 (SLC22A2)As OCT1, OCT2 was cloned first in rat, followed by human. Additionally, a splice variant lacking the last three TMDs was identified in humans. In humans as well as in rodents, OCT2 is strongly expressed in the basolateral membrane of kidney proximal tubules. Furthermore, its expression in lung, placenta, brain, small intestine, thymus, and inner ear has been described. The substrate specificity of OCT1 and OCT2 is broadly overlapping, although significant differences in affinities to several compounds, e.g., dopamine or guanidine were described. Substrates include neurotransmitters such as acetylcholine, epinephrine, norepinephrine, dopamine, histamine, other endogenous compounds such as choline, putrescine, model compounds such as TEA, MPP, ASP or NMN; and drugs such as the antineoplastics cisplatin, oxaliplatin, picoplatin, the antiparkinsonian drug amantadine, the antidemential drug memantine, histamine receptor antagonists cimetidine, famotidine and ramitidine, the antidiabetic metformine as well as the antiviral drugs lamivuidine and zalcitabine. The toxins aflatoxin B1, ethidium bromide, and paraquat are also transported. Furthermore, a transport of inorganic cations such as Cs+ and Cd2+ has been described.OCT2 plays its most important role in the kidney. Here, it is responsible for the cellular uptake of organic cations as the first step of tubular secretion, while the luminal release of cations mainly is mediated by transporters of the multidrug and toxin extrusion family (MATE, SLC47). In several regions of the brain, e.g., hippocampus, it is supposed to be involved in the control of extracellular neurotransmitter concentrations and may facilitate the transport of several drugs across the blood&brain barrier. In the lung, OCT2 may mediate epithelial release of acetylcholine during non-neuronal cholinergic regulation.OCT3 (SLC22A3)OCT3 was cloned in parallel from rat and human. OCT3 is expressed in a broad variety of tissues, including brain, heart, skeletal muscle, liver, lung, kidney, small intestine, skin, mammary gland, and placenta. The substrate spectrum of OCT3 appears to be smaller than that of OCT1 and 2 and includes the neurotransmitters epinephrine, norepinephrine, histamine and agmatine, the model compound MPP and several drugs such as metformin, oxaliplatin, lamivudine, the anaesthetic lidocaine, the antiarrhythmic quinidine, and the antihypotensive drug etilefrine In the liver, OCT3 is expressed at the sinusoidal membrane of hepatocytes and mediates, together with OCT1, the initial step in biliary excretion of organic cations. In brain, OCT3 is expressed in several regions including cortex, hippocampus, and substantia nigra. Here it is supposed to contribute to the regulation of aminergic neurotransmission and a deficiency of OCT3 in the brain of mice lead to behavioral alterations in response to stress and anxiety. In other organs such as lung, skin, or placenta, OCT3 may also be involved in the release of acetylcholine during non-neuronal cholinergic regulations. Although the level of expression of OCT3 is relatively high in heart and skeletal muscle, its function in these tissues remains to be clarified.This subgroup includes four different transporters. Two of them have been identified in both humans and rodents. The other two are only present in humans or rodents, respectively. These transporters are able to translocate organic cations as well as zwitterions and can operate in different transport modes.OCTN1 (SLC22A4)In 1997, human OCTN1 was cloned as the first OCTN (organic cation transporter novel). The mouse ortholog was cloned 3 years later The OCTN1 protein shows only &30% sequence similarity to the OCTs and is expressed in a broad range of tissues including kidney, intestine, spleen, heart, skeletal muscle, brain, mammary gland, thymus, airways, and male reproductive tract. Interestingly, OCTN1 can operate in different modes. Zwitterions may be translocated in a sodium-dependent and a sodium-independent mode while organic cations are transported independently of sodium. Because OCTN1 can also act as a proton/organic cation exchanger it was initially presumed that OCTN1 might be the key player for secretion of organic cations at the luminal membrane of kidney proximal tubules. Meanwhile, it is generally agreed that transporters of the MATE family mainly fulfill this function. However, OCTN1 might also contribute. Its physiological role remains unclear, although it has been proposed that the antioxidant ergotheionine is the most relevant physiological substrate and the sodium-dependent uptake of ergotheionine protects cells from oxidative stress. Furthermore, OCTN1 might also contribute to drug excretion in the kidney as, e.g., quinidine, verapamil, gabapentin, and oxaliplatin are accepted as substrates. Carnitine is only transported with low affinity. Uptake of carnitine therefore seems to be no relevant function of OCTN1. Interestingly, impairment of OCTN1 function has been linked to the development of autoimmune or inflammatory diseases such as Crohn's disease or rheumatoid arthritis, although the exact pathomechanisms have not been clarified yet.OCTN2 (SLC22A5)This transporter was identified in parallel in human and rat shortly after hOCTN1. The protein is expressed in kidney, skeletal muscle, placenta, heart, prostate, and thyroid and to a lesser extent in many other organs including small intestine, liver, lung, or brain. Its predominant function is the cotransport of carnitine and sodium. Carnitine is required for the transport of fatty acids in the mitochondria via a carnitine/acylcarnitine exchanger. In the kidney, OCTN2 is involved in the reabsorption of carnitine in the proximal tubule, in small intestine it contributes to the resorption of carnitine, in heart and skeletal muscle it is needed for the intracellular accumulation of carnitine and in the placenta OCTN2 might contribute to the fetal supply with carnitine. Impairment of OCTN2 function leads to systemic carnitine deficiency (SCD), a disease that might cause cardiomyopathy and progressive skeletal weakness. In addition to sodium-dependent carnitine transport, OCTN2 is also able to work as a sodium-independent uniporter for organic cations including TEA, choline, verapamil, or spironolacton.OCTN3 (Slc22a21)Another carnitine transporter, mOCTN3, was identified in mice, while a human ortholog was not detected. It is highly expressed in the testis and to a lesser extent in the kidney. Carnitine is supposed to be an important factor for sperm maturation and motility and mOCTN3 might be relevant for the supply of sperms with carnitine in the testis. Additionally, mOCTN3 has been identified in the peroxisomal membrane of hepatocytes. Here, it may perform the uptake of carnitine, thus driving the carnitine/acylcarnitine shuttle which is needed for the uptake of fatty acids that are degraded in the peroxisomes. Interestingly, in contrast to mOCTN1 and mOCTN2, carnitine transport by mOCTN3 is Na+-independent.OCT6 (SLC22A16)In humans, an additional carnitine transporter, OCT6 (initially named CT2) was cloned. No ortholog has been identified in rodents. The protein has only 33% sequence similarity to the OCTNs and is therefore not classified as a member of the OCTN subfamily but shows similar functional characteristics as Na+ dependency and a high affinity to carnitine. The strongest expression of OCT6 is observed in testis and epididymis. Additionally, a lower level of expression is seen in embryonic liver, hematopoietic cells, and several cancer cells. In the testis, OCT6 may supply sperms with carnitine, similar to mOCTN3 in mice.Several subtypes of OATs have been identified in different species. Seven subtypes in humans and 10 in rodents have been cloned and functionally characterized.OAT1 (SLC22A6)OAT1 was cloned first in rat and following in human. One functional active and two inactive splice variants were detected additionally. OAT1 is strongly expressed in the kidney, where it is located at the basolateral membrane of proximal tubules. Lower levels of expression have been observed in brain, skeletal muscle, and placenta. In the kidney it acts as an uptake system for organic anions, exchanging them against intracellular glutarate or &-ketoglutarate. Thus, its physiological role mainly is the first step in renal secretion of anions. Additionally, it might also play a role in renal anion reabsorption, as it can mediate bidirectional transport. The substrates of OAT1 include urate, medium chain fatty acids, prostaglandins, cAMP and cGMP, and the glutathione precursor cysteinyl glycine as well as a variety of drugs like tetracycline, acyclovir, cimetidine, ibuprofen, or furosemide. (For a detailed overview on substrates and inhibitors of OATs see Ref .)OAT2 (SLC22A7)OAT2 was first cloned in rat liver and initially named novel liver-specific transport protein (NLT), the human ortholog was identified some years later. This transporter is strongly expressed in liver and kidney and in lower amounts in testis, small intestine, and uterus. However, significant species differences exist in tissue expression. In human kidney it is located in the basolateral membrane of proximal tubule cells, whereas in rodents it has been detected in the apical membrane of proximal tubules. In humans, therefore, the major task in the kidney might be the first step of renal secretion, while in rodents OAT2 appears to be mainly important for tubular reabsorption. OAT2 functions as an anion exchanger and transports glutamate, glutarate, urate, purine, and pyrimidine bases and a broad spectrum of nucleotides and nucleosides, prostaglandins, estrone-3-sulfate (ES) as well as various drugs and toxins including salicylate, tetracycline, methothrexate, and aflatoxin B1. In the liver its physiological role might be the release of glutamate into the sinusoids as well as the uptake of various anions into the hepatocytes as the first step of biliary excretion.OAT3 (SLC22A3)This transporter was cloned from humans and rats in 1999 and characterized during the following years. It is strongly expressed in the kidney and to a low amount in several organs including CNS and the adrenal gland. OAT3 transports a broad range of organic anions and some uncharged substances. This includes endogenous compounds such as urate, ES, dehydroepiandrosterone sulfate (DHEAS), cholate, taurocholate, cAMP, cortisol, prostaglandins, vanilmandelic acid, or glucuronide conj many drugs such as antibiotics (benzylpenicillin, tetracycline), antiviral drugs (valacyclovir, zidovudine, adefovir,&cidofovir), H2 receptor antagonists (cimetidine, famotidine, ranitidine, fexofenadine), diuretics (furosemide, torasemide, bumetanidine), antiinflammatory drugs (indometacin, salicylate, ketoprofen, ibuprofen), cholesterol-lowering drugs (pravastatin, rosuvastatin), cytostatics (methotrexate, topotecan), antihypertensive drugs (quinaprilat), and antidiabetics (sitagliptin). Several toxins as ochratoxin A or aflatoxin B1 are also transported. In the human kidney, where it is located at the basolateral membrane of proximal tubule cells, OAT3 is the most abundantly expressed OAT and therefore plays a crucial role in the initial step of renal secretion of anions such as urate and various anionic drugs.OAT4 (SLC22A11)Human OAT4 was cloned in 2000. A rodent ortholog has not been identified. OAT4 is expressed mainly in kidney and placenta but shows a low level of expression in many tissues including intestine, liver, brain, heart, lung, prostate, skeletal muscle, and testis. It works as an antiporter for organic anions in exchange against dicarboxylates or inorganic anions. Endogenous substrates are ES, urate, prostaglandin E2, and prostaglandin F2. Several anionic drugs such as bumetanide, torasemide, tetracycline, zidovudine, and methotrexate are also transported. Interestingly, OAT4 can operate in different working modes. ES and urate are taken up in exchange against intracellular &-ketoglutarate or hydroxyl ions (influx mode). On the other hand, p-aminohippurate or anionic drugs can be released in exchange against extracellular chloride (efflux mode). Because OAT4 in the kidney is localized at the luminal membrane of proximal tubules, it could contribute to the reabsorption of organic anions from the primary urine as well as to the tubular secretion of anionic drugs. In the placenta, this transporter is located at the basal side of the syncytiotrophoblast where it conducts the uptake of sulfated steroids from the fetal circulation as progenitors for the synthesis of estrogen.OAT5 (SLC22A10/Slc22a19)Human OAT5 has been identified in liver and has not been detected in other organs. Substrates and functions of hOAT5 remain to be clarified. No ortholog of SLC22A10 exists in rodents. In rats and mice an additional anion transporter named Oat5 has been cloned which is encoded by the Slc22a19 gene. No human ortholog to this transporter exists. Oat5 is mainly expressed in the apical membrane of proximal tubules. Endogenous substrates include ES and DHEAS. In rats, Oat5 might be involved in the uptake of ES in exchange against dicarboxylates as succinate, and in mice an exchange of organic anions against dicarboxylates could not be demonstrated.OAT6 (SLC22A20)This protein was cloned first from mouse. Orthologs exist in rat and human, but these proteins have not been functionally characterized. There is evidence that mouse OAT6 exchanges ES against glutarate, suggesting that it acts as an organic anion/dicarboxylate antiporter. Expression is strongest in nasal olfactory mucosa and to a lesser amount in testis. It has been presumed that OAT6 might be involved in olfactation, its precise function, however, remains unclear.OAT7 (SLC22A9)Previously named UST3 or OAT4, the transporter was cloned from human liver where it is mainly expressed in the sinusoidal membrane of hepatocytes. It transports ES and DHEAS and is able to release these compounds in exchange against short chain fatty acids, e.g., butyrate. This might be of some physiological relevance as steroid hormones such as estrogen are sulfated in the liver to increase stability. Additionally, OAT7 might be involved in the hepatic metabolism of short chain fatty acids. No rodent ortholog has been identified.OAT8 (Slc22a24)This protein (previously named Ust1) has been cloned from rats. A human ortholog does not exist. OAT8 is expressed mainly in the intercalated cells of the kidney. In acid-secreting (type A) cells it is located at the luminal membrane and in bicarbonate-secreting (type B) cells it is located at the basolateral membrane. OAT8 is therefore co-localized with the vacuolar-type H+-ATPase which is crucial for acid&base homeostasis. If OAT8 somehow is involved in this function remains to be clarified. Substrates include ES and DHEAS which most likely are exchanged against dicaboxylates as glutarate.OAT9 (Slc22a27)OAT9, which has been identified in rodents but not in humans occurs in a long (OAT9L) and short (OAT9S) splice variant, the latter lacking TMDs 3&6. Surprisingly, only the short variant showed functional activity. OAT9S transports the zwitterionic compound carnitine as well as salicylic acid and cimetidine. The transport mode remains unclarified. The protein has been detected at the luminal membrane of proximal tubules, where it may contribute to carnitine reabsorbtion and at the sinusoidal membrane of hepatocytes.OAT10 (SLC22A13)This was cloned first from humans (previously named ORCTL3). Strongest expression is seen in the kidney where it is located at the luminal membrane of proximal tubules. Additionally, it is expressed in pancreas, skeletal muscle, heart, brain, small and large intestine, placenta, prostate, and testis. It operates as an antiporter, exchanging nicotinate or urate against intracellular lactate, succinate, or glutathione. The immunosuppressive drug cyclosporine A is an additional substrate. The most relevant function is probably the reabsorption of the vitamin nicotinate in the kidney. Rodent orthologs exist but have not been characterized.URAT1 (SLC22A12)The protein (previously named Rst) has been cloned first from mice and later from humans and is expressed most strongly at the luminal membrane of proximal tubules. Human URAT1 is also present in vascular smooth muscle cells and testis, while in mice it has been detected at the blood&brain barrier and the choroid plexus. The main function of URAT1 most likely is the tubular reabsorption of urate which is exchanged against intracellular organic anions, mainly lactate. This is highlighted by the observation, that most patients with idiopathic renal hypouricemia (showing increased renal excretion of urate) have mutations in the SLC22A12 gene. Furthermore, urate can also be exchanged against inorganic anions such as chloride, bromide, iodide, or nitrate. There is evidence that some drugs such as benzylpenicillin or the angiotensin II receptor antagonists candesartan, losartan, olmesartan, and pratosartan are also transported.Analyses of the amino acid sequences and hydropathy plots predicted that all SLC22 transporters show a common membrane topology that includes 12 &-helical TMDs, a large extracellular loop between TMDs 1 and 2 and an intracellular loop between TMDs 6 and 7 (Figure ). The large extracellular loop is glycosylated, contains six conserved cysteins that may form disulfide bridges and plays a pivotal role for the oligomerization which has been demonstrated for rOCT1 and hOCT2. In hOAT2, oligomerization appears to be depending on interactions of the TMDs 6 of two monomers. Oligomerization may be important for membrane trafficking, but does not significantly influence transport activity. The intracellular loop contains phosphorylation sites for protein kinases that are involved in the regulation of SLC22 transporters.Figure&2. Membrane topology of SLC22 transporters by the example of rOCT1. C- and N-terminus are located intracellularly. Between transmembrane domains (TMDs) 1 and 2 a large extracellular loop exists, containing conserved cysteins that are involved in membrane trafficking. The intracellular loop between TMDs 6 and 7 contains several regulatory phosphorylation sites.The understanding of polyspecificity has been a challenging problem because many of these transporters can translocate a variety of substrates with largely differing molecular structures. Numerous approaches were applied to define the requirements for a molecule to be accepted as a substrate of a distinct SLC22 transporter. The majority of the substrates of OCT1, e.g., are monovalent organic cations but the transport of some divalent cations as well as of some uncharged compounds has also been shown. A model has been presented for rOCT2 that suggests a &selectivity filter& for the translocation pathway that is size-dependent and excludes compounds larger than 4&&A from translocation for sterical reasons. Attachment to the substrate binding site, however, is possible also for bigger molecules. This explains why several non-transported compounds inhibit the transport in a competitive fashion. The affinity of binding is positively correlated to the hydrophobicity of compounds.Various attempts have been made to elucidate the structure of the substrate binding site of SLC22 transporters. As no member of the SLC22 family has been crystallized yet, site-directed mutagenesis and homology modeling were the major approaches to these questions. OCT1 and OCT2 are the most intensely investigated subtypes, but there is good evidence that the principal structure of the binding site is common in SLC22 transporters. By sequence analysis, a glutamate residue in the 11th TMD was identified that is conserved in all OCTs while an arginine is present at that position in the OATs. Exchange of this glutamate by neutral or basic amino acids led to a loss of function, indicating that the negative charge in that position is crucial for substrate binding and/or translocation. Exchange of glutamate by aspartate revealed a functional active transporter with a significantly changed affinity for several substrates while the affinity for other substrates (e.g., MPP) remained unchanged. This lead to the idea that OCT1 exhibits a complex binding pocket containing different interaction sites for different substrates and inhibitors. If the arginine that is present at the corresponding position in OATs is exchanged, substrate binding of OATs is also altered, supporting the idea that the SLC22 transporters share a structurally similar binding region.Further mutational analyses supported the concept of a complex binding pocket which is formed by several TMDs. The binding pocket might appear in an inward- or outward-oriented conformation and these conformations can differ in substrate affinity. On the basis of the uptake studies for hOCT2, Harper and Wright suggested a model where two substrates can bind simultaneously to the transporter, although the resulting transporter/substrate1/substrate2 complex cannot perform substrate translocation.As the SLC22 family is a member of the major facilitator superfamily (MFS) and the three-dimensional structures of some MFS transporters have been elucidated by crystal analyses, these structures were taken as templates for the modeling of the structures of SLC22 transporters. The first model presented was a model of rOCT1 in the inward-open conformation that was derived from the crystal structure of the LacY permease of Escherichia coli(Figure ). The model showed a large cleft that is accessible from the aqueous phase. This cleft is formed by eight TMDs and all amino acids that were identified to be involved in substrate binding by mutational analysis appear oriented to the cleft, indicating that the binding pocket is located there. In a similar approach, Zhang et al. modeled the structure of the inward-open rabbit OCT2 using another crystallized MSF protein, the glycerol-3-phosphate transporter (GlpT) as a template. The resulting model was largely consistent with that of rOCT1. GlpT was as well used to generate a model for hOAT1 that also exhibited a large cleft that contained in his inner part the putative binding pocket with a calculated size of 830&&A3. Several amino acids within this pocket that were shown to be involved in substrate binding are homologous to amino acids of OCT1 and OCT2 for which a function in substrate binding also has been demonstrated.Figure&3. Sterical model of rOCT1 (side view) derived from the tertiary structure of LacY. A large cleft that is accessible from the aqueous phase is flanked by eight transmembrane &-helices. This cleft contains an inner cavity with interaction sites for substrate binding. Binding of a corticosterone molecule to the inner cavity is also shown. Mutation of the designated amino acids induces changes in affinity or selectivity for substrates, indicating their involvement in substrate binding. Left: inward- right: outward-open conformation.Recently, rOCT1 was also modeled in the outward-open conformation and the structures of the innermost cavity of the binding pocket in the inward- and outward-open conformation were compared. It was revealed that the general structure of the innermost cavity in both conformations is conserved, but specific differences exist in the position and orientation of single amino acids involved in substrate binding. This may explain the differences in substrate affinities observed on both sides. In addition to the binding sites that mediate transport of substrates, high-affinity binding sites for substrates were described that are not directly involved in transport but may have regulatory functions.At present, it is supposed that OCTs follow an alternating-access transport mode: The substrate binds to the outward-open conformation of the transporter. This induces a conformational change where the substrate&transporter complex passes a transient occluded state to the inward-open conformation. The substrate finally is released to the cytoplasm and the transporter returns to the outward open conformation either in an empty state or together with a substrate molecule that is carried from the cytosol to the extracellular space. The structural changes of OCTs during the transport cycle require a rigid body movement of the six N-terminal TMDs against the six C-terminal TMDs and a hinge domain in TMD 11 is crucial for this movement.Unlike OCTs and OATs, no three-dimensional models have been published in case of OCTNs. There is evidence, however, that OCTNs as well exhibit a complex binding pocket containing different interaction sites. For instance, hOCTN2 is considered a Na+-carnitine cotransporter, but it has been shown that it is capable of transporting carnitine in the absence of sodium as well. The maximum rate of carnitine transport is independent of sodium but the affinity is decreased more than 10-fold without sodium. This indicates that sodium binding to a specific site within the binding pocket changes conformation and affinity of the carnitine binding site. 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