Search

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.

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.sup.+], 3.5 for [tetraethylammonium.sup.+], and 4.0 for [1-methyl-4-phenylpyridinium.sup.+]. 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.sup.+], [K.sup.+], [Ca.sup.2+], [Mg.sup.2+], and/or [Cl.sup.-]. Charge-to-substrate ratios were not influenced by intracellular [succinate.sup.2-] or [glutarate.sup.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.sup.+]-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.sup.+] 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. organic cation uptake; kidney; stoichiometry; charge translocation; membrane potential

Effects of the sulfhydryl reagent methylmethanethiosulfonate (MMTS) on functions of organic cation transporters (OCTs) were investigated. Currents induced by 10 mM choline [[I.sub.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.sub.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.sub.0.5(choline)] values for choline-induced currents were increased. For rOCT1 it was shown that MMTS increased [I.sub.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.sub.50(choline)] for choline to inhibit MPP uptake by rOCTI was increased whereas the [I.sub.0.5(choline)] value for choline-induced current remained unchanged. At variance, in double mutant Cys322Ser, Cys451Met, [I.sub.0.5(choline)] was increased compared with rOCT1 wild-type whereas in the single mutant Cys322Ser [I.sub.0.5(choline)] was not changed. The data suggest that modification of rOCTI 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. sulfhydryl group modification; mutagenesis; substrate affinity; turnover

The product of gene RSC1A1, named RS1, participates in transcriptional and post-transcriptional regulation of the sodium-D-glucose cotransporter SGLT1. Using coexpression in oocytes of Xenopus laevis, post-transcriptional inhibition of human SGLT1 (hSGLT1) and some other transporters by human RS1 (hRS) 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-[[sup.14]C]glucopyranoside ([[sup.14]C]AMG) or hOCT2-mediated uptake of [[sup.14]C] tetraethylammonium ([[sup.14]C]TEA) was measured. Within 30 min after the injection of hRS1 protein, hSGLT1-expressed AMG uptake or hOCT2-expressed TEA uptake was inhibited by ~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 hRSI 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. [Na.sup.+]-D-glucose cotransport; SGLT1; RSl; glucose-dependent regulation; exocytotic pathway; dynamin; brefeldin A

The product of gene RSC1A1, named RS1, is involved in transcriptional and posttranscriptional regulation of sodium-D-glucose cotransporter SGLT1, and removal of RSI in mice led to an increase of SGLT1 expression in small intestine and to obesity (Osswald C, Baumgarten K, Sttimpel 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-[PK.sub.1] 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-[PK.sub.1] 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-[PK.sub.1] cells with brefeldin A induced rapid release of RS1 from the TGN, and confluence of LLC-[PK.sub.1] 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. [Na.sup.+]-D-glucose cotransport; SGLT1; RS1; confluence-dependent regulation; nuclear migration; dynamin; brefeldin A

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. [K.sub.m] values of hOCT2-mediated uptake of 95 [micro]M amiloride and 24 [micro]M ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [[IC.sub.50](in [micro]M) 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 [Ca.sup.2+]/CaM complex (-55 [+ or -] 5%, n = 10 and -63 [+ or -] 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (-54 [+ or -] 5%, n = 14, and -31 [+ or -] 6%, n = 26) and PKA (-16 [+ or -] 5%, n = 16, and -18 [+ or -] 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 [+ or -] 6%, n = 8, and +55 [+ or -] 17%, n = 16). Inhibition of [Ca.sup.2+]/CaM complex resulted in a significant decrease of [V.sub.max] (160-99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (-22 [+ or -] 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 [Ca.sup.2+]/CaM complex causes changes in transport capacity via hOCT2 trafficking. organic cation transport; fluorescence measurement; 4-[4-(dimethylamino)-styryl]-n-methylpyridinium; amiloride

SGLT 1 (SLC5A 1) 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 ~40-kDa protein and a broad ~75-kDa band that exhibited strong zonal (OS > C) and gender differences (F > M). In tissue cryosections, the antibody strongly stained BBM of the $3 PT segments in the OS and medullary rays (F > M) and smooth muscles of the blood vessels and renal capsule (F ~ M) and weakly stained the apical domain of other PT segments in the C (F ~ M). The phlorizin-sensitive uptake of D-[[sup.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. brush-border membrane; immunocytochemistry: kidney; sex differences; sodium-glucose cotransporters: steroid hormones

The rat organic cation transporter (rOCT1) in insect cells is expressed, to purify and reconstitute the transporter in artificial liposomes, and to measure rOCT1 mediated uptake of organic cations. Purification and reconstitution of functional active rOCT1 protein is an important step toward the biophysical characterization and crystallization.

Properties and regulation of the human organic cation (OC) transporter type 2 (hOCT2) expressed in HEK-293 cells were extensively characterized using the fluorescent OC 4-[4-(dimethylamino)styryl]-N-methylpyridinium (AS[P.sup.+]). AS[P.sup.+] uptake was electrogenic and inhibited by TP[A.sup.+] (E[C.sub.50] = 2.7 [micro]M), tetraethylammonium (E[C.sub.50] = 35 [micro]M), cimetidine (E[C.sub.50] = 36 [micro]M), or quinine (E[C.sub.50] = 6.7 [micro]M). Stimulation with carbachol or ATP decreased initial uptake by 44 [+ or -] 3 (n = 14) and 34 [+ or -] 4% (n = 21), respectively, independently of PKC but dependent on phosphatidylinositol 3-kinase (PI3K). PKA stimulation decreased uptake by 18 [+ or -] 4% (n = 40). Inhibition of calmodulin (CAM), [Ca.sup.2+]/CaM-dependent kinase II, or myosin light chain kinase decreased uptake by 63 [+ or -] 2 (n = 15), 40 [+ or -] 4 (n = 30), and 31 [+ or -] 4% (n = 16), respectively. Inhibition of CaM resulted in a significant change in the E[C.sub.50] value for the inhibition of AS[P.sup.+] uptake by tetraethylammonium. In conclusion, we demonstrate that the hOCT2 is inhibited by PI3K and PKA and activated by a CaM-dependent signaling pathway, probably via a change in substrate affinity. organic cation transport; human organic cation transporter type 2; human; calcium/calmodulin; calcium/calmodulin-dependent kinase II; myosin light chain kinase; phospholipase C; protein kinase C; protein kinase A; phosphatidylinositol 3-kinase; electrophysiology; fluorescence microscopy; 4-[4(dimethylamino)styryl]-N-methylpyridinium

For [Beta]-D-glucosylisophosphoramide mustard ([Beta]-D-Glc-IPM), a new alkylating drug in which isophosphoramide mustard is stabilized, a higher selectivity and lower myelotoxicity was observed than for the currently used cytostatic ifosfamide. Because [Beta]-D-Glc-IPM is hydrophilic and does not diffuse passively through the lipid bilayer, we investigated whether a transporter may be involved in the cellular uptake. A variety of cloned Na+-sugar cotransporters were expressed in Xenopus oocytes, and uptake measurements were performed. By tracer uptake and electrical measurements it was found that [Beta]-D-Glc-IPM was transported by the low-affinity [Na.sup.+]-D-glucose cotransporter SAAT1, which had been cloned from pig and is also expressed in humans. At membrane potentials between -50 and -150 mV, a 10-fold higher substrate affinity ([K.sub.m] [approximately equal to] 0.25 mM) and a 10-fold lower [V.sub.max] value were estimated for [Beta]-D-Glc-IPM transport than for the transport of D-glucose or methyl-[Alpha]-D-glucopyranoside (AMG). Transport of [Beta]-D-Glc-IPM and glucose by SAAT1 is apparently performed by the same mechanism because similar sodium dependence, dependence on membrane potential, electrogenicity, and phlorizin inhibition were determined for [Beta]-D-Glc-IPM, D-glucose, and AMG. Transcription of human SAAT1 was demonstrated in various human carcinomas and tumor cell lines. In one of these, the human carcinoma cell line T84, phlorizin inhibitable uptake of [Beta]-D-Glc-IPM was demonstrated with substrate saturation and an apparent [K.sub.m] of 0.4 mM. The data suggest that the [Na.sup.+]-D-glucose cotransporter SAAT1 transports [Beta]-D-Glc-IPM into human tumor cells and may accumulate the drug in the cells. They provide an example for drug targeting by employing a plasma membrane transporter.

A study of an isolated complementary DNA from rat kidney reveals that OCT1, a 556-amino acid membrane protein, influences the elimination of cationic drugs from the kidney. OCT1 is a kind of polyspecific transporter that acts similar to the organic cation uptake in the basolateral membrane in renal proximal tubules and has similar features as cation uptake in hepatocytes. It translocates both hydrophobic and hydrophilic organic cations and is not homologous to other proteins found in kidney.

The cell free (CF) expression and functional reconstitution of the rat organic cation transporters rOct1 and rOct2 and of the rat organic anion transporter rOat1 is described. Functional characterization of purified and reconstituted rOat1 provided evidence that rOat1 is an obligate electroneutral p-aminohippurate/[alpha]-ketoglutarate exchanger which is modulated by a low-affinity chloride binding site.

The rat organic cation transporter (rOCT)-2 was characterized by electrical and tracer flux measurements compared with rOCT1. By applying choline gradients to voltage-clamped Xenopus oocytes expressing rOCT2, potential-dependent currents could be induced in both directions. Tracer flux measurements with seven organic cations revealed similar Michaelis-Menten constant values for both transporters, with the exception of guanidine. In parallel experiments with rOCT2 and rOCT1, inhibition of tetraethylammonium transport by 12 cations, 2 weak bases, corticosterone, and the anions para-amminohippurate, [Alpha]-ketoglutarate, and probenecid was characterized. The [IC.sub.50] values of many inhibitors were similar for both transporters, whereas others were significantly different. Mepiperphenidol and O-methylisoprenaline showed an ~70-fold lower and corticosterone a 38-fold higher affinity for rOCT2. With the use of these inhibitors together with previous information on cation transporters, experimental protocols are proposed to dissect out the individual contributions of rOCT2 and rOCT1 in intact proximal tubule preparations. Inhibition experiments at different pH levels strongly suggest that the weak base quinine passively permeates the plasma membrane at physiological pH and inhibits rOCT2 from the intracellular side. polyspecific cation transporter; rat organic cation transporter; organic cation transporter subtypes; substrate specificity

Karbach, Ulrich, Jorn Kricke, Friederike Meyer-Wentrup, Valentin Gorboulev, Christopher Volk, Dominique Loffing-Cueni, Brigitte Kaissling, Sebastian Bachmann, and Hermann Koepsell. Localization of organic cation transporters OCT1 and OCT2 in rat kidney. Am J Physiol Renal Physiol 279: F679-F687, 2000.--Renal excretion and reabsorption of organic cations are mediated by electrogenic and electroneutral organic cation transporters, which belong to a recently discovered family of polyspecific transporters. These transporters are electrogenic and exhibit differences in substrate specificity. In rat, the renal expression of the polyspecific cation transporters rOCT1 and rOCT2 was investigated. By in situ hybridization, significant amounts of both rOCT1 and rOCT2 mRNA were detected in S1, S2, and S3 segments of proximal tubules. By immunohistochemistry, expression of the rOCT1 protein was mainly observed in S1 and S2 segments of proximal tubules, with lower expression levels in the S3 segments. At variance, rOCT2 protein was mainly expressed in the S2 and S3 segments. Both transporters were localized to the basolateral cell membrane. Neither rOCT1 nor rOCT2 was detected in the vasculature, the glomeruli, and nephron segments other than proximal tubules. The data suggest that rOCT1 and rOCT2 are responsible for basolateral cation uptake in the proximal tubule, which represents the first step in cation secretion. polyspecific cation transporter; organic cation transporter family; gene expression; kidney; immunohistochemisty; in situ hybridization