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, K0.5,succ, of 60 microM. K0.5,succ was independent of membrane potential, suggesting succinate binding at the surface of the fNaDC-3 protein. The maximal succinate-dependent current, deltaImax, increased with hyperpolarization, suggesting that the empty carrier may translocate net charge. Succinate-induced currents showed sigmoidal dependence on Na+ concentration, and K0.5,Na+ decreased with hyperpolarization, suggesting Na+ binding in an ion well. Lowering the external Na+ concentration to 20 mM increased K0.5,succ approximately threefold. Succinate-induced currents were inhibited by Li+ with an Ki,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.
Burckhardt
B C
BC
Zentrum Physiologie und Pathophysiologie, Abt Vegetative Physiologie und Pathophysiologie, Georg-August Universität Göttingen, Germany. bcburckhardt@veg-physiol.med.uni-goettingen.de
Steffgen
J
J
Langheit
D
D
Müller
G A
GA
Burckhardt
G
G
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D029382:Dicarboxylic Acid Transporters; D004553:Electric Conductivity; D005260:Female; D005432:Flounder / Q000378:metabolism*; D015870:Gene Expression; D007668:Kidney / Q000737:chemistry*; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D008536:Meglumine / Q000494:pharmacology; D008564:Membrane Potentials; D009865:Oocytes / Q000502:physiology; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D019802:Succinic Acid / Q000378:metabolism / Q000494:pharmacology; D014982:Xenopus laevis
D002352:Carrier Proteins; D029382:Dicarboxylic Acid Transporters; D008536:Meglumine; D008094:Lithium; D012964:Sodium; D019802:Succinic Acid