A KINETIC-MODEL WITH ORDERED CYTOPLASMIC DISSOCIATION FOR SUC1, AN ARABIDOPSIS H+ SUCROSE COTRANSPORTER EXPRESSED IN XENOPUS OOCYTES/

To elucidate the kinetic properties of the Arabidopsis H+/sucrose cotr ansporter, SUC1, with respect to transmembrane voltage and ligand conc entrations, the transport system was heterologously expressed in Xenop us laevis oocytes. Steady-state plasma membrane currents associated wi th 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 concent rations, and this enabled determination of values for the maximum curr ents in the presence of each ligand (i(max)(H),i(max)(S) for H+ and su crose) and of the ligand concentrations eliciting half-maximal current s (K-m(H),K-m(S)). The i(max)(H) and i(max)(S) exhibited marked and st atistically significant increases as a function of increasingly negati ve membrane potential. However, the K-m(H) and K-m(S) m decreased with increasingly negative membrane potential. Furthermore, at any given v oltage, i(max)(S) increased max and K-m(S) decreased as a function of the external H+ concentration. Eight six-state carrier models-which co mprised the four possible permutations of intracellular and extracellu lar ligand binding order, each with charge translocation on the sugar- loaded or -unloaded forms of the carrier-were analyzed algebraically w ith 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 t he carrier) exhibit sufficient kinetic flexibility to describe the obs ervations. Combining these two, a single model emerges in which the bi nding on the external side can be random, but it can only be ordered o n the inside, with the sugar dissociating before the proton.