Effect of substrate on the pre-steady-state kinetics of the Na +/glucose cotransporter

Abstract

When measuring Na+/glucose cotransporter (SGLT1) activity in Xenopus oocytes with the two-electrode voltage-clamp technique, pre-steady-state currents dissipate completely in the presence of saturating α-methyl-glucose (αMG, a nonhydrolyzable glucose analog) concentrations. In sharp contrast, two SGLT1 mutants (C255A and C511A) that lack a recently identified disulfide bridge express the pre-steady-state currents in the presence of αMG. The dose-dependent effects of αMG on pre-steady-state currents were studied for wild-type (wt) SGLT1 and for the two mutants. Increases in αMG concentration reduced the total transferred charge (partially for the mutants, totally for wt SGLT1), shifted the transferred charge versus membrane potential (Q-V) curve toward positive potentials, and significantly modified the time constants of the pre-steady-state currents. A five-state kinetic model is proposed to quantitatively explain the effect of αMG on pre-steady-state currents. This analysis reveals that the reorientation of free transporter is the slowest step for wt SGLT1 either in the presence or in the absence of αMG. In contrast, the conformational change of the fully loaded mutant transporters constitutes their rate-limiting step in the presence of substrate and explains the persistence of pre-steady-state currents in this situation. © 2007 by the Biophysical Society.