Downregulation of the Na(+)- D-glucose cotransporter SGLT1 by protein RS1 (RSC1A1) is dependent on dynamin and protein kinase C.

We have previously shown that the regulatory protein RS1, cloned from pig, rabbit and human (RSC1A1), is localized intracellularly and inhibits the transcription of the Na(+)- D-glucose cotransporter SGLT1 in LLC-PK(1) cells. We also reported that transport activities of human SGLT1 (hSGLT1) and human organic cation transporter hOCT2 expressed in Xenopus oocytes were decreased upon co-expression of human RS1 (hRS1). The present paper indicates that the glucose transporter GLUT1 and the peptide transporter PEPT1 are not influenced by hRS1. Voltage-clamp experiments in oocytes expressing hSGLT1 demonstrated that hRS1 reduced the maximal substrate-induced currents but did not change substrate activation, membrane potential dependence, Na(+) dependence or substrate selectivity of hSGLT1. Co-expression experiments with a dominant-negative dynamin mutant showed that the posttranslational inhibition of hSGLT1 by hRS1 was dependent on the function of dynamin. Finally, we observed that hRS1 changed the short-term effect of protein kinase C (PKC) on hSGLT1. Whereas the PKC activators phorbol-12-myristate-13-acetate (PMA) and sn-1,2-dioctanoyl glycerol (DOG) increased alpha-methyl glucose (AMG) uptake expressed by hSGLT1 alone as described earlier, PMA and DOG decreased AMG uptake mediated by hSGLT1 when hRS1 was co-expressed. Taken together, these data indicate that hRS1 modulates dynamin-dependent trafficking of intracellular vesicles containing hSGLT1 in Xenopus oocytes, and modulates PKC-dependent short-term regulation of this transporter.

The transport modifier RS1 is localized at the inner side of the plasma membrane and changes membrane capacitance.

Previously we cloned membrane associated (M(r) 62000-67000) polypeptides from pig (pRS1), rabbit (rbRS1) and man (hRS1) which modified transport activities that were expressed in Xenopus laevis oocytes by the Na(+)-D-glucose cotransporter SGLT1 and/or the organic cation transporter OCT2. These effects were dependent on the species of RS1 and on the target transporters. hRS1 and rbRS1 were shown to be intronless single copy genes which are expressed in various tissues and cell types. Earlier immunohistochemical data with a monoclonal IgM antibody suggested an extracellular membrane association of RS1. In the present paper antibodies against recombinant pRS1 were raised and the distribution and membrane localization of RS1 reevaluated. After subcellular fractionation of renal cortex RS1 was found associated with brush border membranes and an about 1:200 relation between RS1 and SGLT1 protein was estimated. Also after overexpression in X. laevis oocytes RS1 was associated with the plasma membrane, however, at variance to the kidney it was also observed in the cytosol. Labeling experiments with covalently binding lipid-permeable and lipid-impermeable biotin analogues showed that RS1 is localized at the inner side of the plasma membrane. Western blots with plasma membranes from Xenopus oocytes revealed that SGLT1 protein in the plasma membrane was reduced when hRS1 was coexpressed with human SGLT1 which leads to a reduction in V(max) of expressed glucose transport. Measurements of membrane capacitance and electron microscopic inspection showed that the expression of hRS1 leads to a reduction of the oocyte plasma membrane surface. The data suggest that RS1 is an intracellular regulatory protein that associates with the plasma membrane. Overexpression of RS1 may effect the incorporation and/or retrieval of transporters into the plasma membrane.

Cloning and characterization of the transport modifier RS1 from rabbit which was previously assumed to be specific for Na+-D-glucose cotransport.

Previously we cloned membrane associated polypeptides from pig and man (pRS1, hRS1) which altered rate and glucose dependence of Na+-d-glucose cotransport expressed by SGLT1 from rabbit and man. This paper describes the cloning of a related cDNA sequence from rabbit intestine (rbRS1) which encodes a gene product with about 65% amino acid identity to pRS1 and hRS1. Hybridization of endonuclease-restricted genomic DNA with cDNA fragments of rbRS1 showed that there is only one gene with similarity to rbRS1 in rabbit, and genomic PCR amplifications revealed that the rbRS1 gene is intronless. Comparing the transcription of rbRS1 and rbSGLT1 in various tissues and cell types, different mRNA patterns were obtained for both genes. In Xenopus oocytes the Vmax of expressed Na+-d-glucose cotransport was increased or decreased when rbRS1 was coexpressed with rbSGLT1 or hSGLT1, respectively. After coexpression with hSGLT1 the glucose dependence of the expressed transport was changed. By coexpression of rbRS1 with the human organic cation transporter hOCT2 the expressed cation uptake was not altered; however, the expressed cation uptake was drastically decreased when hRS1 was coexpressed with hOCT2. The data show that RS1 can modulate the function of transporters with non-homologous primary structures.

Transport of the new chemotherapeutic agent beta-D-glucosylisophosphoramide mustard (D-19575) into tumor cells is mediated by the Na+-D-glucose cotransporter SAAT1.

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+-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 (Km approximately 0.25 mM) and a 10-fold lower Vmax 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 Km of 0.4 mM. The data suggest that the Na+-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.

The human gene of a protein that modifies Na(+)-D-glucose co-transport.

Recently, a cDNA (pRS1) was cloned from pig kidney cortex that encodes a membrane-associated protein involved in Na(+)-coupled sugar transport. pRS1 alters sugar transport by SGLT1 from rabbit intestine or by SMIT from dog kidney which is homologous to SGLT1. In contrast, pRS1 does not influence transporters from other genetic families. We report the cloning of the intronless human gene hRS1 (6,743 bp), which encodes a 617-amino-acid protein with 74% amino acid identity to pRS1. By fluorescence in situ hybridization, hRS1 was localized to chromosome 1p36.1. The localization to one chromosome and Southern blot analysis of restricted genomic DNA suggest that there is only one RS1-homologous gene in humans. Functionality of hRS1 was demonstrated by co-expression experiments of hRS1 and SGLT1 from human intestine in oocytes from Xenopus laevis. They show that hRS1-protein inhibits Na(+)-D-glucose co-transport expressed by human SGLT1 by decreasing both the Vmax and the apparent Km value of the transporter. The analysis of the 5'-noncoding sequence of hRS1 revealed different enhancer consensus sequences that are absent in the SGLT1 gene, e.g., several consensus sequences for steroid-binding proteins.

Drug excretion mediated by a new prototype of polyspecific transporter.

Cationic drugs of different types and structures (antihistaminics, antiarrhythmics, sedatives, opiates, cytostatics and antibiotics, for example) are excreted in mammals by epithelial cells of the renal proximal tubules and by hepatocytes in the liver. In the proximal tubules, two functionally disparate transport systems are involved which are localized in the basolateral and luminal plasma membrane and are different from the previously identified neuronal monoamine transporters and ATP-dependent multidrug exporting proteins. Here we report the isolation of a complementary DNA from rat kidney that encodes a 556-amino-acid membrane protein, OCT1, which has the functional characteristics of organic cation uptake over the basolateral membrane of renal proximal tubules and of organic cation uptake into hepatocytes. OCT1 is not homologous to any other known protein and is found in kidney, liver and intestine. As OCT1 translocates hydrophobic and hydrophilic organic cations of different structures, it is considered to be a new prototype of polyspecific transporters that are important for drug elimination.

Cloning of a membrane-associated protein which modifies activity and properties of the Na(+)-D-glucose cotransporter.

An expression library from porcine kidney cortex was screened with a monoclonal antibody (R4A6) which stimulates high-affinity phlorizin binding in kidney and intestine but does not react with the membrane protein (SGLT1) which mediates Na(+)-coupled transport of D-glucose (Hediger, M.A., Coady, M.J., Ikeda, T.S., and Wright, E.M. (1987) Nature 330, 379-381). A cDNA (RS1) was obtained which codes for a hydrophilic M(r) 66,832 polypeptide and contains a predicted hydrophobic alpha-helix at the COOH terminus. After expression in Xenopus oocytes RS1 protein was found associated with the plasma membrane. RS1-homologous mRNAs were detected in renal outer cortex and outer medulla, small intestine, liver, and LLCPK1 cells, but not in skeletal muscle, heart muscle, Madin-Darby canine kidney (MDCK) cells, renal inner medulla, and Xenopus oocytes. After nondenaturing gel electrophoresis of renal brush-border membranes comigration of RS1- and SGLT1-homologous proteins as a high molecular weight complex was demonstrated. RS1 altered the expression of Na(+)-glucose cotransport by SGLT1 in Xenopus oocytes. There was no effect on the expression of the nonhomologous transporters for Na(+)-gamma-aminobutyric acid cotransport and for Na(+)-independent glucose transport. However, RS1 also changed the expression of the SGLT1-homologous Na(+)-myo-inositol cotransporter from MDCK cells. The Vmax of methyl-alpha-D-glucopyranoside (AMG) transport expressed after injection of a small amount of SGLT1-cRNA was increased 40-fold when a stoichiometric amount of RS1-cRNA was coinjected. In addition the voltage and glucose dependence of expressed AMG uptake and the concentration dependence of transport inhibition by phlorizin were changed when stoichiometric amounts of RS1-cRNA were coinjected with SGLT1-cRNA. Thus with SGLT1 one apparent transport site (K0.5 about 100 microM) and one apparent phlorizin inhibition site (Ki about 5 microM) was observed whereas with SGLT1 plus RS1 two apparent transport sites (K0.5(1) about 20 microM, K0.5(2) about 1 mM) and two apparent phlorizin inhibition sites (Ki(1) about 0.3 microM, Ki(2) about 30 microM) were found as has been described in brush-border membrane vesicles of kidney and intestine (see e.g. Koepsell, H., Fritzsch, G., Korn, K., and Madrala, A. (1990) J. Membr. Biol. 114, 113-132). The data suggest that the Na(+)-D-glucose cotransporter and possibly also other SGLT1-type Na(+)-cotransporters contain RS1-type regulatory subunits.

Blood-brain barrier characteristic enzymatic properties in cultured brain capillary endothelial cells.

Isolated brain capillary endothelial cells contain high activity levels of the blood-brain barrier (BBB) marker enzymes gamma-glutamyltranspeptidase (gamma-GT) and alkaline phosphatase (ALP). In primary culture the activities of these specific enzymes decrease with increasing cell proliferation to a constant low value characteristic for a confluent monolayer. However, activities are retained in non-proliferating cells. After passage of cells from a confluent cell monolayer a further reduction of enzyme activity was observed which corresponds to the newly triggered cell proliferation. Culture of cerebral endothelial cells on structural components of the native vascular basement membrane-like type IV collagen, fibronectin, laminin or a commercially available basement membrane cannot prevent the activity decrease of both gamma-GT and ALP. Antiserum raised against a native renal dog gamma-GT binds to the cerebral endothelial gamma-GT and suppresses its activity. The relative activity decrease induced by a given amount of anti-gamma-GT-antiserum is constant at all times in culture. This result clearly shows that the observed decrease in gamma-GT activity in proliferating cells in culture correlates to a decreased number of enzyme molecules per cell and not to an inhibition of expressed enzymes. Possibly the de novo synthesis of this enzyme is prevented in vitro. In contrast to the loss of the activity of the BBB marker enzymes gamma-GT and ALP, the activity of angiotensin-converting enzyme (ACE), a marker for all vascular endothelial cells, is highly preserved in cultured cerebral endothelial cells.