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 >or= 6-deoxy-d-glucose > d-glucose > d-galactose >or= alpha-methyl glucoside; 3-deoxy-d-glucose, d-xylose, and l-glucose did not measurably affect binding. These results were different from those of competitive alpha-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.

C-terminal Loop 13 of Na+/Glucose Cotransporter 1 Contains Both Stereospecific and Non-stereospecific Sugar Interaction Sites

“…Stereospecificity of interactions between extracellularly located parts of the whole SGLT1 and glucose has been shown previously (10). For example, the binding probability of 1-thio-␤-D-glucose, coupled to the flexible cross-linker on an atomic force microscopy tip by a thio-glycosidic bond at the sugar's C1 position, was decreased significantly if (in the presence of sodium) free D-glucose was applied during force distance cycles using SGLT1-transfected Chinese hamster ovary cells, whereas only a slight effect could be observed in the presence of L-glu- Table 3.…”

“…4 and 8) and functional asymmetry (sugar selectivity seems to be different between the extracellular and the cytoplasmic face) (9). Recently we have shown that there are at least two sugar interaction sites on rabbit SGLT1, one representing the initial binding site and one related to the translocation pathway (10). SGLT1 can be inhibited by several glucosides with either aromatic or aliphatic aglucone residues (11,12).…”

Forces and Dynamics of Glucose and Inhibitor Binding to Sodium Glucose Co-transporter SGLT1 Studied by Single Molecule Force Spectroscopy

“…New SGLT1 Sensors-In the current study new coupling chemistry for tethering thioglucose, hexyl-glucose, and aminophlorizin to the AFM tip was developed. Previously, AFM studies on SGLT1 had been performed mainly with a vinylsulfone linker between the PEG chain and thioglucose or a modified 3Ј aminophlorizin (19,20,48). By using acrylamide as the thioreactive group to attach thioglucose to PEG 5000 , the size (and charge) of the thioester linkage is reduced compared with vinylsulfone-PEG 800.…”

“…Thus, it might be assumed that at 37°C some of the primarily not accessible binding sites further down in the translocation pathway are reached by thioglucose on AA-PEG 5000 (21). A sequential multistep mechanism for sugar recognition and translocation has also been proposed based on prior AFM investigations (20).…”

“…They form a vestibule for the entry of the sugar into the translocation pathway and contain the first of several sugar recognition sites. This vestibule is accessible to the sugar only in the presence of sodium (20,21). Phlorizin acts as a competitive inhibitor of SGLT1 with an apparent K i of 1 M (22).…”