Mechanistic insights and functional determinants of the transport cycle of the ascorbic acid transporter SVCT2: Activation by sodium and absolute dependence on bivalent cations

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 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.