A Kinetic Model with Ordered Cytoplasmic Dissociation for SUC1, an Arabidopsis H⁺/Sucrose Cotransporter Expressed in Xenopus Oocytes

Abstract.

To elucidate the kinetic properties of the Arabidopsis H⁺/sucrose cotransporter, SUC1, with respect to transmembrane voltage and ligand concentrations, the transport system was heterologously expressed in Xenopus laevis oocytes. Steady-state plasma membrane currents associated with transport of sucrose were measured with two-electrode voltage clamp over the voltage range −180 to +40 mV as a function of extracellular pH and sugar concentrations. At any given voltage, currents exhibited hyperbolic kinetics with respect to extracellular H⁺ and sugar concentrations, and this enabled determination of values for the maximum currents in the presence of each ligand (i ^H _max , i ^S _max for H⁺ and sucrose) and of the ligand concentrations eliciting half-maximal currents (K ^H _m , K ^S _m ). The i ^H _max and i ^S _max exhibited marked and statistically significant increases as a function of increasingly negative membrane potential. However, the K ^H _m and K ^S _m decreased with increasingly negative membrane potential. Furthermore, at any given voltage, i ^S _max increased and K ^S _m decreased as a function of the external H⁺ concentration. Eight six-state carrier models—which comprised the four possible permutations of intracellular and extracellular ligand binding order, each with charge translocation on the sugar-loaded or -unloaded forms of the carrier—were analyzed algebraically with respect to their competence to account for the ensemble of kinetic observations. Of these, two models (first-on, first-off and last-on, first-off with respect to sucrose binding as it passes from outside to inside the cell and with charge translocation on the loaded form of the carrier) exhibit sufficient kinetic flexibility to describe the observations. Combining these two, a single model emerges in which the binding on the external side can be random, but it can only be ordered on the inside, with the sugar dissociating before the proton.