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1.
Mammalian multidrug and toxic compound extrusion 1 (MATE1) are polyspecific H+-coupled exporters of organic cations (OCs) and responsible for excretion of metabolic waste products and xenobiotics. Here, we report a novel variant of mouse MATE1, mMATE1b, that has a long carboxyl terminal hydrophobic tail homologous to other MATE1 transporter proteins. Mouse MATE1b mediates tetraethylammonium (TEA) uptake with properties similar to that of mMATE1 and is localized in renal brush border membranes. Thus, mMATE1b is a functional variant of mMATE1 and seems to be the true counterpart to other MATE1 transporters.  相似文献   

2.
MATE1 was the first mammalian example of the multidrug and toxin extrusion (MATE) protein family to be identified. Human MATE1 (hMATE1) is predominantly expressed and localized to the luminal membranes of the urinary tubules and bile canaliculi and mediates H+-coupled electroneutral excretion of toxic organic cations (OCs) into urine and bile (Otsuka M, Matsumoto T, Morimoto R, Arioka S, Omote H, and Moriyama Y. Proc Natl Acad Sci USA 102: 17923–17928, 2005). mMATE1, a mouse MATE ortholog, is also predominantly expressed in kidney and liver, although its transport properties are not yet characterized. In the present study, we investigated the transport properties and localization of mMATE1. Upon expression of this protein in HEK-293 cells, mMATE1 mediated electroneutral H+/tetraethylammonium exchange and showed a substrate specificity similar to that of hMATE1. Immunological techniques with specific antibodies against mMATE1 combined with RT-PCR revealed that mMATE1 is also expressed in various cells, including brain glia-like cells and capillaries, pancreatic duct cells, urinary bladder epithelium, adrenal gland cortex, cells of the islets of Langerhans, Leydig cells, and vitamin A-storing Ito cells. These results indicate that mMATE1 is a polyspecific H+/OC exchanger. The unexpectedly wide distribution of mMATE1 suggests involvement of this transporter protein in diverse biological functions other than excretion of OCs from the body. multidrug and toxin extrusion; multidrug transport; hydrophobic cation  相似文献   

3.
Human multidrug and toxic compound extrusion 2 (hMATE2) is a kidney-specific isoform of hMATE1, an exporter of toxic organic cations (OCs) of exogenous and endogenous origins at the final excretion step in the kidneys and liver (Otsuka et al., 2005), and contains a splicing variant, MATE2K, that has an exon of hMATE2 deleted (Masuda et al., 2006). In the present study, we characterized the degree of expression and the transport properties of hMATE2. Quantitative PCR analysis with probes specific for hMATE2 indicated the presence of hMATE2 mRNA in the kidneys, which corresponded to 39% of total mRNA encoding both hMATE2 and hMATE2K. hMATE2-specific antibodies immunostained the renal urinary tubules. Upon expression in HEK293 cells, hMATE2 was localized in intracellular vesicular structures, and thus transport activity of tetraethylammonium (TEA), a typical substrate for MATE transporters, by the cells was not detected. The hMATE2 protein was purified and reconstituted into liposomes. An artificially imposed pH gradient (ΔpH) across the proteoliposomal membrane drove the uptake of TEA. Dissipation of ΔpH by ammonium sulfate effectively inhibited the TEA uptake, while that of the membrane potential by valinomycin had little effect. The profiles of cis-inhibition of TEA transport by hMATE2 and hMATE2K are similar to each other. Thus, both hMATE2 and hMATE2K equally operate in the human kidneys to extrude OCs into the urine.  相似文献   

4.
The polyspecific organic cation transporters 1 and 2 (Oct1 and -2) transport a broad range of substrates, including drugs, toxins, and endogenous compounds. Their strategic localization in the basolateral membrane of epithelial cells in the liver, intestine (Oct1), and kidney (Oct1 and Oct2) suggests that they play an essential role in removing noxious compounds from the body. We previously showed that in Oct1(-/-) mice, the hepatic uptake and intestinal excretion of organic cations are greatly reduced. Since Oct1 and Oct2 have extensively overlapping substrate specificities, they might be functionally redundant. To investigate the pharmacologic and physiologic roles of these proteins, we generated Oct2 single-knockout and Oct1/2 double-knockout mice. Oct2(-/-) and Oct1/2(-/-) mice are viable and fertile and display no obvious phenotypic abnormalities. Absence of Oct2 in itself had little effect on the pharmacokinetics of tetraethylammonium (TEA), but in Oct1/2(-/-) mice, renal secretion of this compound was completely abolished, leaving only glomerular filtration as a TEA clearance mechanism. As a consequence, levels of TEA were substantially increased in the plasma of Oct1/2(-/-) mice. This study shows that Oct1 and Oct2 together are essential for renal secretion of (small) organic cations. A deficiency in these proteins may thus result in increased drug sensitivity and toxicity.  相似文献   

5.
6.
The polyspecific organic cation transporter 1 (OCT1 [SLC22A1]) mediates facilitated transport of small (hydrophilic) organic cations. OCT1 is localized at the basolateral membrane of epithelial cells in the liver, kidney, and intestine and could therefore be involved in the elimination of endogenous amines and xenobiotics via these organs. To investigate the pharmacologic and physiologic role of this transport protein, we generated Oct1 knockout (Oct1(-/-)) mice. Oct1(-/-) mice appeared to be viable, healthy, and fertile and displayed no obvious phenotypic abnormalities. The role of Oct1 in the pharmacology of substrate drugs was studied by comparing the distribution and excretion of the model substrate tetraethylammonium (TEA) after intravenous administration to wild-type and Oct1(-/-) mice. In Oct1(-/-) mice, accumulation of TEA in liver was four to sixfold lower than in wild-type mice, whereas direct intestinal excretion of TEA was reduced about twofold. Excretion of TEA into urine over 1 h was 53% of the dose in wild-type mice, compared to 80% in knockout mice, probably because in Oct1(-/-) mice less TEA accumulates in the liver and thus more is available for rapid excretion by the kidney. In addition, we found that absence of Oct1 leads to decreased liver accumulation of the anticancer drug metaiodobenzylguanidine and the neurotoxin 1-methyl-4-phenylpyridium. In conclusion, our data show that Oct1 plays an important role in the uptake of organic cations into the liver and in their direct excretion into the lumen of the small intestine.  相似文献   

7.
The barrier function of the human mammary gland collapses if challenged with cationic drugs, causing their accumulation in milk. However, underlying molecular mechanisms are not well understood. To gain insight into the mechanism, we characterized transport of organic cations in the MCF12A human mammary gland epithelial cells, using carnitine and tetraethylammonium (TEA) as representative nutrient and xenobiotics probes, respectively. Our results show that the mammary gland cells express mRNA and proteins of human (h) novel organic cation transporters (OCTN) 1 and hOCTN2 (a Na+-dependent carnitine carrier with Na+-independent xenobiotics transport function), which belong to the solute carrier superfamily (SLC) of transporters. Other SLC OCTs such as hOCT1 and extraneuronal monoamine transporter (EMT)/hOCT3 are also expressed at mRNA levels, but hOCT2 was undetectable. We further showed mRNA expression of ATB0+ (an amino acid transporter with a Na+/Cl(-)-dependent carnitine transport activity), and Fly-like putative transporter 2/OCT6 (a splice variant of carnitine transporter 2: a testis-specific Na+-dependent carnitine transporter). TEA uptake was pH dependent. Carnitine uptake was dependent on Na+, and partly on Cl-, compatible with hOCTN2 and ATB0+ function. Modeling analyses predicted multiplicity of the uptake mechanisms with the high-affinity systems characterized by K(m) of 5.1 microM for carnitine and 1.6 mM for TEA, apparently similar to the reported hOCTN2 parameter for carnitine, and that of EMT/hOCT3 for TEA. Verapamil, cimetidine, carbamazepine, quinidine, and desipramine inhibited the carnitine uptake but required supratherapeutic concentrations, suggesting robustness of the carnitine uptake systems against xenobiotic challenge. Our findings suggest functional roles of a network of multiple SLC organic cation/nutrient transporters in human mammary gland drug transfer.  相似文献   

8.
Many large organic cations are potent blockers of K(+) channels and other cation-selective channels belonging to the P-region superfamily. However, the mechanism by which large hydrophobic cations enter and exit the narrow pores of these proteins is obscure. Previous work has shown that a conserved Lys residue in the DEKA locus of voltage-gated Na(+) channels is an important determinant of Na(+)/K(+) discrimination, exclusion of Ca(2+), and molecular sieving of organic cations. In this study, we sought to determine whether the Lys(III) residue of the DEKA locus interacts with internal tetra-alkylammonium cations (TAA(+)) that block Na(+) channels in a voltage-dependent fashion. We investigated block by a series of TAA(+) cations of the wild-type rat muscle Na(+) channel (DEKA) and two different mutants of the DEKA locus, DEAA and DERA, using whole-cell recording. TEA(+) and larger TAA(+) cations block both wild-type and DEAA channels. However, DEAA exhibits dramatic relief of block by large TAA(+) cations as revealed by a positive inflection in the macroscopic I-V curve at voltages greater than +140 mV. Paradoxically, relief of block at high positive voltage is observed for large (e.g., tetrapentylammonium) but not small (e.g., TEA(+)) symmetrical TAA(+) cations. The DEKA wild-type channel and the DERA mutant exhibit a similar relief-of-block phenomenon superimposed on background current rectification. The results indicate: (a) hydrophobic TAA(+) cations with a molecular diameter as large as 15 A can permeate Na(+) channels from inside to outside when driven by high positive voltage, and (b) the Lys(III) residue of the DEKA locus is an important determinant of inward rectification and internal block in Na(+) channels. From these observations, we suggest that hydrophobic interfaces between subunits, pseudosubunits, or packed helices of P-region channel proteins may function in facilitating blocker access to the pore, and may thus play an important role in the blocking and permeation behavior of large TAA(+) cations and potentially other kinds of local anesthetic molecules.  相似文献   

9.
Many organic cations are transported across the apical membrane of the proximal tubule by specific saturable mechanisms. The goal of this study was to determine if the transporter for tetraethylammonium (TEA) in the brush border membrane of an established opossum kidney (OK) cell line is glycosylated and to elucidate the function of this glycosylation. The uptake of TEA was determined in OK cell monolayers treated with tunicamycin (TM), a compound that prevents synthesis of the core oligosaccharide precursor molecules. TM exposure significantly decreased the incorporation of [3H]mannose in OK cell proteins and significantly reduced TEA uptake in a time and a concentration dependent manner. No effect of TM exposure on cellular protein synthesis, DNA content, cell viability, or on [3H]proline uptake was observed. The transport of TEA in control cells was characterized by a Km of 26.9 +/- 16.4 microM and a Vmax of 378 +/- 39 pmol/mg of protein/min. TM treatment (1 microgram/ml for 21 h) significantly increased the Km by over 4-fold to 111.5 +/- 18.4 microM while not affecting the Vmax. The apparent KI values of other organic cations known to interact with this transport system were also significantly increased by TM exposure. Estimated KI values of N1-methylnicotinamide, cimetidine, and mepiperphenidol increased by 6-fold, 4-fold, and 2-fold, respectively, after exposure of OK cells to TM. An increased KI for protons was also observed. Additional inhibitors of the N-linked glycosylation pathway, castanospermine, deoxynojirimycin, and deoxymannojirimycin significantly decreased TEA transport, whereas swainsonine had no effect. Our results suggest that the organic cation transporter is glycosylated. The N-linked oligosaccharide side chain appears to be of the hybrid type, and it either directly or indirectly affects the binding site of the transporter for both organic cations and protons. This is the first report describing the importance of glycosylation in the function of the organic cation transporter in the apical membrane of OK cells.  相似文献   

10.
11.
Human multidrug and toxic compound extrusion 1 (hMATE1) is an electroneutral H(+)/organic cation exchanger responsible for the final excretion step of structurally unrelated toxic organic cations in kidney and liver. To elucidate the molecular basis of the substrate recognition by hMATE1, we substituted the glutamate residues Glu273, Glu278, Glu300, and Glu389, which are conserved in the transmembrane regions, for alanine or aspartate and examined the transport activities of the resulting mutant proteins using tetraethylammonium (TEA) and cimetidine as substrates after expression in human embryonic kidney 293 (HEK-293) cells. All of these mutants except Glu273Ala were fully expressed and present in the plasma membrane of the HEK-293 cells. TEA transport activity in the mutant Glu278Ala was completely absent. Both Glu300Ala and Glu389Ala and all aspartate mutants exhibited significantly decreased activity. Glu273Asp showed higher affinity for cimetidine, whereas it has reduced affinity to TEA. Glu278Asp showed decreased affinity to cimetidine. Both Glu300Asp and Glu389Asp had lowered affinity to TEA, whereas the affinity of Glu389Asp to cimetidine was fourfold higher than that of the wild-type transporter with about a fourfold decrease in V(max) value. Both Glu273Asp and Glu300Asp had altered pH dependence for TEA uptake. These results suggest that all of these glutamate residues are involved in binding and/or transport of TEA and cimetidine but that their individual roles are different.  相似文献   

12.
Organic cation transporters   总被引:1,自引:0,他引:1  
Over the last 15 years, a number of transporters that translocate organic cations were characterized functionally and also identified on the molecular level. Organic cations include endogenous compounds such as monoamine neurotransmitters, choline, and coenzymes, but also numerous drugs and xenobiotics. Some of the cloned organic cation transporters accept one main substrate or structurally similar compounds (oligospecific transporters), while others translocate a variety of structurally diverse organic cations (polyspecific transporters). This review provides a survey of cloned organic cation transporters and tentative models that illustrate how different types of organic cation transporters, expressed at specific subcellular sites in hepatocytes and renal proximal tubular cells, are assembled into an integrated functional framework. We briefly describe oligospecific Na+- and Cl--dependent monoamine neurotransmitter transporters (SLC6-family), high-affinity choline transporters (SLC5-family), and high-affinity thiamine transporters (SLC19-family), as well as polyspecific transporters that translocate some organic cations next to their preferred, noncationic substrates. The polyspecific cation transporters of the SLC22 family including the subtypes OCT1-3 and OCTN1-2 are presented in detail, covering the current knowledge about distribution, substrate specificity, and recent data on their electrical properties and regulation. Moreover, we discuss artificial and spontaneous mutations of transporters of the SLC22 family that provide novel insight as to the function of specific protein domains. Finally, we discuss the clinical potential of the increasing knowledge about polymorphisms and mutations in polyspecific organic cation transporters.  相似文献   

13.
Na(+),K(+)-ATPase, a basolateral transporter responsible for tubular reabsorption of Na(+) and for providing the driving force for vectorial transport of various solutes and ions, can also act as a signal transducer in response to the interaction with steroid hormones. At nanomolar concentrations ouabain binding to Na(+),K(+)-ATPase activates a signaling cascade that ultimately regulates several membrane transporters including Na(+),K(+)-ATPase. The present study evaluated the long-term effect of ouabain on Na(+),K(+)-ATPase activity (Na(+) transepithelial flux) and expression in opossum kidney (OK) cells with low (40) and high (80) number of passages in culture, which are known to overexpress Na(+),K(+)-ATPase (Silva et al., 2006, J Membr Biol 212, 163-175). Activation of a signal cascade was evaluated by quantification of ERK1/2 phosphorylation by Western blot. Na(+),K(+)-ATPase activity was determined by electrophysiological techniques and expression by Western blot. Incubation of cells with ouabain induced activation of ERK1/2. Long-term incubation with ouabain induced an increase in Na(+) transepithelial flux and Na(+),K(+)-ATPase expression only in OK cells with 80 passages in culture. This increase was prevented by incubation with inhibitors of MEK1/2 and PI-3K. In conclusion, ouabain-activated signaling cascade mediated by both MEK1/2 and PI-3K is responsible for long-term regulation of Na(+) transepithelial flux in epithelial renal cells. OK cell line with high number of passages is suggested to constitute a particular useful model for the understanding of ouabain-mediated regulation of Na(+) transport.  相似文献   

14.
Multidrug resistance poses grand challenges to the effective treatment of infectious diseases and cancers. Integral membrane proteins from the multidrug and toxic compound extrusion (MATE) family contribute to multidrug resistance by exporting a wide variety of therapeutic drugs across cell membranes. MATE proteins are conserved from bacteria to humans and can be categorized into the NorM, DinF and eukaryotic subfamilies. MATE transporters hold great appeal as potential therapeutic targets for curbing multidrug resistance, yet their transport mechanism remains elusive. During the past 5 years, X-ray structures of 4 NorM and DinF transporters have been reported and guided biochemical studies to reveal how MATE transporters extrude different drugs. Such advances, although substantial, have yet to be discussed collectively. Herein I review these structures and the unprecedented mechanistic insights that have been garnered from those structure-inspired studies, as well as lay out the outstanding questions that present exciting opportunities for future work.  相似文献   

15.
The human organic cation transporter type 1 (hOCT1) is an important transport system for small organic cations in the liver. Organic cation transporters are regulated by different signaling pathways, but the regulation of hOCT1 has not yet been studied. In this work, we have for the first time investigated the regulation of hOCT1. hOCT1 was expressed in Chinese hamster ovary cells (CHO-hOCT1) and in human embryonic kidney cells (HEK293-hOCT1). Its activity was monitored using microfluorimetry with the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)) as substrate. hOCT1 expressed in CHO-cells was inhibited by protein kinase A (PKA) activation (1 microM forskolin, -58 +/- 6%, n = 12), calmodulin inhibition (0.1 microM calmidazolium, -68 +/- 3%, n = 6; 10 microM ophiobolin A, -48 +/- 10%, n = 7), calmodulin-dependent kinase II inhibition (1 microM KN62, -78 +/- 4%, n = 12), and inhibition of p56(lck) tyrosine kinase (10 microM aminogenistein, -35 +/- 7%, n = 12). The apparent affinities for TEA(+) were lower in CHO-hOCT1 than in HEK293-hOCT1, while those for TPA(+) and quinine were almost identical; the rank order of EC(50) values (TPA(+) > quinine > TEA(+)) was independent of the expression system. EC(50) values for TEA(+) in CHO-hOCT1 or HEK293-hOCT1 were increased under calmidazolium incubation (6.3 and 1.4 mM, respectively). hOCT1 was inhibited by PKA and endogenously activated by calmodulin, calmodulin-dependent kinase II, and p56(lck) tyrosine kinase. Regulation pathways were the same in the two expression systems. Since apparent substrate affinities depend on activity of regulatory pathways, the expression system plays a role in determining the substrate affinities.  相似文献   

16.
Turgor pressure is a cellular parameter, important for a range of physiological processes in plants, like cell elongation, gas exchange and gravitropic/phototropic bending. Regulation of turgor pressure involves ion and water transport at the expense of metabolic energy (ATP). The primary pump in the plasma membrane (the H(+)-ATPase) is a key player in turgor regulation since it provides the driving force for ion uptake, followed by water influx through osmosis. Using the phytotoxin fusicoccin (a well-known activator of the ATPase) as a tool, 14-3-3 proteins were identified as regulators of the H(+)-ATPase. Since fusicoccin has a dramatic effect on K(+) accumulation and cellular respiration as well, we studied whether 14-3-3 proteins play a role in the regulation of the mitochondrial F(0)F(1)-ATP synthase and ion channels in the vacuolar and plasma membranes. Besides the plasma membrane H(+)-ATPase, we have identified thus far at least four other transport proteins that are regulated by 14-3-3 proteins. The mechanism of regulation will be described and the possibility that 14-3-3 proteins act as coordinators of ion transporters with varied but interdependent functions will be discussed.  相似文献   

17.
Polyspecific organic anion transporters (OATs) and organic cation transporters (OCTs) of the SLC22 transporter family play a pivotal role in absorption, distribution, and excretion of drugs. Polymorphisms in these transporters influence therapeutic effects. On the basis of functional characterizations, homology modeling, and mutagenesis, hypotheses for how OCTs bind and translocate structurally different cations were raised, assuming functionally competent monomers. However, homo-oligomerization has been described for OATs and OCTs. In the present study, evidence is provided that the large extracellular loops (EL) of rat Oct1 (rOct1) and rat Oat1 (rOat1) mediate homo- but not hetero-oligomerization. Replacement of the cysteine residues in the EL of rOct1 by serine residues (rOct1(6ΔC-l)) or breaking disulfide bonds with dithiothreitol prevented oligomerization. rOct1 chimera containing the EL of rOat1 (rOct1(rOat1-l)) showed oligomerization but reduced transporter amount in the plasma membrane. For rOct1(6ΔC-l) and rOct1(rOat1-l), similar K(m) values for 1-methyl-4-phenylpyridinium(+) (MPP(+)) and tetraethylammonium(+) (TEA(+)) were obtained that were higher compared with rOct1 wild type. The increased K(m) of rOct1(rOat1-l) indicates an allosteric effect of EL on the cation binding region. The similar substrate affinity of the oligomerizing and non-oligomerizing loop mutants suggests that oligomerization does not influence transport function. Independent transport function of rOct1 monomers was also demonstrated by showing that K(m) values for MPP(+) and TEA(+) were not changed after treatment with dithiothreitol and that a tandem protein with two rOct1 monomers showed about 50% activity with unchanged K(m) values for MPP(+) and TEA(+) when one monomer was blocked. The data help to understand how OCTs work and how mutations in patients may affect their functions.  相似文献   

18.
Basolateral transport of the prototypical type I organic cation tetraethylammonium (TEA) by the Malpighian tubules of Drosophila melanogaster was studied using measurements of basolateral membrane potential (V(bl)) and uptake of [(14)C]-labeled TEA. TEA uptake was metabolically dependent and saturable (maximal rate of mediated TEA uptake by all potential transport processes, reflecting the total transport capacity of the membrane, 0.87 pmol.tubule(-1).min(-1); concentration of TEA at 0.5 of the maximal rate of TEA uptake value, 24 muM). TEA uptake in Malpighian tubules was inhibited by a number of type I (e.g., cimetidine, quinine, and TEA) and type II (e.g., verapamil) organic cations and was dependent on V(bl). TEA uptake was reduced in response to conditions that depolarized V(bl) (high-K(+) saline, Na(+)-free saline, NaCN) and increased in conditions that hyperpolarized V(bl) (low-K(+) saline). Addition of TEA to the saline bathing Malpighian tubules rapidly depolarized the V(bl), indicating that TEA uptake was electrogenic. Blockade of K(+) channels with Ba(2+) did not block effects of TEA on V(bl) or TEA uptake indicating that TEA uptake does not occur through K(+) channels. This is the first study to provide physiological evidence for an electrogenic carrier-mediated basolateral organic cation transport mechanism in insect Malpighian tubules. Our results also suggest that the mechanism of basolateral TEA uptake by Malpighian tubules is distinct from that found in vertebrate renal tubules.  相似文献   

19.
20.
Vacuoles release sucrose via tonoplast-localised SUC4-type transporters   总被引:1,自引:0,他引:1  
Arabidopsis thaliana has seven genes for functionally active sucrose transporters. Together with sucrose transporters from other dicot and monocot plants, these proteins form four separate phylogenetic groups. Group-IV includes the Arabidopsis protein SUC4 (synonym SUT4) and related proteins from monocots and dicots. These Group-IV sucrose transporters were reported to be either tonoplast- or plasma membrane-localised, and in heterologous expression systems were shown to act as sucrose/H(+) symporters. Here, we present comparative analyses of the subcellular localisation of the Arabidopsis SUC4 protein and of several other Group-IV sucrose transporters, studies on tissue specificity of the Arabidopsis SUC4 promoter, phenotypic characterisations of Atsuc4.1 mutants and AtSUC4 overexpressing (AtSUC4-OX) plants, and functional comparisons of Atsuc4.1 and AtSUC4-OX vacuoles. Our data show that SUC4-type sucrose transporters from different plant families (Brassicaceae, Cucurbitaceae and Solanaceae) localise exclusively to the tonoplast, demonstrating that vacuolar sucrose transport is a common theme of all SUC4-type proteins. AtSUC4 expression is confined to the stele of Arabidopsis roots, developing anthers and meristematic tissues in all aerial parts. Analyses of the carbohydrate content of WT and mutant seedlings revealed reduced sucrose content in AtSUC4-OX seedlings. This is in line with patch-clamp analyses of AtSUC4-OX vacuoles that characterise AtSUC4 as a sucrose/H(+) symporter directly in the tonoplast membrane.  相似文献   

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