Datensatz

Zusammenfassung

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 V_max 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 (K_0.5 about 100 microM) and one apparent phlorizin inhibition site (K_i about 5 microM) was observed whereas with SGLT1 plus RS1 two apparent transport sites (K_0.5(1) about 20 microM, K_0.5(2) about 1 mM) and two apparent phlorizin inhibition sites (K_i(1) about 0.3 microM, K_i(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.