4.7 Article

Bridging the gap between structure and kinetics of human SGLT1

Journal

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
Volume 302, Issue 9, Pages C1293-C1305

Publisher

AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpcell.00397.2011

Keywords

human SGLT1; vSGLT; stoichiometry; alternating access; kinetics; human SGLT1 homology models

Funding

  1. National Institutes of Health [DK19567, GM78844]
  2. American Heart Association [630258N]

Ask authors/readers for more resources

Sala-Rabanal M, Hirayama BA, Loo DDF, Chaptal V, Abramson J, Wright EM. Bridging the gap between structure and kinetics of human SGLT1. Am J Physiol Cell Physiol 302: C1293-C1305, 2012. First published December 7, 2011; doi:10.1152/ajpcell.00397.2011.-The Na+-glucose cotransporter hSGLT1 is a member of a class of membrane proteins that harness Na+ electrochemical gradients to drive uphill solute transport. Although hSGLT1 belongs to one gene family (SLC5), recent structural studies of bacterial Na+ cotransporters have shown that Na+ transporters in different gene families have the same structural fold. We have constructed homology models of hSGLT1 in two conformations, the inward-facing occluded (based on vSGLT) and the outward open conformations (based on Mhp1), mutated in turn each of the conserved gates and ligand binding residues, expressed the SGLT1 mutants in Xenopus oocytes, and determined the functional consequences using biophysical and biochemical assays. The results establish that mutating the ligand binding residues produces profound changes in the ligand affinity (the half-saturation concentration, K-0.5); e. g., mutating sugar binding residues increases the glucose K-0.5 by up to three orders of magnitude. Mutation of the external gate residues increases the Na+ to sugar transport stoichiometry, demonstrating that these residues are critical for efficient cotransport. The changes in phlorizin inhibition constant (K-i) are proportional to the changes in sugar K-0.5, except in the case of F101C, where phlorizin Ki increases by orders of magnitude without a change in glucose K-0.5. We conclude that glucose and phlorizin occupy the same binding site and that F101 is involved in binding to the phloretin group of the inhibitor. Substituted-cysteine accessibility methods show that the cysteine residues at the position of the gates and sugar binding site are largely accessible only to external hydrophilic methanethiosulfonate reagents in the presence of external Na+, demonstrating that the external sugar (and phlorizin) binding vestibule is opened by the presence of external Na+ and closes after the binding of sugar and phlorizin. Overall, the present results provide a bridge between kinetics and structural studies of cotransporters.

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