Potential-dependent steady-state kinetics of a dicarboxylate transporter cloned from winter flounder kidney

The two-electrode voltage-clamp technique in combination with tracer uptake experiments was used to investigate the dependence of dicarboxylate transport kinetics on membrane potential in Xenopus laevis oocytes expressing the flounder renal high-affinity-type sodium dicarboxylate cotransporter (fNaDC-3). Steady-state succinate-dependent currents in the presence of Na+ were saturable with an apparent affinity constant for succinate, K-0.5,K-succ. of 60 muM. K-0,K-5.succ was independent of membrane potential, suggesting succinate binding at the surface of the fNaDC-3 protein. The maximal succinate-dependent current, DeltaI(max), increased with hyperpolarization, suggesting that the empty carrier may translocate net charge. Succinate-induccd currents showed sigmoidal dependence on Na+ concentration, and K-0.5,K-Na+ decreased with hyperpolarization, suggesting Na+ binding in an ion well. Lowering the external Na+ concentration to 20 mM increased K-0.5,K-succ approximately threefold. Succinate-induced currents were inhibited by Li+ with an K-i,K-Li+ of approximately 0.5 mM, and a Hill coefficient of below unity indicating the interaction of one Li+ ion with an inhibitory site at fNaDC-3.