Identification of a region critically involved in the interaction of phlorizin with the rabbit sodium-D-glucose cotransporter SGLT1

In order to define potential interaction sites of SGLT1 with the transport inhibitor phlorizin, mutagenesis studies were performed in a hydrophobic re gion of loop 13 (aa 604-610), located extracellularly, close to the C-termi nus. COS 7 cells were transiently transfected with the mutants and the kine tic parameters of alpha -methyl-D-glucopyranoside (AMG) uptake into the cel ls were investigated. Replacement of the respective an-Lino acids with lysi ne reduced the maximal uptake rate: Y604K showed 2.2%, L606K 48.4%, F607K 1 5.1%, C608K 13.1%, G609K 14.1%, and L610K 17.2% of control. In all mutants the apparent K, for phlorizin increased at least by a factor of 5 compared to the wild-type K-i of 4.6 +/- 0.7 mu mol/l; most striking changes were ob served for Y604K (K-i = 75.3 +/- 19.0 mu mol/l) and C608K (K-i = 83.6 +/- 1 3.9 mu mol/l). Replacement of these amino acids with a nonpolar amino acid instead of lysine such as in Y604F, Y604G and C608A showed markedly higher affinities for phlorizin. In cells expressing the mutants the apparent affi nity of AMG uptake for the sugar was not statistically different from that of the wild type (K-m = 0.8 +/- 0.2 mmol/l). These studies suggest that the region between amino acids 604 and 610 is in volved in the interaction between SGLT1 and phlorizin, probably by providin g a hydrophobic pocket for one of the aromatic rings of the aglucone moiety of the glycoside.