Mechanistic insights and functional determinants of the transport cycle of the ascorbic acid transporter SVCT2

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 a Na+-dependent transporter. The properties of SVCT2 are modulated by Ca2+/Mg2+ 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 Ca2+/Mg2+ 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 Ca2+/Mg2+. 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 Ca2+/Mg2+-dependent transporter.