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 m onoclonal antibody (R4A6) which stimulates high-affinity phlorizin bin ding in kidney and intestine but does not react with the membrane prot ein (SGLT1) which mediates Na+-coupled transport Of D-glucose (Hediger , M. A., Coady, M. J., Ikeda, T. S., and Wright, E. M. (1987) Nature 3 30, 379-381). A cDNA (RS1) was obtained which codes for a hydrophilic M(r) 66,832 polypeptide and contains a predicted hydrophobic alpha-hel ix at the COOH terminus. After expression in Xenopus oocytes RS1 prote in was found associated with the plasma membrane. RS1-homologous mRNAs were detected in renal outer cortex and outer medulla, small intestin e, liver, and LLCPK1 cells, but not in skeletal muscle, heart muscle, Madin-Darby canine kidney (MDCK) cells, renal inner medulla, and Xenop us oocytes. After nondenaturing gel electrophoresis of renal brush-bor der membranes comigration of RS1- and SGLT1-homologous proteins as a h igh molecular weight complex was demonstrated. RS1 altered the express ion 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 tr ansport. However, RS1 also changed the expression of the SGLT1-homolog ous Na+-myo-inositol cotransporter from MDCK cells. The V(max) of meth yl-alpha-D-glucopyranoside (AMG) transport expressed after injection o f a small amount of SGLT1-cRNA was increased 40-fold when a stoichiome tric amount of RS1-cRNA was coinjected. In addition the voltage and gl ucose dependence of expressed AMG uptake and the concentration depende nce of transport inhibition by phlorizin were changed when stoichiomet ric amounts of RS1-cRNA were coinjected with SGLT1-cRNA. Thus with SGL T1 one apparent transport site (K0.5 about 100 muM) and one apparent p hlorizin inhibition site (K(i) about 5 pm) was observed whereas with S GLT1 plus RS1 two apparent transport sites (K0.5(1) about 20 muM, K0.5 (2) about 1 mM) and two apparent phlorizin inhibition sites (K(i)(1) a bout 0.3 muM, K(i)(2) about 30 muM) were found as has been described i n brush-border membrane vesicles of kidney and intestine (see eg. Koep sell, H., Fritzsch, G., Korn, K., and Madrala, A. (1990) J. Membr. Bio l. 114, 113-132). The data suggest that the Na+-D-glucose cotransporte r and possibly also other SGLT1-type Na+-cotransporters contain RS1-ty pe regulatory subunits.