Abstract

We characterized the human Na +-ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that it functions as aNa +-dependent transporter. The properties of SVCT2 are modulated by Ca 2+/Mg 2+ and a reciprocal functional interaction between Na + and ascorbic acid that defines the substrate binding order and the transport stoichiometry. Na + increased the ascorbic acid transport rate in a cooperative manner, decreasing the transport K m without affecting the V max, thus converting a low affinity form of the transporter into a high affinity transporter. Inversely, ascorbic acid affected in a bimodal and concentration-dependent manner the Na + cooperativity, with absence of cooperativity at low and high ascorbic acid concentrations. Our data are consistent with a transport cycle characterized by a Na +:ascorbic acid stoichiometry of 2:1 and a substrate binding order of the type Na +:ascorbic acid:Na +. However, SVCT2 is not electrogenic. SVCT2 showed an absolute requirement for Ca 2+/Mg 2+ for function, with both cations switching the transporter from an inactive into an active conformation by increasing the transport V max without affecting the transport K m or the Na + cooperativity. Our data indicate that SVCT2 may switch between a number of states with characteristic properties, including an inactive conformation in the absence of Ca 2+/Mg 2+. At least three active states can be envisioned, including a low affinity conformation at Na + concentrations below 20 mM and two high affinity conformations at elevated Na + concentrations whose Na + cooperativity is modulated by ascorbic acid. Thus, SVCT2 is a Ca 2+/Mg 2+-dependent transporter. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.