Substrate specificity of sugar transport by rabbit SGLT1: Single-molecule atomic force microscopy versus transport studies

Abstract

In the apical membrane of epithelial cells from the small intestine and the kidney, the high-affinity Na+/D-glucose cotransporter SGLT1 plays a crucial role in selective sugar absorption and reabsorption. How sugars are selected at the molecular level is, however, poorly understood. Here atomic force microscopy (AFM) was employed to investigate the substrate specificity of rbSGLT1 on the single-molecule level, while competitive-uptake assays with isotope-labeled sugars were performed in the study of the stereospecificity of the overall transport. rbSGLT1-transfected Chinese hamster ovary (CHO) cells were used for both approaches. Evidence of binding of D-glucose to the extracellular surface of rbSGLT1 could be obtained using AFM tips carrying 1-thio-D-glucose coupled at the C1 position to a PEG linker via a vinylsulfon group. Competition experiments with monosaccharides in solution revealed the following selectivity ranking of binding: 2-deoxy-D-glucose ≥ 6-deoxy-D-glucose > D-glucose > D-galactose ≥ α-methyl glucoside; 3-deoxy-D-glucose, D-xylose, and L-glucose did not measurably affect binding. These results were different from those of competitive α-methyl glucoside transport assays, where the ranking of inhibition was as follows: D-glucose > D-galactose > 6-deoxy-D-glucose; no uptake inhibition by D-xylose, 3-deoxy-D-glucose, 2-deoxy-D-glucose, or L-glucose was observed. Taken together, these results suggest that the substrate specificity of SGLT1 is determined by different recognition sites: one possibly located at the surface of the transporter and others located close to or within the translocation pathway. © 2007 American Chemical Society.