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 a Na(+)-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.