Conformational dynamics of hSGLT1 during Na +/glucose cotransport

Abstract

This study examines the conformations of the Na +/glucose cotransporter (SGLT1) during sugar transport using charge and fluorescence measurements on the human SGLT1 mutant G507C expressed in Xenopus oocytes. The mutant exhibited similar steady-state and presteady-state kinetics as wild-type SGLT1, and labeling of Cys507 by tetramethylrhodamine-6-maleimide had no effect on kinetics. Our strategy was to record changes in charge and fluorescence in response to rapid jumps in membrane potential in the presence and absence of sugar or the competitive inhibitor phlorizin. In Na + buffer, step jumps in membrane voltage elicited presteady-state currents (charge movements) that decay to the steady state with time constants τ med (3-20 ms, medium) and τ slow (15-70 ms, slow). Concurrently, SGLT1 rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages (ΔF). The charge vs. voltage (Q-V) and fluorescence vs. voltage (ΔF-V) relations (for medium and slow components) obeyed Boltzmann relations with similar parameters: zδ (apparent valence of voltage sensor) ≈ 1; and V 0.5 (midpoint voltage) between -15 and -40 mV. Sugar induced an inward current (Na +/glucose cotransport), and reduced maximal charge (Q max) and fluorescence (ΔF max) with half-maximal concentrations (K 0.5) of 1 mM. Increasing [αMDG] o also shifted the V 0.5 for Q and ΔF to more positive values, with K 0.5's ≈ 1 mM. The major difference between Q and ΔF was that at saturating [αMDG] o, the presteady-state current (and Q max) was totally abolished, whereas ΔF max was only reduced 50%. Phlorizin reduced both Q max and ΔF max (K i ≈ 0.4 μM), with no changes in V 0.5's or relaxation time constants. Simulations using an eight-state kinetic model indicate that external sugar increases the occupancy probability of inward-facing conformations at the expense of outward-facing conformations. The simulations predict, and we have observed experimentally, that presteady-state currents are blocked by saturating sugar, but not the changes in fluorescence. Thus we have isolated an electroneutral conformational change that has not been previously described. This rate-limiting step at maximal inward Na +/sugar cotransport (saturating voltage and external Na + and sugar concentrations) is the slow release of Na + from the internal surface of SGLT1. The high affinity blocker phlorizin locks the cotransporter in an inactive conformation. © The Rockefeller University Press.