d-Glucose-Recognition and Phlorizin-Binding Sites in Human Sodium/d-Glucose Cotransporter 1 (hSGLT1):  A Tryptophan Scanning Study

Abstract

In order to gain a better understanding of the structure-function relation in hSGLT1, single Trp residues were introduced into a functional hSGLT1 mutant devoid of Trps at positions that previously had been postulated to be involved in sugar recognition/translocation and/or phlorizin binding. The mutant proteins were expressed in Pichia pastoris, purified, and reconstituted into liposomes. In transport experiments the putative sugar binding site mutants W457hSGLT1 and W460hSGLT1 showed a drastic decrease in affinity toward alpha-methyl-d-glucopyranoside with Km values of 13.3 and 5.26 mM compared to 0.4 mM of the Trp-less hSGLT1. In addition, a strong decrease in the inhibitory effect of phlorizin was observed. In Trp fluorescence studies the position of the emission maxima of the mutants, their sensitivity to N-bromosuccinimide oxidation, and their interaction with water soluble quenchers demonstrate that Trp457 and Trp460 are in contact with the hydrophilic extravesicular environment. In both mutants Trp fluorescence was quenched significantly, but differently, by various glucose analogues. They also show significant protection by d-glucose and phlorizin against acrylamide, KI, or TCE quenching. W602hSGLT1 and W609hSGLT1, the putative aglucone binding site mutants, exhibit normal sugar and phlorizin affinity, and show fluorescence properties which indicate that these residues are located in a very hydrophilic environment. Phlorizin and phloretin, but not d-glucose, protect both mutants against collisional quenchers. Depth-calculations using the parallax method suggest a location of Trp457 and Trp460 at an average distance of 10.8 A and 7.4 A from the center of the bilayer, while Trp602 and Trp609 are located outside the membrane. These results suggest that in the native carrier residues Gln at position 457 and Thr at position 460 reside in a hydrophilic access pathway extending 5-7 A into the membrane to which sugars as well as the sugar moiety of inhibitory glucosides bind. Residues Phe602 and Phe609 contribute by their hydrophobic aromatic residues toward binding of the aglucone part of phlorizin. Thereby in the phlorizin-carrier complex a close vicinity between these two subdomains of the transporter is established creating a phlorizin binding pocket with the previously estimated dimensions of 10 x 17 x 7 A.

Identification of phlorizin binding domains in sodium-glucose cotransporter family: SGLT1 as a unique model system

“…The side view helix H1 is shown to illustrate the positioning of Pz with regards to aglucone and gluoside binding pockets. Such interaction leads to a condensed state of loop 13 followed by major conformational changes [10], similar to what has been observed in the intact human SGLT1 carrier [22]. Evidence for specific conformational arrangements of the aglucone binding site was also obtained in AFM studies on intact cells [23].…”

Sodium glucose cotransporter SGLT1 as a therapeutic target in diabetes mellitus

“…Human SGLT1, encoded by the SLC5A1 gene on chromosome 22q13.1, is a 664-amino-acid protein, which contains 14 transmembrane α-helical domains [5, 22]. The glucose-binding domain of human SGLT1 is supposed to include amino acid residues 457-460 [23]. SGLT1 has a high-affinity for glucose (K 0.5 of 0.5-2 mM) and transports sodium and glucose with a 2:1 stoichiometry which enhances its concentrating power [5, 23-25].…”

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

“…Panayotova-Heiermann et al (16,17) have shown that the C-terminal half of the protein is responsible for D-glucose binding and translocation. Several mutagenesis studies and tryptophan scanning analysis of the isolated transporter have identified a variety of amino acids in the transmembrane helix between aa 454 and aa 460 (18,19). There is also evidence from single molecule recognition studies on brush border membrane vesicles using PAN3-2-poised cantilevers that D-glucose interacts with loop 13 of the transporter.…”

“…In previous studies we have shown that phlorizin binds to loop 13 of rabbit SGLT1 (aa 541-638) via the aromatic aglucone moiety inducing conformational changes of the loop, predominantly in the region between aa 600 and aa 621 (4,6) of the C terminus. Mutagenesis experiments provided also data on individual amino acids critically important for the interaction with the inhibitors (6,13,14).…”