Membrane topology of loop 13-14 of the Na+/glucose cotransporter (SGLT1): a SCAM and fluorescent labelling study.
The accessibility of the hydrophilic loop between putative transmembrane segments XIII and XIV of the Na+/glucose cotransporter (SGLT1) was studied in Xenopus oocytes, using the substituted cysteine accessibility method (SCAM) and fluorescent labelling. Fifteen cysteine mutants between positions 565 and 664 yielded cotransport currents of similar amplitude than the wild-type SGLT1 (wtSGLT1). Extracellular, membrane-impermeant MTSES(-) and MTSET(+) had no effect on either cotransport or Na+ leak currents of wtSGLT1 but 9 mutants were affected by MTSES and/or MTSET. We also performed fluorescent labelling on SGLT1 mutants, using tetramethylrhodamine-5-maleimide and showed that positions 586, 588 and 624 were accessible. As amino acids 604 to 610 in SGLT1 have been proposed to form part of a phlorizin (Pz) binding site, we measured the K(i)(Pz) and K(m)(alphaMG) for wtSGLT1 and for cysteine mutants at positions 588, 605-608 and 625. Although mutants A605C, Y606C and D607C had slightly higher K(i)(Pz) values than wtSGLT1 with minimal changes in K(m)((alpha)MG), the effects were modest and do not support the original hypothesis. We conclude that the large, hydrophilic loop near the carboxyl terminus of SGLT1 is thus accessible to the external solution but does not appear to play a major part in the binding of phlorizin.
Gagnon
Dominique G
DG
Groupe d'étude des protéines membranaires (GEPROM), Université de Montréal, C.P. 6128, succ. centre-ville, Montréal, Québec H3C 3J7, Canada.
Holt
Andrea
A
Bourgeois
Francis
F
Wallendorff
Bernadette
B
Coady
Michael J
MJ
Lapointe
Jean-Yves
JY
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D005456:Fluorescent Dyes / Q000494:pharmacology*; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry*; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010695:Phlorhizin / Q000737:chemistry; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012235:Rhodamines / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1; D014982:Xenopus laevis / Q000378:metabolism
D018076:DNA, Complementary; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D010455:Peptides; D020033:Protein Isoforms; D012235:Rhodamines; C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; C005358:tetramethylrhodamine; D012964:Sodium; D010695:Phlorhizin; D005947:Glucose; D003545:Cysteine