Two-step mechanism of phlorizin binding to the SGLT1 protein in the kidney.

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.