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,succ , of 60 µM. K 0.5,succ was independent of membrane potential, suggesting succinate binding at the surface of the fNaDC-3 protein. The maximal succinate-dependent current, Δ I max , increased with hyperpolarization, suggesting that the empty carrier may translocate net charge. Succinate-induced currents showed sigmoidal dependence on Na + concentration, and K 0.5,Na+ decreased with hyperpolarization, suggesting Na + binding in an ion well. Lowering the external Na + concentration to 20 mM increased K 0.5,succ approximately threefold. Succinate-induced currents were inhibited by Li + with an K i,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.