Simulated annealing reveals the kinetic activity of SGLT1, a member of the LeuT structural family

Abstract

The Na+/glucose cotransporter (SGLT1) is the archetype of membrane proteins that use the electrochemical Na+ gradient to drive uphill transport of a substrate. The crystal structure recently obtained for vSGLT strongly suggests that SGLT1 adopts the inverted repeat fold of the LeuT structural family for which several crystal structures are now available. What is largely missing is an accurate view of the rates at which SGLT1 transits between its different conformational states. In the present study, we used simulated annealing to analyze a large set of steady-state and pre–steady-state currents measured for human SGLT1 at different membrane potentials, and in the presence of different Na+ and α-methyl-d-glucose (αMG) concentrations. The simplest kinetic model that could accurately reproduce the time course of the measured currents (down to the 2 ms time range) is a seven-state model (C1 to C7) where the binding of the two Na+ ions (C4→C5) is highly cooperative. In the forward direction (Na+/glucose influx), the model is characterized by two slow, electroneutral conformational changes (59 and 100 s−1) which represent reorientation of the free and of the fully loaded carrier between inside-facing and outside-facing conformations. From the inward-facing (C1) to the outward-facing Na-bound configuration (C5), 1.3 negative elementary charges are moved outward. Although extracellular glucose binding (C5→C6) is electroneutral, the next step (C6→C7) carries 0.7 positive charges inside the cell. Alignment of the seven-state model with a generalized model suggested by the structural data of the LeuT fold family suggests that electrogenic steps are associated with the movement of the so-called thin gates on each side of the substrate binding site. To our knowledge, this is the first model that can quantitatively describe the behavior of SGLT1 down to the 2 ms time domain. The model is highly symmetrical and in good agreement with the structural information obtained from the LeuT structural family.