Kinetic Mechanisms of Inhibitor Binding: Relevance to the Fast-Acting Slow-Binding Paradigm

Abstract

Although phlorizin inhibition of Na+-glucose cotransport occurs within a few seconds, 3H-phlorizin binding to the sodium-coupled glucose transport protein(s) requires several minutes to reach equilibrium (the fast-acting slow-binding paradigm). Using kinetic models of arbitrary dimension that can be reduced to a two-state diagram according to Cha's formalism, we show that three basic mechanisms of inhibitor binding can be identified whereby the inhibitor binding step either (A) represents, (B) precedes, or (C) follows the rate-limiting step in a binding reaction. We demonstrate that each of mechanisms A-C is associated with a set of unique kinetic properties, and that the time scale over which one may expect to observe mechanism C is conditioned by the turnover number of the catalytic cycle. In contrast, mechanisms A and B may be relevant to either fast-acting or slow-binding inhibitors. However, slow-binding inhibition according to mechanism A may not be compatible with a fast-acting behavior on the steady-state time scale of a few seconds. We conclude that the recruitment hypothesis (mechanism C) cannot account for slow phlorizin binding to the sodium-coupled glucose transport protein(s), and that mechanism B is the only alternative that may explain the fast-acting slow-binding paradigm.

The Actual Ionic Nature of the Leak Current through the Na+/Glucose Cotransporter SGLT1

“…This shift represents only 24% of the predicted shift in E Na and is indicative of Na þ being only one of several permeant ions present. A portion of the Na þ effect on the leak current amplitude certainly reflects the requirement of Na þ for Pz binding as is observed commonly for wt SGLT1 (25,26).…”

“…3, inset), other cations must participate in the leak current. Because external Na þ is required for Pz binding (25,26) (that in turn is required for leak current measurement), we replaced 75 mM Na þ with NMDG þ , keeping 15 mM Na þ present in the external solution to permit sufficient Pz binding. With the C292A mutant expressed at the plasma membrane, the leak current averaged À56.9 5 4.2 nA at À130 mV (Fig.…”