Residue 457 Controls Sugar Binding and Transport in the Na+/Glucose Cotransporter

Abstract

The Na ؉ /glucose cotransporter (SGLT1) is highly selective for its natural substrates, D-glucose and D-galactose. We have investigated the structural basis of this sugar selectivity on the human isoform of SGLT1, single site mutants of hSGLT1, and the pig SGLT3 isoform, expressed in Xenopus oocytes using electrophysiological methods and the effects of cysteine-specific reagents. Kinetics of transport of glucose analogues, each modified at one position of the pyranose ring, were determined for each transporter. Correlation of kinetics with amino acid sequences indicates that residue Gln-457 sequentially interacts with O1 of the pyranose in the binding site, and with O5 in the translocation pathway. Furthermore, correlation of the selectivity characteristics of the SGLT isoforms (SGLT1 transports both glucose and galactose, but SGLT2 and SGLT3 transport only glucose) with amino acid sequence differences, suggests that residue 460 (threonine in SGLT1, and serine in SGLT2 and SGLT3) are involved in hydrogen bonding to O4 of the pyranose. In addition, the results show that substrate specificity of binding is not correlated to substrate specificity of transport, suggesting there are at least two steps in the sugar translocation process.The Na ϩ /glucose cotransporters (SGLTs) 1 transform the energy of the Na ϩ electrochemical gradient into mechanical work to drive sugar across the membrane, potentially against its concentration gradient (e.g. Refs. 1-3). Previously we have used the interaction of large aromatic glycosides to probe the vestibule to the sugar-binding site and the translocation pathway. These studies determined essential aglycone-protein interactions of human SGLT1 (hSGLT1) and pig SGLT3 (pS-GLT3) and provided a minimum size for the vestibule and sugar transport "pore" (e.g. Refs. 4 -6). In this study we focus our attention on the importance of sugar-protein hydrogen bonding in substrate specificity of transport. Determination of interactions between the sugar and the protein is an important step in elucidating the structural factors that determine specificity in the sugar-binding site and will allow future studies on characterization of the translocation pathway.Our strategy was to: 1) determine the importance of the interactions at each position of the sugar for transport through hSGLT1; 2) test the proposed interactions in cotransporters mutated in a single residue identified through sequence analysis; and 3) compare the interactions of these sugars in an SGLT isoform with known differences in sugar recognition (pSGLT3).In this work we have taken advantage of advances in gene technology and application of biophysical methods to cotransport to extend classic studies of the selectivity of sugar cotransport to the molecular level. We studied sugar specificity in four clones: hSGLT1, two hSGLT1 mutants, Q457C and Q457E, and pSGLT3. Interactions of sugars with the SGLTs were studied by analyzing the transport of sugars that differ in only one position of the ring. The importance of each position in the gl...

Kinetics of the Reverse Mode of the Na+/Glucose Cotransporter

“…Similar results were obtained for sugar competition of αMDG uptake into inside-out membrane vesicles expressing hSGLT1 (αMDG > 3-O-methyl-glucose ~ D-galactose > D-glucose; Quick & Tomasevic & Wright, 2003). In contrast, the relative affinities for sugar cotransport in the forward direction by rabbit, rat and human isoforms of SGLT1 is αMDG ~ D-glucosẽ D-galactose > 3-O-methyl-glucose (Ikeda et al, 1989;Birnir et al, 1991;PanayatovaHeiermann, Loo & Wright, 1995;Hirayama et al, 1996;Díez-Sampedro et al, 2001). The differences in sugar affinity and selectivity for cotranport in the two directions under the same driving forces suggest differences in the architecture of the outward-and inwardfacing sugar binding sites.…”

“…5. Assuming that the maximum rate for Na + /sugar cotransport in the outward direction is identical for these sugars, as is the case for cotransport in the forward direction (Birnir et al, 1991;Hirayama, Loo & Wright, 1997;Díez-Sampedro, Wright & Hirayama, 2001), the relative affinities (for rSGLT1) are αMDG > D-galactose > 3-O-methyl-glucose > D-glucose > > L-glucose. Similar results were obtained for sugar competition of αMDG uptake into inside-out membrane vesicles expressing hSGLT1 (αMDG > 3-O-methyl-glucose ~ D-galactose > D-glucose; Quick & Tomasevic & Wright, 2003).…”

Molecular Analysis of the SGLT2 Gene in Patients with Renal Glucosuria

“…Many of the missense mutations (T51P, R137H, G272R, K311R, and R499C) affect residues that are highly conserved across the human SGLT family and across SGLT of other species (Mus musculus [mouse], Rattus norvegicus [rat], Oryctolagus cuniculus [rabbit]), which underlines their pathogenic role. Other missense mutations (G449D and F453L) lie in very close vicinity to Q457, the residue interacting with O1 and O5 of the pyranose in SGLT1 (30). SGLT2 mutations seem to be randomly distributed along the coding sequence of the gene, but, interestingly, no SGLT2 mutation was detected in regions 289 to 304 and 369 to 405, 1 considered to be mutational hot spots in SGLT1 (31).…”

Addition of NMDA-receptor antagonist MK801 during oxygen/glucose deprivation moderately attenuates the upregulation of glucose uptake after subsequent reoxygenation in brain endothelial cells

“…Addition of 10 µM MK801 reduced glucose uptake to 845 ± 20 % measured directly after OGD (statistically not significant) and significantly decreased it to 163 ± 9 % after the additional reoxygenation phase (p<0.05). To elucidate the roles of glut1 and sglt1 in the glucose uptake rates phloretin, a glut1 inhibitor [7], and galactose, a sglt1 inhibitor [3], were added during the uptake studies. Directly after OGD 10 µM phloretin significantly reduced the uptake of glucose to 170 ± 4 % (p<0.05), whereas supplementation of 20 mM galactose revealed a moderate, but also significant decrease to 657 ± 14 % (p<0.05).…”