Publications (32)139.14 Total impact
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Article: A Substrate Binding Hinge Domain Is Critical for Transport-related Structural Changes of Organic Cation Transporter 1.
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ABSTRACT: Organic cation transporters are membrane potential-dependent facilitative diffusion systems. Functional studies, extensive mutagenesis, and homology modeling indicate the following mechanism. A transporter conformation with a large outward-open cleft binds extracellular substrate, passes a state in which the substrate is occluded, turns to a conformation with an inward-open cleft, releases substrate, and subsequently turns back to the outward-open state. In the rat organic cation transporter (rOct1), voltage- and ligand-dependent movements of fluorescence-labeled cysteines were measured by voltage clamp fluorometry. For fluorescence detection, cysteine residues were introduced in extracellular parts of cleft-forming transmembrane α-helices (TMHs) 5, 8, and 11. Following expression of the mutants in Xenopus laevis oocytes, cysteines were labeled with tetramethylrhodamine-6-maleimide, and voltage-dependent conformational changes were monitored by voltage clamp fluorometry. One cysteine was introduced in the central domain of TMH 11 replacing glycine 478. This domain contains two amino acids that are involved in substrate binding and two glycine residues (Gly-477 and Gly-478) allowing for helix bending. Cys-478 could be modified with the transported substrate analog [2-(trimethylammonium)-ethyl]methanethiosulfonate but was inaccessible to tetramethylrhodamine-6-maleimide. Voltage-dependent movements at the indicator positions of TMHs 5, 8, and 11 were altered by substrate applications indicating large conformational changes during transport. The G478C exchange decreased transporter turnover and blocked voltage-dependent movements of TMHs 5 and 11. [2-(Trimethylammonium)-ethyl]methanethiosulfonate modification of Cys-478 blocked substrate binding, transport activity, and movement of TMH 8. The data suggest that Gly-478 is located within a mechanistically important hinge domain of TMH 11 in which substrate binding induces transport-related structural changes.Journal of Biological Chemistry 07/2012; 287(37):31561-73. · 4.77 Impact Factor -
Article: Response to Comment on: Gorboulev et al. Na+-D-glucose Cotransporter SGLT1 Is Pivotal for Intestinal Glucose Absorption and Glucose-Dependent Incretin Secretion. Diabetes 2012;61:187-196.
Diabetes 06/2012; 61(6):e5. · 8.29 Impact Factor -
Article: Na(+)-D-glucose cotransporter SGLT1 is pivotal for intestinal glucose absorption and glucose-dependent incretin secretion.
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ABSTRACT: To clarify the physiological role of Na(+)-D-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1(-/-) mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1(-/-) mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1(-/-) mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose-free diet. In wild-type mice, passage of D-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1(-/-) mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2.Diabetes 11/2011; 61(1):187-96. · 8.29 Impact Factor -
Article: The large extracellular loop of organic cation transporter 1 influences substrate affinity and is pivotal for oligomerization.
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ABSTRACT: 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.Journal of Biological Chemistry 09/2011; 286(43):37874-86. · 4.77 Impact Factor -
Article: Novel shuttling domain in a regulator (RSC1A1) of transporter SGLT1 steers cell cycle-dependent nuclear location.
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ABSTRACT: The gene product of RSC1A1, RS1, participates in the regulation of the Na(+)-D-glucose cotransporter SGLT1. RS1 inhibits release of SGLT1 from the trans Golgi network. In subconfluent LLC-PK(1) cells, RS1 migrates into the nucleus and modulates transcription of SGLT1, whereas most confluent cells do not contain RS1 in the nuclei. We showed that confluence-dependent nuclear location of RS1 is because of different phases of the cell cycle and identified a RS1 nuclear shuttling domain (RNS) with an associated protein kinase C (PKC) phosphorylation site (RNS-PKC) that mediates cell cycle-dependent nuclear location. RNS-PKC contains a novel non-conventional nuclear localization signal interacting with importin beta1, a nuclear export signal mediating export via protein CRM1 and a Ca(2+)-dependent calmodulin binding site. PKC and calmodulin compete for binding to RNS-PKC. Mutagenesis experiments and analyses of the phosphorylation status suggest the following sequences of events. Subconfluent cells without and with synchronization to the G2/M phase contain non-phosphorylated RNS-PKC that mediates nuclear import of RS1 but not its export. During confluence or synchronization of subconfluent cells to the G2/M phase, phosphorylation of RNS-PKC mediates rapid nuclear export of RS1.Traffic 09/2009; 10(11):1599-618. · 4.92 Impact Factor -
Article: Five amino acids in the innermost cavity of the substrate binding cleft of organic cation transporter 1 interact with extracellular and intracellular corticosterone.
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ABSTRACT: We have shown previously that Leu447 and Gln448 in the transmembrane helix (TMH) 10 of rat organic cation transporter rOCT1 are critical for inhibition of cation uptake by corticosterone. Here, we tested whether the affinity of corticosterone is different when applied from the extracellular or intracellular side. The affinity of corticosterone was determined by measuring the inhibition of currents induced by tetraethylammonium(+) (TEA(+)) in Xenopus laevis oocytes expressing rOCT1. Either corticosterone and TEA(+) were added to the bath simultaneously or the oocytes were preincubated with corticosterone, washed, and TEA(+)-induced currents were determined subsequently. In mutant L447Y, K(i) values for extracellular and intracellular corticosterone were decreased, whereas in mutant Q448E, only the K(i) for intracellular corticosterone was changed. Modeling of the interaction of corticosterone with rOCT1 in the inward- or outward-facing conformation predicted direct binding to Leu447, Phe160 (TMH2), Trp218 (TMH4), Arg440 (TMH10), and Asp475 (TM11) from both sides. In mutant F160A, affinities for extracellular and intracellular corticosterone were increased, whereas maximal inhibition was reduced in W218F and R440K. In stably transfected epithelial cells, the affinities for inhibition of 1-methyl-4-phenyl-pyridinium(+) (MPP(+)) uptake by extracellular and intracellular corticosterone were decreased when Asp475 was replaced by glutamate. In mutants F160A, W218Y, R440K, and L447F, the affinities for MPP(+) uptake were changed, and in mutant D475E, the affinity for TEA(+) uptake was changed. The data suggest that Phe160, Trp218, Arg440, Leu447, and Asp475 are located within an innermost cavity of the binding cleft that is alternatingly exposed to the extracellular or intracellular side during substrate transport.Molecular pharmacology 06/2009; 76(2):275-89. · 4.53 Impact Factor -
Article: Charge-to-substrate ratio during organic cation uptake by rat OCT2 is voltage dependent and altered by exchange of glutamate 448 with glutamine.
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ABSTRACT: Uptake of substrate and electric charge was measured simultaneously in voltage-clamped Xenopus laevis oocytes expressing rat organic cation transporter 2 (rOCT2). At 0 mV, saturating substrate concentrations induced uptake of more positive elementary charges than monovalent organic cations, with charge-to-substrate ratios of 1.5 for guanidinium(+), 3.5 for tetraethylammonium(+), and 4.0 for 1-methyl-4-phenylpyridinium(+). At negative holding potentials, the charge-to-substrate ratios decreased toward unity. At 0 mV, charge-to-substrate ratios higher than unity were observed at different extracellular pH and after replacement of extracellular Na(+), K(+), Ca(2+), Mg(2+), and/or Cl(-). Charge-to-substrate ratios were not influenced by intracellular succinate(2-) or glutarate(2-). The effects of membrane potential and ion substitution strongly suggest that the surplus of transported positive charge is not generated by passive ion permeabilities. Rather, we hypothetize that small cations are taken up together with organic cation substrates whereas the outward reorientation of the empty transporter is electroneutral. Nonselective cotransport of small cations was supported by the three-dimensional structures of rOCT2 in its inward-facing and outward-facing conformations, which we determined by homology modeling based on known corresponding structures of H(+)-lactose permease of E. coli, and by functional analysis of OCT mutants. In our model, the innermost cavity of the outward-open binding cleft is negatively charged by Glu448 and Asp475, whereas the inward-open innermost cavity is electroneutral, containing Asp379, Asp475, Lys215, and Arg440. Substitution of Glu448 by glutamine reduced the charge-to-TEA(+) ratio at 0 mV to unity. The observed charge excess associated with organic cation uptake into depolarized cells may contribute to tubular damage in renal failure.American journal of physiology. Renal physiology 03/2009; 296(4):F709-22. · 3.68 Impact Factor -
Article: Transport of lamivudine [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine] and high-affinity interaction of nucleoside reverse transcriptase inhibitors with human organic cation transporters 1, 2, and 3.
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ABSTRACT: Nucleoside reverse transcriptase inhibitors (NRTIs) need to enter cells to act against the HIV-1. Human organic cation transporters (hOCT1-3) are expressed and active in CD4+ T cells, the main target of HIV-1, and have been associated with antiviral uptake in different tissues. In this study, we examined whether NRTIs interact and are substrates of hOCT in cells stably expressing these transporters. Using [(3)H]N-methyl-4-phenylpyridinium, we found a high-affinity interaction among abacavir [[(1S,4R)-4-[2-amino-6-(cyclopropylamino)purin-9-yl]-cyclopent-2-enyl]methanol sulfate] (ABC); <0.08 nM], azidothymidine [3'-azido-3'-deoxythymidine (AZT); <0.4 nM], tenofovir disoproxil fumarate (<1.0 nM), and emtricitabine (<2.5 nM) and hOCTs. Using a wide range of concentrations of lamivudine [(-)-beta-L-2',3'-dideoxy-3'-thiacyitidine (3TC)], we determined two different binding sites for hOCTs: a high-affinity site (K(d1) = 12.3-15.4 pM) and a low-affinity site (K(d2) = 1.9-3.4 mM). Measuring direct uptake of [(3)H]3TC and inhibition with hOCT substrates, we identified 3TC as a novel substrate for hOCT1, 2, and 3, with hOCT1 as the most efficient transporter (K(m) = 1.25 +/- 0.1 mM; V(max) = 10.40 +/- 0.32 nmol/mg protein/min; V(max)/K(m) = 8.32 +/- 0.40 microl/mg protein/min). In drug-drug interaction experiments, we analyzed cis-inhibition of [(3)H]3TC uptake by ABC and AZT and found that 40 to 50% was inhibited at low concentrations of the drugs (K(i) = 22-500 pM). These data reveal that NRTIs experience a high-affinity interaction with hOCTs, suggesting a putative role for these drugs as modulators of hOCT activity. Finally, 3TC is a novel substrate for hOCTs and the inhibition of its uptake at low concentrations of ABC and AZT could have implications for the pharmacokinetics of 3TC.Journal of Pharmacology and Experimental Therapeutics 01/2009; 329(1):252-61. · 3.83 Impact Factor -
Article: Cell free expression and functional reconstitution of eukaryotic drug transporters.
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ABSTRACT: Polyspecific organic cation and anion transporters of the SLC22 protein family are critically involved in absorption and excretion of drugs. To elucidate transport mechanisms, functional and biophysical characterization of purified transporters is required and tertiary structures must be determined. Here, we synthesized rat organic cation transporters OCT1 and OCT2 and rat organic anion transporter OAT1 in a cell free system in the absence of detergent. We solubilized the precipitates with 2% 1-myristoyl-2-hydroxy- sn-glycero-3-[phospho- rac-(1-glycerol)] (LMPG), purified the transporters in the presence of 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or octyl glucoside, and reconstituted them into proteoliposomes. From 1 mL reaction vessels 0.13-0.36 mg of transporter proteins was purified. Thus, from five to ten 1 mL reaction vessels sufficient protein for crystallization was obtained. In the presence of 1% LMPG and 0.5% CHAPS, OCT1 and OAT1 formed homo-oligomers but no hetero-oligomers. After reconstitution of OCT1, OCT2, and OAT1 into proteoliposomes, similar Michaelis-Menten K m values were measured for uptake of 1-methyl-4-phenylpyridinium and p-aminohippurate (PAH (-)) by the organic cation and anion transporters, respectively, as after expression of the transporters in cells. Using the reconstituted system, evidence was obtained that OAT1 operates as obligatory and electroneutral PAH (-)/dicarboxylate antiporter and contains a low-affinity chloride binding site that stimulates turnover. PAH (-) uptake was observed only with alpha-ketoglutarate (KG (2-)) on the trans side, and trans-KG (2-) increased the PAH (-) concentration in voltage-clamped proteoliposomes transiently above equilibrium. The V max of PAH (-)/KG (2-) antiport was increased by Cl (-) in a manner independent of gradients, and PAH (-)/KG (2-) antiport was independent of membrane potential in the absence or presence of Cl (-).Biochemistry 05/2008; 47(15):4552-64. · 3.42 Impact Factor -
Article: High-affinity cation binding to organic cation transporter 1 induces movement of helix 11 and blocks transport after mutations in a modeled interaction domain between two helices.
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ABSTRACT: Voltage-clamp fluorometry was performed with a cysteine-deprived mutant of rat organic cation transporter 1 (rOCT1) in which Phe483 in transmembrane alpha-helix (TMH) 11 close to the extracellular surface was replaced by cysteine and labeled with tetramethylrhodamine-6-maleimide. Potential-dependent fluorescence changes were observed that were sensitive to presence of substrates choline, tetraethylammonium (TEA), and 1-methyl-4-phenylpyridinium (MPP) and of the nontransported inhibitor tetrabutylammonium (TBuA). Using potential-dependent fluorescence changes as readout, one high-affinity binding site per substrate and two high-affinity binding sites for TBuA were identified in addition to the previously described single interaction sites. In a structure model of rOCT1 with an inward open cleft that was derived from a known crystal structure of lacY permease, Phe483 is close to Trp147 in TMH 2. In contrast, in a model with an outward open cleft these amino acids are far apart. After replacement of Phe483 or Trp147 by cysteine or serine, high-affinity binding of TBuA leads to inhibition of MPP or TEA uptake, whereas it has no effect on cation uptake by wild-type rOCT1. Coexisting high-affinity cation binding sites in organic cation transporters may collect low concentration xenobiotics and drugs; however, translocation including transitions between outward- and inward-oriented conformations may only be induced when a low-affinity cation binding site is loaded. We propose that cations bound to high-affinity sites may be translocated together with cations bound to low-affinity sites or that they may block the translocation mechanism.Molecular pharmacology 02/2008; 73(1):50-61. · 4.53 Impact Factor -
Article: Identification of cysteines in rat organic cation transporters rOCT1 (C322, C451) and rOCT2 (C451) critical for transport activity and substrate affinity.
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ABSTRACT: Effects of the sulfhydryl reagent methylmethanethiosulfonate (MMTS) on functions of organic cation transporters (OCTs) were investigated. Currents induced by 10 mM choline [I(max(choline))] in Xenopus laevis oocytes expressing rat OCT1 (rOCT1) were increased four- to ninefold after 30-s incubation with 5 mM MMTS whereas I(max(choline)) by rat OCT2 was 70% decreased. MMTS activated the rOCT1 transporter within the plasma membrane without changing stoichiometry between translocated charge and cation. After modification of oocytes expressing rOCT1 or rOCT2 with MMTS, I(0.5(choline)) values for choline-induced currents were increased. For rOCT1 it was shown that MMTS increased I(0.5) values for different cations by different degrees. Mutagenesis of individual cysteine residues in rOCT1 revealed that modification of cysteine 322 in the large intracellular loop, and of cysteine 451 at the transition of the transmembrane alpha-helix (TMH) 10 to the short intracellular loop between the TMH 10 and 11 is responsible for the observed effects of MMTS. After replacement of cysteine 451 by methionine, the IC(50(choline)) for choline to inhibit MPP uptake by rOCT1 was increased whereas the I(0.5(choline)) value for choline-induced current remained unchanged. At variance, in double mutant Cys322Ser, Cys451Met, I(0.5(choline)) was increased compared with rOCT1 wild-type whereas in the single mutant Cys322Ser I(0.5(choline)) was not changed. The data suggest that modification of rOCT1 at cysteines 322 and 451 leads to an increase in turnover. They indicate that cysteine 451 in rOCT1 interacts with the large intracellular loop and that cysteine 451 in both rOCT1 and rOCT2 is critical for the affinity of choline.American journal of physiology. Renal physiology 10/2007; 293(3):F767-79. · 3.68 Impact Factor -
Article: Tripeptides of RS1 (RSC1A1) inhibit a monosaccharide-dependent exocytotic pathway of Na+-D-glucose cotransporter SGLT1 with high affinity.
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ABSTRACT: The human gene RSC1A1 codes for a 67-kDa protein named RS1 that mediates transcriptional and post-transcriptional regulation of Na(+)-D-glucose cotransporter SGLT1. The post-transcriptional regulation occurs at the trans-Golgi network (TGN). We identified two tripeptides in human RS1 (Gln-Cys-Pro (QCP) and Gln-Ser-Pro (QSP)) that induce posttranscriptional down-regulation of SGLT1 at the TGN leading to 40-50% reduction of SGLT1 in plasma membrane. For effective intracellular concentrations IC(50) values of 2.0 nM (QCP) and 0.16 nm (QSP) were estimated. Down-regulation of SGLT1 by tripeptides was attenuated by intracellular monosaccharides including non-metabolized methyl-alpha-D-glucopyranoside and 2-deoxyglucose. In small intestine post-transcriptional regulation of SGLT1 may contribute to glucose-dependent regulation of liver metabolism and intestinal mobility. QCP and QSP are transported by the H(+)-peptide cotransporter PepT1 that is colocated with SGLT1 in small intestinal enterocytes. Using coexpression of SGLT1 and PepT1 in Xenopus oocytes or polarized Caco-2 cells that contain both transporters we demonstrated that the tripeptides were effective when applied to the extracellular compartment. After a 1-h perfusion of intact rat small intestine with QSP, glucose absorption was reduced by 30%. The data indicate that orally applied tripeptides can be used to down-regulate small intestinal glucose absorption, e.g. in diabetes mellitus.Journal of Biological Chemistry 10/2007; 282(39):28501-13. · 4.77 Impact Factor -
Article: RS1 (RSC1A1) regulates the exocytotic pathway of Na+-D-glucose cotransporter SGLT1.
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ABSTRACT: The product of gene RSC1A1, named RS1, participates in transcriptional and posttranscriptional regulation of the sodium-d-glucose cotransporter SGLT1. Using coexpression in oocytes of Xenopus laevis, posttranscriptional inhibition of human SGLT1 (hSGLT1) and some other transporters by human RS1 (hRS1) was demonstrated previously. In the present study, histidine-tagged hRS1 was expressed in oocytes or Sf9 cells and purified using nickel(II)-charged nitrilotriacetic acid-agarose. hRS1 protein was injected into oocytes expressing hSGLT1 or the human organic cation transporter hOCT2, and the effect on hSGLT1-mediated uptake of methyl-alpha-D-[14C]glucopyranoside ([14C]AMG) or hOCT2-mediated uptake of [14C]tetraethylammonium ([14C]TEA) was measured. Within 30 min after the injection of hRS1 protein, hSGLT1-expressed AMG uptake or hOCT2-expressed TEA uptake was inhibited by approximately 50%. Inhibition of AMG uptake was decreased when a dominant negative mutant of dynamin I was coexpressed and increased after stimulation of PKC. Inhibition remained unaltered when endocytosis was inhibited by chlorpromazine, imipramine, or filipin but was prevented when exocytosis was inhibited by botulinum toxin B or when the release of vesicles from the TGN and endosomes was inhibited by brefeldin A. Inhibition of hSGLT1-mediated AMG uptake and hOCT2-mediated TEA uptake by hRS1 protein were decreased at an enhanced intracellular AMG concentration. The data suggest that hRS1 protein exhibits glucose-dependent, short-term inhibition of hSGLT1 and hOCT2 by inhibiting the release of vesicles from the trans-Golgi network.American journal of physiology. Renal physiology 01/2007; 291(6):F1213-23. · 3.68 Impact Factor -
Article: Transporter regulator RS1 (RSC1A1) coats the trans-Golgi network and migrates into the nucleus.
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ABSTRACT: The product of gene RSC1A1, named RS1, is involved in transcriptional and posttranscriptional regulation of sodium-d-glucose cotransporter SGLT1, and removal of RS1 in mice led to an increase of SGLT1 expression in small intestine and to obesity (Osswald C, Baumgarten K, Stümpel F, Gorboulev V, Akimjanova M, Knobeloch K-P, Horak I, Kluge R, Joost H-G, and Koepsell H. Mol Cell Biol 25: 78-87, 2005). Previous data showed that RS1 inhibits transcription of SGLT1 in LLC-PK1 cells derived from porcine kidney. A decrease of the intracellular amount of RS1 protein was observed during cell confluence, which was paralleled by transcriptional upregulation of SGLT1. In the present study, the subcellular distributions of endogenously expressed RS1 and SGLT1 were compared in LLC-PK1 cells and human embryonic kidney (HEK)-293 cells using immunofluorescence microscopy. RS1 was located at the plasma membrane, at the entire trans-Golgi network (TGN), and within the nucleus. Treatment of LLC-PK1 cells with brefeldin A induced rapid release of RS1 from the TGN, and confluence of LLC-PK1 cells was accompanied by reduction of nuclear location of RS1; 84-90% of subconfluent cells and 5-34% of confluent cells contained RS1 in the nuclei. This suggests that confluence-dependent transcriptional inhibition by RS1 is partially regulated by nuclear migration. Furthermore, we assigned SGLT1 to microtubule-associated tubulovesicular structures and dynamin-containing parts of the TGN. The data indicate that RS1 inhibits the dynamin-dependent release of SGLT1-containing vesicles from the TGN.American journal of physiology. Renal physiology 01/2007; 291(6):F1201-12. · 3.68 Impact Factor -
Article: Characterization of regulatory mechanisms and states of human organic cation transporter 2.
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ABSTRACT: Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37 degrees C. After subtraction of unspecific uptake determined in WT at 37 degrees C or in hOCT2 at 8 degrees C saturable specific uptake of both substrates was measured. Km values of hOCT2-mediated uptake of 95 microM amiloride and 24 microM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC50 (in microM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca2+/CaM complex (-55 +/- 5%, n = 10 and -63 +/- 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (-54 +/- 5%, n = 14, and -31 +/- 6%, n = 26) and PKA (-16 +/- 5%, n = 16, and -18 +/- 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 +/- 6%, n = 8, and +55 +/- 17%, n = 16). Inhibition of Ca2+/CaM complex resulted in a significant decrease of Vmax (160-99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (-22 +/- 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca2+/CaM complex causes changes in transport capacity via hOCT2 trafficking.AJP Cell Physiology 07/2006; 290(6):C1521-31. · 3.54 Impact Factor -
Article: Rat renal glucose transporter SGLT1 exhibits zonal distribution and androgen-dependent gender differences.
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ABSTRACT: SGLT1 (SLC5A1) mediates a part of glucose and galactose reabsorption in the mammalian proximal tubule (PT), but the detailed localization of the transporter along the tubule is still disputable. Here, we used several methods to localize rat SGLT1 (rSGLT1) in the kidneys of intact and variously treated male (M) and female (F) rats. In immunoblots of isolated cortical (C) and outer stripe (OS) brush-border membranes (BBM), a peptide-specific polyclonal antibody for rSGLT1 labeled a sharp inzone-, and gender-dependent approximately 40-kDa protein and a broad approximately 75-kDa band that exhibited strong zonal (OS > C) and gender differences (F > M). In tissue cryosections, the antibody strongly stained BBM of the S3 PT segments in the OS and medullary rays (F > M) and smooth muscles of the blood vessels and renal capsule (F approximately M) and weakly stained the apical domain of other PT segments in the C (F approximately M). The phlorizin-sensitive uptake of d-[(3)H]galactose in BBM vesicles, as well as the tissue abundance of rSGLT1-specific mRNA, matched the immunoblotting data related to the 75-kDa protein and the immunostaining in S3, proving zonal and gender differences in the functional transporter. Ovariectomy had no effect, castration upregulated, whereas treatment of castrated rats with testosterone, but not with estradiol or progesterone, downregulated the 75-kDa protein and the immunostaining in S3. We conclude that in the rat kidney, the expression of SGLT1 is represented by a 75-kDa protein localized largely in the PT S3 segments, where it exhibits gender differences (F > M) at both the protein and mRNA levels that are caused by androgen inhibition.American journal of physiology. Renal physiology 04/2006; 290(4):F913-26. · 3.68 Impact Factor -
Article: Purification and functional reconstitution of the rat organic cation transporter OCT1.
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ABSTRACT: The rat organic cation transporter rOCT1 with six histidine residues added to the C-terminus was expressed in Sf9 insect cells, and expression of organic cation transport was demonstrated. To purify rOCT1 protein, Sf9 cells were lysed with 1% (w/v) CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], centrifuged, and subjected to sequential affinity chromatography using lentil-lectin Sepharose and nickel(II)-charged nitrilotriacetic acid-agarose. This procedure yielded approximately 70 microg of purified rOCT1 protein from 10 standard culture plates. Using a freeze-thaw procedure, purified rOCT1 was reconstituted into proteoliposomes formed from phosphatidylcholine, phosphatidylserine, and cholesterol. Proteoliposomes exhibited uptake of [3H]-1-Methyl-4-phenylpyridinium ([3H]MPP) that was inhibited by quinine and stimulated by an inside-negative membrane potential. MPP uptake was saturable with an apparent K(m) of 30 +/- 17 microM. MPP uptake (0.1 microM) was inhibited by tetraethylammonium, tetrabutylammonium, and tetrapentylammonium with IC50 values of 197 +/- 11, 19 +/- 1, and 1.8 +/- 0.03 microM, respectively. With membrane potential clamped to 0 mV using valinomycin in the presence of 100 mM potassium on both sides of the membrane, uptake of 0.1 microM MPP was trans stimulated 3-fold by 2.5 mM intracellular choline, and efflux of 0.1 microM MPP was trans stimulated 4-fold by 9.5 mM extracellular choline. The data show that rOCT1 is capable and sufficient to mediate transport of organic cations. The observed trans stimulation under voltage-clamp conditions shows that rOCT1 operates as a transporter rather than a channel. Purification and reconstitution of functional active rOCT1 protein is an important step toward the biophysical characterization and crystallization.Biochemistry 10/2005; 44(36):12253-63. · 3.42 Impact Factor -
Article: Individual PKC-phosphorylation sites in organic cation transporter 1 determine substrate selectivity and transport regulation.
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ABSTRACT: To elucidate the molecular mechanisms underlying stimulation of rat organic cation transporter type 1 (rOCT1) by protein kinase C (PKC) activation, functional properties and regulation of rOCT1 stably expressed in HEK293 cells after site-directed mutagenesis of putative PKC phosphorylation-sites were compared with wild-type (WT) rOCT1 using microfluorometric measurements with the fluorescence organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)). Either substitutions of single (S286A, S292A, T296A, S328A, and T550A) or of all five PKC-sites (5x-PKC) with alanine suppressed PKC-induced stimulation of ASP(+) uptake, whereas regulation by p56(lck) tyrosine kinase was conserved in all mutants. Remarkably, the apparent affinities for TEA(+), TPA(+), and quinine were changed differently in each mutant (EC(50) in WT, S286A, S292A, T296A, S328A, T550A, and 5x-PKC in mumol: TEA(+): 105, 153, 56, 1135, 484, 498, 518; TPA(+): 0.1, 2.1, 0.3, 1.0, 43, 0.3, 2.2; quinine: 1.5, 3.0, 2.5, 4.8, 81, 7.6, 8.9, respectively). After mutations, no effects of PKC activation on apparent affinity of rOCT1 for these substrates could be detected, in contrast to what was observed in WT. PKC activation had no significant effect on rOCT1 trafficking from intracellular pools to the cell membrane. Substitution of all PKC sites suppressed PKC-induced phosphorylation of rOCT1. In conclusion, it was found that the presence of all five potential PKC phosphorylation sites is necessary for the PKC-induced stimulation of rOCT1. The different effects on the EC(50) values by the different mutations suggest that the large intracellular loop participates in building the substrate binding pocket of rOCT1 or specifically modulates its structure.Journal of the American Society of Nephrology 07/2005; 16(6):1562-70. · 9.66 Impact Factor -
Article: Amino acids critical for substrate affinity of rat organic cation transporter 1 line the substrate binding region in a model derived from the tertiary structure of lactose permease.
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ABSTRACT: To identify functionally relevant amino acids in the rat organic cation transporter 1 (rOCT1), 18 consecutive amino acids in the presumed fourth transmembrane alpha helix (TMH) were mutated and functionally characterized after expression in Xenopus laevis oocytes. After mutation of three amino acids on successive turns of the alpha helix, K(m) values for tetraethylammonium (TEA) and/or 1-methyl-4-phenylpyridinium (MPP) were decreased. After replacement of Trp218 by tyrosine (W218Y) and Tyr222 by leucine (Y222L), the K(m) values for both TEA and MPP were decreased. In mutants Y222F and T226A, only the K(m) values for TEA and MPP were decreased, respectively. The data suggest that amino acids Trp218 and Tyr222 participate in the binding of both TEA and MPP, whereas Thr226 is only involved in the binding of MPP. Using the crystal structure of the lactose permease LacY from Escherichia coli that belongs to the same major facilitator superfamily as rOCT1, we modeled the tertiary structure of the presumed 12 transmembrane alpha helices. The validity of the model was suggested because seven amino acids that have been shown to participate in the binding of cations by mutagenesis experiments [fourth TMH Trp218, Tyr222, and Thr226 (this work); 10th TMH Ala443, Leu447, and Gln448 (companion work in this issue of Molecular Pharmacology); 11th TMH Asp475 (previous report)] are located in one region surrounding a large cleft that opens to the intracellular side. The dimensions of TEA in comparison with the interacting amino acids in the modeled cleft suggest that more than one TEA molecule can bind in parallel to the modeled conformation of the transporter.Molecular Pharmacology 06/2005; 67(5):1600-11. · 4.88 Impact Factor -
Article: Subtype-specific affinity for corticosterone of rat organic cation transporters rOCT1 and rOCT2 depends on three amino acids within the substrate binding region.
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ABSTRACT: The affinity of corticosterone to organic cation transporters (OCTs) is subtype- and species-dependent. For example, the IC50 values for corticosterone inhibition of cation uptake by transporters rOCT1 and rOCT2 are approximately 150 and approximately 4 microM, respectively. By introducing domains and amino acids from rOCT2 into rOCT1, we found that the exchange of three amino acids in the presumed 10th transmembrane alpha helix is sufficient to increase the affinity of rOCT1 for corticosterone to that of rOCT2. Replacement of these amino acids in rOCT2 decreased the affinity for corticosterone. These amino acids (Ala443, Leu447, and Gln448 in rOCT1 and Ile443, Tyr447, and Glu448 in rOCT2) are probably located within the substrate binding region because in rOCT1 mutants, the K(m) values for uptake of tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP) were decreased in parallel with a decrease of the IC50 values for the inhibition of cation uptake by corticosterone. In mutant rOCT1(L447Y/Q448E), the IC50 value for the inhibition of [3H]MPP (0.1 microM) uptake by corticosterone (24 +/- 4 microM) was significantly higher compared with the IC50 value for inhibition of [14C]TEA (10 microM) uptake (5.3 +/- 1.7 microM). This finding suggests an allosteric interaction between transported cation and corticosterone. Because this substrate-specific effect cannot be explained by differential replacement of corticosterone by MPP versus TEA and was observed after point mutations within the presumed substrate region, the data suggest that MPP or TEA bind to the substrate binding region simultaneously with corticosterone and cause a short-range allosteric effect on the corticosterone binding site.Molecular Pharmacology 06/2005; 67(5):1612-9. · 4.88 Impact Factor
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Institutions
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2005–2012
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Universität Würzburg
- Institute for Anatomy and Cell Biology
Würzburg, Bavaria, Germany
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2005–2009
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Institute of Anatomy and Cell Biology
Heidelberg, Baden-Wuerttemberg, Germany
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2006
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Institute for Medical Research and Occupational Health
Zagreb, Grad Zagreb, Croatia
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2003
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University of California, Los Angeles
- Department of Physiology
Los Angeles, CA, USA
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