Cys351 and Cys361 of the Na+/glucose cotransporter are important for both function and cell-surface expression.

Here, we identify Cys351 and Cys361 as novel residues critical for the function and plasma membrane targeting of the Na+/glucose transporter-1 (SGLT1). HEK-293 cells expressing the C351A and C361A mutants showed no detectable Na(+)-coupled uptake for alpha-methyl glucoside (AMG). Cell-surface biotinylation and Western blot revealed that the two mutants were overexpressed in 293 cells; however, none of them exhibited normal cell-surface expression. When reconstituted in proteoliposomes, mutant SGLT1s demonstrated significantly lower affinity for AMG compared with the wild-type transporter. Incubation with the reducing agent dithiothreitol did not alter the catalytic activity of wild-type protein, but surprisingly, it nearly restored the ability of SGLT1-C351A and -C361A to bind and translocate AMG. Thus, the C351A and C361A mutations might cause a global reorganization of the disulfide bonds of SGLT1. Furthermore, we showed that a double mutation (C351A/C361A) restored the cell-surface expression of the single C-to-A mutants (C351A and C361A).

The endogenous CXXC motif governs the cadmium sensitivity of the renal Na+/glucose co-transporter.

Cadmium (Cd2+) poisoning causes severe renal disorders manifested by defects in reabsorptive transport of various compounds. It is reported here that the renal brush-border membrane Na+/glucose co-transporter-1 (SGLT1) is a molecular target for Cd2+ toxicity. In micromolar concentrations, Cd2+ acted as a noncompetitive, partial inhibitor of methyl-D-glucopyranoside uptake in vesicles from COS-7 cells transiently expressing SGLT1. In contrast, only a modest effect in the closely related Na+/myo-inositol co-transporter-1 (SMIT1) was observed. The factor responsible for this difference was the CXXC motif (X can be any residue) at the cytoplasmic end of the eighth transmembrane segment (TM8) of SGLT1. Thus, a mutational transfer of this motif conveyed Cd2+ sensitivity to SMIT1. Moreover, mimicking the inhibitory effect of Cd2+, the biarsenical molecule FlAsH-EDT2 strongly inhibited the SGLT1 that had an engineered tetracysteine motif at the cytoplasmic end of TM8. The experiments also showed that covalent binding of the sulfhydryl reactive biotin-PEO-maleimide to the SGLT1 wild type but not to the mutant lacking the CXXC motif was suppressed by Cd2+. Taken together, these results suggest that in SGLT1, Cd2+ binding to the CXXC motif induces conformational changes that cause a partial inhibition of d-glucose transport.