Coupled Sodium/Glucose Cotransport by SGLT1 Requires a Negative Charge at Position 454
- 25 September 2004
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 43 (41), 13175-13184
- https://doi.org/10.1021/bi048652d
Abstract
Na+/glucose cotransport by SGLT1 is a tightly coupled process that is driven by the Na+ electrochemical gradient across the plasma membrane. We have previously proposed that SGLT1 contains separate Na+- and glucose-binding domains, that A166 (in the Na+ domain) is close to D454 (in the sugar domain), and that interactions between these residues influence sugar specificity and transport. We have now expressed the mutant D454C in Xenopus laevis oocytes and examined the role of charge on residue 454 by replacing the Asp with Cys or His, and by chemical mutation of D454C with alkylating reagents of different charge (MTSES-, MTSET+, MMTS0, MTSHE0, and iodoacetate-). Functional properties were examined by measuring sugar transport and cotransporter currents. In addition, D454C was labeled with fluorescent dyes and the fluorescence of the labeled transporter was recorded as a function of voltage and ligand concentration. The data shows that (1) aspartate 454 is critically important for the normal trafficking of the protein to the plasma membrane; (2) there were marked changes in the functional properties of D454C, i.e., a reduction in turnover number and a loss of voltage sensitivity, although there were no alterations in sugar selectivity or sugar and Na+ affinity; (3) a negative charge on residue 454 increased Na+ and sugar transport with a normal stoichiometry of 2 Na+:1 sugar. A positive charge on residue 454, in contrast, uncoupled Na+ and sugar transport, indicating the importance of the negative charge in the coordination of the cotransport mechanism.Keywords
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