Five Transmembrane Helices Form the Sugar Pathway through the Na+/Glucose Cotransporter

• Published in: The Journal of biological chemistry 1997-08, Vol.272 (33), p.20324-20327
• Main Authors: Panayotova-Heiermann, Mariana, Eskandari, Sepehr, Turk, Eric, Zampighi, Guido A., Wright, Ernest M.
• Format: Article
• Language: English
• Subjects: Animals; Carbohydrate Metabolism; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - physiology; Methylglucosides - metabolism; Monosaccharide Transport Proteins - chemistry; Monosaccharide Transport Proteins - physiology; Peptide Fragments - chemistry; Phlorhizin - pharmacology; Protein Structure, Secondary; Rabbits; Recombinant Proteins - chemistry; Sodium-Glucose Transporter 1; Xenopus laevis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.33.20324

To test the hypothesis that the C-terminal half of the Na+/glucose cotransporter (SGLT1) contains the sugar permeation pathway, a cDNA construct (C5) coding for rabbit SGLT1 amino acids 407–662, helices 10–14, was expressed inXenopus oocytes. Expression and function of C5was followed by Western blotting, electron microscopy, radioactive tracer, and electrophysiological methods. The C5 protein was synthesized in 20-fold higher levels than SGLT1. The particle density in the protoplasmic face of the oocyte plasma membrane increased 2-fold after C5-cRNA injection compared with noninjected oocytes. The diameters of the C5 particles were heterogeneous (4.8 ± 0.3, 7.1 ± 1.2, and 10.3 ± 0.8 nm) in contrast to the endogenous particles (7.6 ± 1.2 nm). C5 increased the α-methyl-d-glucopyranoside (αMDG) uptake up to 20-fold above that of noninjected oocytes and showed an apparent K0.5αMDGof 50 mm and a turnover of ∼660 s−1. Influx was independent of Na+ with transport characteristics similar to those of SGLT1 in the absence of Na+: 1) selective (αMDG >d-glucose >d-galactose ≫l-glucose ≈ d-mannose), 2) inhibited by phloretin, KiPT= ∼500 μm, and 3) insensitive to phlorizin. These results indicate that C5 behaves as a specific low affinity glucose uniporter. Preliminary studies with three additional constructs, hC5 (the human equivalent of C5), hC4 (human SGLT1 amino acids 407–648, helices 10–13), and hN13 (amino acids 1–648, helices 1–13), further suggest that helices 10–13 form the sugar permeation pathway for SGLT1.