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Probing the Structure of the hSGLT1 Sugar Binding Site
Author(s) -
Gorraitz Edurne,
Loo Donald D.F.,
Hirayama Bruce A.,
Jiang Xuan,
Wright Ernest M.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.694.12
Subject(s) - chemistry , fluorescence , biophysics , sugar , binding site , cotransporter , sodium , biochemistry , biology , organic chemistry , physics , quantum mechanics
The crystal structure of vSGLT shows that sugar is bound in the center of the protein separated from the extra‐ and intracellular compartments by gates. We have investigated the sugar binding vestibule of the human Na + /glucose cotransporter hSGLT1 by labeling residues in the sugar binding site with tetramethylrhodamine methanethiosulfonate (TMR‐MTS). H83, T287, and Y290 were individually mutated to cysteine, expressed in Xenopus oocytes, and their access to external TMR‐MTS was monitored by simultaneous fluorescence and electrophysiological recordings. Treatment with TMR‐MTS completely inhibited sugar transport but only in the presence of external Na + . Voltage‐jumps elicited changes in rhodamine fluorescence which followed the presteady state charge movements for T287C and Y290C. Labeling blocked the H83C capacitive currents. There was no effect of external sugar on the fluorescence but, in contrast, rhodamine fluorescence changes were dependent on the external Na + . In summary, external TMR‐MTS gains access to the sugar binding site and blocks sugar binding but not sodium‐dependent partial reactions. We conclude that external Na + triggers a conformational change in hSGLT1 to open the external gate and permit sugar binding. This large conformational change can accommodate molecules with a volume up to 1,700 Å 3 , i.e. TMR‐MTS or >50 water molecules. Support by NIH grant DK 19567 to E.M.W.