Effect of substrate on the presteady state kinetics of the Na⁺/glucose cotransporter

¹ Université de Montréal

^* To whom correspondence should be addressed. E-mail: jean-yves.lapointe{at}umontreal.ca.

Submitted on June 26, 2006
Revised on July 29, 2006
Accepted on 5 October 2006

	Abstract

When measuring Na⁺/glucose cotransporter (SGLT1) activity in Xenopus oocytes with the two-electrode voltage-clamp technique, presteady state currents dissipate completely in the presence of saturating methyl-glucose (MG, a nonhydrolysable glucose analogue) concentrations. In sharp contrast, two SGLT1 mutants (C255A and C511A) which lack a recently identified disulfide bridge (Gagnon et al., JGP, 2006, 127:145-158), express the presteady state currents in the presence of MG. The dose-dependent effects of MG on presteady state currents were studied for wild type (wt) SGLT1 and for the two mutants. Increases in MG 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 presteady state currents. A five-state kinetic model is proposed to quantitatively explain the effect of MG on presteady 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 MG. 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 presteady state currents in this situation.

Key Words: SGLT1, kinetic model, mutagenesis, presteady state currents, transferred charge, two-electrode voltage-clamp