Two-Step Mechanism of Phlorizin Binding to the SGLT1 Protein in the Kidney

Abstract

The relationships between phlorizin binding and Na+-glucose cotransport were addressed in rabbit renal brush-border membrane vesicles. At pH 6.0 and 8.6, high affinity phlorizin binding followed single exponential kinetics. With regard to phlorizin concentrations, the binding data conformed to simple Scatchard kinetics with lower apparent affinities of onset binding (Kdi = 12-30 microM) compared to steady-state binding (Kde = 2-5 microM), and the first-order rate constants demonstrated a Michaelis-Menten type of dependence with Km values identical to Kdi. Phlorizin dissociation from its receptor sites also followed single exponential kinetics with time constants insensitive to saturating concentrations of unlabeled phlorizin or D-glucose, but directly proportional to Na+ concentrations. These results prove compatible with homogeneous binding to SGLT1 whereby fast Na+ and phlorizin addition on the protein is followed by a slow conformation change preceding further Na+ attachment, thus occluding part of the phlorizin-bound receptor complexes. This two-step mechanism of inhibitor binding invalidates the recruitment concept as a possible explanation of the fast-acting slow-binding paradigm of phlorizin, which can otherwise be resolved as follows: the rapid formation of an initial collision complex explains the fast-acting behavior of phlorizin with regard to its inhibition of glucose transport; however, because this complex also rapidly dissociates in a rapid filtration assay, the slow kinetics of phlorizin binding are only apparent and reflect its slow isomerization into more stable forms.

Ligands on the string: single-molecule AFM studies on the interaction of antibodies and substrates with the Na+-glucose co-transporter SGLT1 in living cells

“…Phlorizin, an aromatic ␤-glucoside, is a well-known potent competitive inhibitor of the SGLT1 transporter (Diedrich, 1966). It is postulated that phlorizin is supposed to bind with a two-step mechanism to both the sugar-binding site of the transporter (mainly through hydrogen bonds) and to a binding site for the aglucone moiety (phloretin) mainly through hydrophobic interactions Oulianova and Berteloot, 1996;Oulianova et al, 2001). Site-directed mutagenesis studies and studies using reconstituted peptide in vitro have shown that the C-terminal loop 13 is involved in the binding of phlorizin (Novakova et al, 2001;Raja et al, 2003;Xia et al, 2003).…”

Phlorizin Recognition in a C-terminal Fragment of SGLT1 Studied by Tryptophan Scanning and Affinity Labeling

“…Phlorizin, a ␤-glucoside of the aromatic compound phloretin, is the most potent competitive inhibitor, with an apparent K i of 1 M (7). It is proposed that the phlorizin binding to SGLT1 is a two-step process: rapid formation of an initial collision complex, followed by a slow isomerization process that occludes phlorizin within its receptor site (8). Phlorizin is thereby supposed to bind to both the sugar-binding site and the aglucon-binding site, the latter with a hydrophobic/aromatic surface (9,10).…”

4-Acetoxyscirpendiol of Paecilomyces tenuipes Inhibits Na+/D-Glucose Cotransporter Expressed in Xenopus laevis Oocytes

“…The present study shows that 4-ASD and its analogs are potential inhibitors of SGLT-1. As the inhibitory steps involving glucosides are well understood (Oulianova et al, 2001), inhibition by 4-ASD, despite the chemical structural differences of the 4-ASD analogues, strongly implies that additional inhibitory pathways reduce the overall performance of SGLTs. Further in vivo study of SGLT family gene expression and of the biochemical pathways affecting by the presence of 4-ASD may be necessary, since the present study used Xenopus oocytes which heterologously express SGLT-1.…”