Active site voltage clamp fluorometry of the sodium glucose cotransporter hSGLT1

Proceedings of the National Academy of Sciences of the United States of America
Edurne GorraitzDonald D F Loo

Abstract

In the human sodium glucose cotransporter (hSGLT1) cycle, the protein undergoes conformational changes where the sugar-binding site alternatively faces the external and internal surfaces. Functional site-directed fluorometry was used to probe the conformational changes at the sugar-binding site. Residues (Y290, T287, H83, and N78) were mutated to cysteines. The mutants were expressed in Xenopus laevis oocytes and tagged with environmentally sensitive fluorescent rhodamines [e.g., tetramethylrhodamine (TMR)-thiols]. The fluorescence intensity was recorded as the mutants were driven into different conformations using voltage jumps. Sugar binding and transport by the fluorophore-tagged mutants were blocked, but Na+ binding and the voltage-dependent conformational transitions were unaffected. Structural models indicated that external Na+ binding opened a large aqueous vestibule (600 Å3) leading to the sugar-binding site. The fluorescence of TMR covalently linked to Y290C, T287C, and H83C decreased as the mutant proteins were driven from the inward to the outward open Na+-bound conformation. The time courses of fluorescence changes (milliseconds) were close to the SGLT1 capacitive charge movements. The quench in rhodamine fluorescen...Continue Reading

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Citations

Aug 23, 2018·Diabetologia·Chiara GhezziErnest M Wright
Dec 12, 2018·Nature Communications·Paola BisignanoMichael Grabe
Nov 15, 2019·Nature·Gabriel A FitzgeraldScott C Blanchard
Jan 30, 2021·Function·Ernest M Wright, Donald D F Loo
Apr 4, 2021·International Journal of Molecular Sciences·Jeff Abramson, Ernest M Wright
May 15, 2019·Biochimica Et Biophysica Acta. Biomembranes·Hans EbelJörg D Schulzke

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Methods Mentioned

BETA
X-ray
Fluorescence

Software Mentioned

SigmaPlot
pClamp
Maestro Molecular Modeling Interface
Molsoft ICM - Pro
UCSF Chimera
Coot
PyMol
PRODRG
Internal Coordinates Mechanics (

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