Phenylglucosides and the Na+/glucose cotransporter (SGLT1): Analysis of interactions

Abstract

Phenylglucosides are transported by the intestinal Na+/glucose cotransporter (SGLT1) and phlorizin, the classical competitive inhibitor of SGLT1, is also a phenylglucoside. To investigate the structural requirements for binding of substrates to SGLT1, we have studied the interactions between phenylglucosides and the cotransporter expressed in Xenopus oocytes using tracer uptake and electrophysiological methods. Some phenylglucosides inhibited the Na(+)-dependent uptake of 14C-alpha-methyl-D-glucopyranoside (alpha MDG) with apparent Kis in the range 0.1 to 20 mM, while others had no effect. Electrophysiological experiments indicated that phenylglucosides can act either as: (1) transported substrates, e.g., arbutin; (2) nontransported inhibitors, e.g., glucosylphenyl-isothiocyanate; or (3) noninteracting sugars, e.g., salicin. The transported substrates (glucose, arbutin, phenylglucoside and helicin) induced different maximal currents, and computer simulations showed that this may be explained by a difference in the translocation rates of the sugar and Na(+)-loaded transporter. Computational chemistry indicated that all these beta-phenylglucosides have similar 3-D structures. Analysis showed that among the side chains in the para position of the phenyl ring the -OH group (arbutin) facilitates transport, but the -NCS (glucosylphenyl-isothiocyanate) inhibits transport. In the ortho position, -CH2OH (salicin) prevents interaction, but the aldehyde (helicin) permits the molecule to be transported. Studies such as these may help to understand the geometry and nature of glucoside binding to SGLT1.

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

“…1DOglc had a K 0.5 of 26 mM, three times higher than glucose, and the sugar K 0.5 was decreased by adding a methyl group to glucose in the axial configuration, ␣MDG (4.8 mM). This may be due to a favorable interaction of the methyl group with a hydrophobic residue in the binding site (4,5). The inability of pSGLT3 to bind 3OMglc suggests that a hydrogen bond donation to the protein is important in the third position in the sugar, although we cannot eliminate the possibility that -CH 3 is too big to fit in the binding site.…”

“…1 The abbreviations used are: SGLT, Na ϩ /glucose cotransporter; 1DOglc, 1-deoxy-D-glucopyranoside; 2DOglc, 2-deoxy-D-glucopyranoside; 2F2DOglc, 2-fluoro-2-deoxy-D-glucopyranoside; 3F3DOglc, 3-fluoro-3-deoxy-D-glucopyranoside; 3OMglc, 3-O-methyl-D-glucopyranoside; 4F4DOglc, 4-fluoro-4-deoxy-D-glucopyranoside; 5thioglc, 5-thio-Dglucopyranoside; 6DOglc, 6-deoxy-D-glucopyranoside; 6F6DOglc, 6-fluoro-6-deoxy-D-glucopyranoside; ␣MDG, ␣-methyl-D-glucopyranoside; ␤MDG, ␤-methyl-D-glucopyranoside; Glc, glucose; MeMTS, methyl methanethiosulfonate; MTS, methanethiosulfonate; MTSEA, 2-aminoethyl methanethiosulfonate; MTSHE; 2-hydroxyethyl methanethiosulfonate; hSGLT1, Na ϩ /glucose cotransporter, isoform 1, human; pSGLT3, Na ϩ /glucose cotransporter, isoform 3, pig; WT, wild-type hSGLT1; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; GLUT, facilitated glucose transporter. sion chamber (4). For the experiments, the oocytes were bathed in Na ϩ buffer composed of (in millimolar) 100 NaCl, 2 KCl, 1 MgCl 2 , 1 CaCl 2 , 10 HEPES/Tris, pH 7.4, and in the Na ϩ -free buffer, choline-Cl replaced NaCl.…”

“…This residue lies on the same face of the helix as 457, and the separation distance is compatible with simultaneous interactions with both O1 and O4. In addition, the sugar would be positioned with the 1 position facing the extracellular medium, as predicted by experiments using glycosides (4,5).…”

“…The ␣ or ␤ methyl group may also make hydrophobic interactions that compensate for the lost of hydrogen bond donation at position 1. The additional mass of the methyl group is easily accommodated, because our previous studies have shown that large structures can be added to the pyranose ring, especially in the ␤-configuration (4,5).…”

Arginine‐427 in the Na+/glucose cotransporter (SGLT1) is involved in trafficking to the plasma membrane

“…Xenopus laevis oocytes were isolated, injected with cRNA coding for wild-type (WT) or R427A rbSGLTI and 50 ,uM [t4C]-c~-methyl-D-glucoside (c~MDG) uptake was measured 3-7 days later [3]. SGLT1 membrane currents were measured using the two-microelectrode voltage clamp method [3].…”