In order to define potential interaction sites of SGLT1 with the transport inhibitor phlorizin, mutagenesis studies were performed in a hydrophobic region of loop 13 (aa 604–610), located extracellularly, close to the C-terminus. COS 7 cells were transiently transfected with the mutants and the kinetic parameters of α-methyl-d-glucopyranoside (AMG) uptake into the cells were investigated. Replacement of the respective amino acids with lysine reduced the maximal uptake rate: Y604K showed 2.2%, L606K 48.4%, F607K 15.1%, C608K 13.1%, G609K 14.1%, and L610K 17.2% of control. In all mutants the apparent K i for phlorizin increased at least by a factor of 5 compared to the wild-type K i of 4.6 ± 0.7 μmol/l; most striking changes were observed for Y604K (K i = 75.3 ± 19.0 μmol/l) and C608K (K i = 83.6 ± 13.9 μmol/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 affinity of AMG uptake for the sugar was not statistically different from that of the wild type (K m = 0.8 ± 0.2 mmol/l).
The Journal of Membrane Biology – Springer Journals
Published: Nov 1, 2001
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