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 transp ort kinetics on membrane potential in Xenopus laevis oocytes expressing the flounder renal high-affinity-type sodium dicarboxylate cotransporter (fNaD C-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 s uccinate binding at the surface of the fNaDC-3 protein. The maximal succina te-dependent current, DeltaI(max), increased with hyperpolarization, sugges ting that the empty carrier may translocate net charge. Succinate-induccd c urrents showed sigmoidal dependence on Na+ concentration, and K-0.5,K-Na+ d ecreased with hyperpolarization, suggesting Na+ binding in an ion well. Low ering the external Na+ concentration to 20 mM increased K-0.5,K-succ approx imately 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 i ndicating the interaction of one Li+ ion with an inhibitory site at fNaDC-3 .