Transport model of the human Na+-coupled L-ascorbic acid (vitamin C) transporter SVCT1.
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 (K(0.5) approximately 70 microM), temperature dependent (Q(10) approximately 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 (Q(max)) was approximately 25 nC, around a midpoint (V(0.5)) at -9 mV, and with apparent valence approximately -1. Q(max) was conserved upon progressive removal of Na(+), whereas V(0.5) 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.
Mackenzie
Bryan
B
Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, PO Box 670576, Cincinnati, Ohio 45267-0576, USA. bryan.mackenzie@uc.edu
Illing
Anthony C
AC
Hediger
Matthias A
MA
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D001693:Biological Transport, Active / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism*; D004574:Electromagnetic Fields; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000235:genetics; D008954:Models, Biological; D009865:Oocytes; D029363:Organic Anion Transporters, Sodium-Dependent / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011110:Polymorphism, Genetic / Q000235:genetics; D012964:Sodium / Q000378:metabolism; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014981:Xenopus
D029363:Organic Anion Transporters, Sodium-Dependent; C555162:SLC23A1 protein, human; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters; D012964:Sodium; D001205:Ascorbic Acid