title: Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding.
abstract: Investigation of the structure/function relationships of the sodium-glucose transporter (SGLT1) is crucial to understanding the cotransporter mechanism. In the present study, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulfonate (MTS) derivatives to test whether predicted transmembrane IV participates in sugar binding. Five charged and polar residues (K139, Q142, T156, K157, and D161) and two glucose/galactose malabsorption missense mutations (I147 and S159) were replaced with cysteine. Mutants I147C, T156C, and K157C exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. I147C was similar in function to wild-type and was not studied further. Mutation of lysine-157 to cysteine (K157C) causes loss of phloridzin and alpha-methyl-D-glucopyranoside (alphaMG) binding. These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA). Mutation of threonine-156 to cysteine (T156C) reduces the affinity of alphaMG and phloridzin for T156C by approximately 5-fold and approximately 20-fold, respectively. In addition, phloridzin protects cysteine-156 in T156C from alkylation by MTSEA. Therefore, the presence of a positive charge or a polar residue at 157 and 156, respectively, affects sugar binding and sugar-induced Na(+) currents.
authors.affiliation: Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Tiemin
authors.initials: T
authors.lastname: Liu
authors.forename: Bryan
authors.initials: B
authors.lastname: Lo
authors.forename: Pam
authors.initials: P
authors.lastname: Speight
authors.forename: Mel
authors.initials: M
authors.lastname: Silverman
mesh_terms: D000478:Alkylation; D000818:Animals; D019556:COS Cells; D050260:Carbohydrate Metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D008698:Mesylates / Q000494:pharmacology; D008757:Methylglucosides / Q000378:metabolism; D020125:Mutation, Missense; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D010695:Phlorhizin / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051269:Sodium-Glucose Transporter 1 / Q000235:genetics / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D008698:Mesylates; D008757:Methylglucosides; D051269:Sodium-Glucose Transporter 1; C093171:methanethiosulfonate ethylammonium; C045880:methanesulfonic acid; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; C027020:methylglucoside; D005020:Ethyl Methanesulfonate; D010695:Phlorhizin; D003545:Cysteine