Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Abstract

Mackenzie B, Illing AC, Hediger MA. Transport model of the human Na ϩ -coupled L-ascorbic acid (vitamin C) transporter SVCT1. Am J Physiol Cell Physiol 294: C451-C459, 2008. First published December 19, 2007 doi:10.1152/ajpcell.00439.2007.-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 Ϸ 70 M), temperature dependent (Q10 Ϸ 5), and energized by the Na ϩ electrochemical potential gradient. We obtained a Na ϩ -L-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. L-Ascorbic acid and Na ϩ saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered Na ϩ , L-ascorbic acid, Na ϩ . 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 ϩ , maximal charge translocation (Qmax) was Ϸ25 nC, around a midpoint (V0.5) at Ϫ9 mV, and with apparent valence ϷϪ1. Qmax was conserved upon progressive removal of Na ϩ , 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 ϩ 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. cotransporters; sodium dependent; intestinal absorption; model simulation; renal reabsorption; Xenopus oocyte VITAMIN C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant scavenger of free radicals and as a cofactor in many enzymatic reactions (3,6,10,26,29). It cannot be synthesized in Homo sapiens and must be derived from the diet. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial Na ϩ -dependent Lascorbic acid cotransporter SVCT1 (reviewed in Refs. 32 and 35), the product of the SLC23A1 gene. Selective sorting of SVCT1 protein to the apical membrane has been demonstrated in cultured cell lines (Caco2 and MDCK) of intestinal and renal origin (2, 23).We and others previously demonstrated that human SVCT1 is a Na ϩ -dependent transporter that favors L-ascorbic acid over D-isoascorbic acid and that the oxidized form of vitamin C, dehydroascorbic acid, is excluded (5, 34). Here we explored its molecular mechanisms by testing the hypothesis that the Na ϩ dependence and rheogenicity of SVCT1 arise from...

Cardioprotection against ischaemia/reperfusion by vitamins C and E plus n−3 fatty acids: molecular mechanisms and potential clinical applications

“…It should be noted that most human cells have vitamin C transporters coupled with sodium transport at a ratio of 2:1 (sodium/ascorbate), with a K 0.5 of 70 μmol/l. Approximately 90 % of vitamin C is cytosolic [96]. When vitamin C is administered orally, the relationship between the dose and plasma concentration is sigmoidal.…”

Prevention of Postoperative Atrial Fibrillation: Novel and Safe Strategy Based on the Modulation of the Antioxidant System

“…In addition, recent in vitro studies have also been successful to study the effects in vitro of oxidative stress with and without this vitamin C concentration, thus validating ascorbate to counteract the effects of oxidative stress (Virdis et al, 2009). It should be noted that most human cells have vitamin C transporters, coupled with sodium transport at a ratio Na:ascorbate = 2:1, having a K 0.5 by 70 μmol/L, being distributed about 90% in the cytosol (Mackenzie et al, 2008). When vitamin C is given by mouth, the relationship between oral dose and plasma concentration is sigmoidal.…”

Effects of Na+ and H+ on steady-state and presteady-state currents of the human concentrative nucleoside transporter 3 (hCNT3)

“…There was no reduction in Q max with decreasing extracellular Na + or H + concentrations, similarly to rGAT1, pSGLT3, rNIS, and hSVCT1 [4,7,34,40]. Therefore, the charge movements in hCNT3 are primarily due to the conformational changes of the empty transporter between the inward-and outward-facing conformations.…”

“…In plants, prokaryotes and lower eukaryotes, cotransporters commonly couple the transport of substrates to the electrochemical gradient of protons, as the H + /hexose cotransporter SPT1 from Arabidopsis thaliana [1], the lactose permease Lac Y from Escherichia coli [2] or the proton/myo-inositol cotransporter from Leishmania donovani [3]. In contrast, in most animal cells, the evolution has led to utilization of the transmembrane Na + gradient as the ion-motive force, as reported for the rat sodium-iodide symporter NIS [4], the mouse γ-aminobutyric acid transporter GAT3 [5], the mouse NaPi-IIb transporter [6] or the human ascorbic acid transporter SVCT1 [7]. However, in endomembranes and at the plasma membrane of specialized cells the H + -coupled cotransport is still present, e.g., the human oligopeptide transporters PEPT1 and PEPT2 [8][9][10] or the human aminoacid symporter PAT1 [11].…”