Vitamin C (l-ascorbic acid) is an essential micronutrient that serves as an antioxidant and as a cofactor in many enzymatic reactions. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial apical l-ascorbic acid cotransporter SVCT1 (SLC23A1). We explored the molecular mechanisms of SVCT1-mediated l-ascorbic acid transport using radiotracer and voltage-clamp techniques in RNA-injected Xenopus oocytes. l-Ascorbic acid transport was saturable ( K0.5 ≈ 70 μM), temperature dependent ( Q10 ≈ 5), and energized by the Na+ electrochemical potential gradient. We obtained a Na+-l-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. l-Ascorbic acid and Na+ saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered Na+, l-ascorbic acid, Na+. In the absence of l-ascorbic acid, SVCT1 mediated pre-steady-state currents that decayed with time constants 3–15 ms. Transients were described by single Boltzmann distributions. At 100 mM Na+, maximal charge translocation ( Qmax) was ≈25 nC, around a midpoint ( V0.5) at −9 mV, and with apparent valence ≈−1. Qmax was conserved upon progressive removal of Na+, whereas V0.5 shifted to more hyperpolarized potentials. Model simulation predicted that the pre-steady-state current predominantly results from an ion-well effect on binding of the first Na+ partway within the membrane electric field. We present a transport model for SVCT1 that will provide a framework for investigating the impact of specific mutations and polymorphisms in SLC23A1 and help us better understand the contribution of SVCT1 to vitamin C metabolism in health and disease.
Ascorbic acid transport in brain microvascular pericytes
