Mechanistic Insights and Functional Determinants of the Transport Cycle of the Ascorbic Acid Transporter SVCT2

Abstract

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 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 Km without affecting the Vmax, 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 Vmax without affecting the transport Km 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. 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 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 Km without affecting the Vmax, 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 Vmax without affecting the transport Km 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. Vitamin C exists in two chemically distinct forms: the reduced ionizable form (ascorbic acid) and the oxidized nonionic form (dehydroascorbic acid) (1Rose R.C. Biochim. Biophys. Acta. 1988; 947: 335-366Crossref PubMed Scopus (173) Google Scholar). Human cells acquire vitamin C using two different transporter systems that differ in structural as well as functional terms. One transporter system is a bidirectional, low affinity, high capacity system that includes several members of the facilitative glucose transporter (GLUT) 4The abbreviations used are: GLUT, facilitative glucose transporter; RT, reverse transcription; GFP, green fluorescent protein. family, shows absolute specificity for oxidized vitamin C, and transports dehydroascorbic acid down a substrate concentration gradient. Fourteen glucose transporter isoforms (GLUT1-GLUT14) have been molecularly characterized (2Joost H.G. Thorens B. Mol. Membr. Biol. 2001; 18: 247-256Crossref PubMed Scopus (573) Google Scholar, 3Joost H.G. Bell G.I. Best J.D. Birnbaum M.J. Charron M.J. Chen Y.T. Doege H. James D.E. Lodish H.F. Moley K.H. Moley J.F. Mueckler M. Rogers S. Schurmann A. Seino S. Thorens B. Am. J. Physiol. 2002; 282: E974-E976Crossref PubMed Scopus (336) Google Scholar), and there is evidence obtained from expression studies in Xenopus laevis oocytes indicating that GLUT1, GLUT3, and GLUT4 (4Rumsey S.C. Kwon O. Xu G.W. Burant C.F. Simpson I. Levine M. J. Biol. Chem. 1997; 272: 18982-18989Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 5Rumsey S.C. Daruwala R. Al-Hasani H. Zarnowski M.J. Simpson I.A. Levine M. J. Biol. Chem. 2000; 275: 28246-28253Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 6Vera J.C. Rivas C.I. Fischbarg J. Golde D.W. Nature. 1993; 364: 79-82Crossref PubMed Scopus (450) Google Scholar, 7Vera J.C. Rivas C.I. Zhang R.H. Farber C.M. Golde D.W. Blood. 1994; 84: 1628-1634Crossref PubMed Google Scholar, 8Vera J.C. Rivas C.I. Velasquez F.V. Zhang R.H. Concha II Golde D.W. J. Biol. Chem. 1995; 270: 23706-23712Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 9Maulen N.P. Henriquez E.A. Kempe S. Carcamo J.G. Schmid-Kotsas A. Bachem M. Grunert A. Bustamante M.E. Nualart F. Vera J.C. J. Biol. Chem. 2003; 278: 9035-9041Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) are dehydroascorbic acid transporters. The evidence regarding GLUT2 is still controversial, and there are no data regarding the capacity of GLUT8, GLUT10, and GLUT12-GLUT14 to transport dehydroascorbic acid. GLUT5 is a fructose transporter unable to transport dehydroascorbic acid, which is probably also the situation with GLUT7, GLUT9, and GLUT11, although these last transporters have been less studied from a functional perspective. A second vitamin C transport system is a high affinity, low capacity system, the sodium-ascorbic acid co-transporters (SVCTs), composed of two members that show absolute specificity for ascorbic acid and transport the substrate down the electrochemical sodium gradient (10Liang W.J. Johnson D. Jarvis S.M. Mol. Membr. Biol. 2001; 18: 87-95Crossref PubMed Scopus (214) Google Scholar, 11Tsukaguchi H. Tokui T. Mackenzie B. Berger U.V. Chen X.Z. Wang Y. Brubaker R.F. Hediger M.A. Nature. 1999; 399: 70-75Crossref PubMed Scopus (731) Google Scholar). SVCT1 and SVCT2 have been expressed in heterologous systems, but only SVCT1 has been functionally characterized in detail. SVCT1 has an apparent transport Km in the range of 50-200 μm, is electrogenic, and shows a for Na+ H. Tokui T. Mackenzie B. Berger U.V. Chen X.Z. Wang Y. Brubaker R.F. Hediger M.A. Nature. 1999; 399: 70-75Crossref PubMed Scopus (731) Google Scholar). SVCT2 has been to in heterologous systems at high and the data from these studies not show a high of H. Tokui T. Mackenzie B. Berger U.V. Chen X.Z. Wang Y. Brubaker R.F. Hediger M.A. Nature. 1999; 399: 70-75Crossref PubMed Scopus (731) Google Scholar, B. Biophys. 1999; PubMed Scopus Google Scholar, R. J. S.C. Levine M. 1999; PubMed Scopus Google Scholar, Y. Mackenzie B. H. S. Hediger M.A. Biophys. 2000; PubMed Scopus Google Scholar). a of these there is no regarding the of basic functional as the transport Km to the to the Na+:ascorbic acid the substrate binding or the by which cations We a of the ascorbic acid transporter SVCT2 of human cells and a basic model for the transport Our data from and and and expression in and transport and electrochemical studies indicate that SVCT2 is a high affinity transporter of Na+ and ascorbic acid that shows cooperativity for Na+ and has an absolute requirement for Ca2+/Mg2+ for The transport cycle is characterized by a 2:1 Na+:ascorbic acid transport stoichiometry and a substrate binding order of the type Na+:ascorbic acid:Na+. cells in mm with and Golde D.W. Nualart F. Vera J.C. 1997; Google Scholar). using the and reverse using the N.P. Henriquez E.A. Kempe S. Carcamo J.G. Schmid-Kotsas A. Bachem M. Grunert A. Bustamante M.E. Nualart F. Vera J.C. J. Biol. Chem. 2003; 278: 9035-9041Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). of used as in a of the R. J. S.C. Levine M. 1999; PubMed Scopus Google Scholar), with the at of at at and at and at The by and with cells in and in for at in the or and with and the with and F. A. 1999; PubMed Scopus Google Scholar, M.A. M. A. J.C. Vera J.C. Nualart F. J. 2001; PubMed Scopus Google Scholar). as using in J.C. Rivas C.I. Velasquez F.V. Zhang R.H. Concha II Golde D.W. J. Biol. Chem. 1995; 270: 23706-23712Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, Golde D.W. Nualart F. Vera J.C. 1997; Google Scholar). The transport mm mm mm mm mm and mm in with with transport and by the with transport of acid and at the concentrations in the by mm mm mm mm mm mm The and in mm and the by The data are as the and to a of three in using the and by using the of of and ascorbic acid for of the acid The transport a of sodium transport mm mm and mm and of or ascorbic acid. of SVCT2 and in SVCT1 and SVCT2 obtained by using that the and of SVCT1 and B. Biophys. 1999; PubMed Scopus Google Scholar). using of of and the of at of at at and at and at The by with and from with the in the and by at the at and using in the and in cells using the in the using an with an mm mm mm mm mm and mm and the cells at for cells and the with a of The at and and for transport data for with the data with the a to using from obtained from cells and for SVCT1 or SVCT2 of the for SVCT2 but not for SVCT1 and The specificity of the by using human that for SVCT1 and for SVCT2 and human that for SVCT2 and for SVCT1 and and data not are in with studies of the of SVCT1 and in and cells N.P. Henriquez E.A. Kempe S. Carcamo J.G. Schmid-Kotsas A. Bachem M. Grunert A. Bustamante M.E. Nualart F. Vera J.C. J. Biol. Chem. 2003; 278: 9035-9041Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 11Tsukaguchi H. Tokui T. Mackenzie B. Berger U.V. Chen X.Z. Wang Y. Brubaker R.F. Hediger M.A. Nature. 1999; 399: 70-75Crossref PubMed Scopus (731) Google Scholar, B. Biophys. 1999; PubMed Scopus Google Scholar). of SVCT2 at the by and with or The using and from cells showed with an of which is consistent with the of SVCT2 from the of the but there no with an in from cells that SVCT1 not The expression of SVCT2 in the but for SVCT1 of the by using cells and to the different transporters. Human cells for SVCT1 and for SVCT1 and for and cells for SVCT2 and for SVCT1 not which is consistent with data the expression of SVCT1 and SVCT2 in different and cells H. Tokui T. Mackenzie B. Berger U.V. Chen X.Z. Wang Y. Brubaker R.F. Hediger M.A. Nature. 1999; 399: 70-75Crossref PubMed Scopus (731) Google Scholar, H. B. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar). studies that the cells expressed an ascorbic acid transporter with functional properties to of SVCT2 of ascorbic acid that the cells ascorbic acid at a rate of cells for at least not showed that transport at ascorbic acid and of the transport data with the a that is of the of a functional for which an apparent Km of and a Vmax of cells studies of SVCT1 and SVCT2 have indicating that may show different Km the expression system used and also the or from the transporters SVCT1 and SVCT2 expressed in laevis oocytes showed transport Km of and for the human isoforms the Km and H. Tokui T. Mackenzie B. Berger U.V. Chen X.Z. Wang Y. Brubaker R.F. Hediger M.A. Nature. 1999; 399: 70-75Crossref PubMed Scopus (731) Google Scholar, B. Biophys. 1999; PubMed Scopus Google Scholar, R. J. S.C. Levine M. 1999; PubMed Scopus Google Scholar, Y. Mackenzie B. H. S. Hediger M.A. Biophys. 2000; PubMed Scopus Google Scholar, H. B. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar, M. T. A. Vera J.C. O. Nualart F. J. 2001; PubMed Scopus Google Scholar). in human SVCT1 and SVCT2 have transport Km of and μm, N.P. Henriquez E.A. Kempe S. Carcamo J.G. Schmid-Kotsas A. Bachem M. Grunert A. Bustamante M.E. Nualart F. Vera J.C. J. Biol. Chem. 2003; 278: 9035-9041Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The may be to of the Thus, two different transport Km have been for the transporter from the and expressed in the by two different R. J. S.C. Levine M. 1999; PubMed Scopus Google Scholar, Y. Mackenzie B. H. S. Hediger M.A. Biophys. 2000; PubMed Scopus Google Scholar). We have the properties of SVCT2 expressed in different cells and and that in the transport Km is to 20 not the functional properties of the ascorbic acid transporter expressed in the cells are with as Na+ and the of the ascorbic acid transporters of the type is by sodium of ascorbic acid by the cells the of sodium as by a in the rate of to less the Na+ in the with or transport of ascorbic acid, a concentration at which SVCT2 is in the of concentrations of A in the ascorbic acid transport rate as a of increasing Na+ concentration with the rate increasing at least the concentration of Na+ from to a that characterized by a Na+ concentration that increased the transport rate to of the of mm and a between rate and Na+ that the of Na+ the transport rate cooperative the data to the the obtained showed an of which is consistent with the of at least two sodium cooperativity. of can be as indicating that the binding of Na+ to the the affinity of the second Na+ and not the ascorbic acid stoichiometry A. A. of Scholar, J. Am. J. Physiol. PubMed Google Scholar). However, obtained in expression studies of which a cooperative Na+ with an have been as indicating a 2:1 Na+:ascorbic acid stoichiometry B. Biophys. 1999; PubMed Scopus Google Scholar, H. B. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar). We by the transport of in the and in the absence of ascorbic acid and it with the transport of ascorbic acid in the and in the absence of mm these the ascorbic transport rate and the ascorbic acid transport rate Thus, at ascorbic acid and mm the transport of of ascorbic acid to the transport of two Na+ in a 2:1 acid stoichiometry Na+ by the the by which Na+ the rate of ascorbic acid At and mm the and the transporter still not at ascorbic acid, indicating that SVCT2 of ascorbic acid at low Na+ but with a transport Km in the range A and A of the transport using Na+ concentrations to that Na+ increased the transport rate by decreasing the transport Km for ascorbic acid in a and manner Thus, there an in the ascorbic acid transport from mm to as Na+ increased from to 20 by a in which the transport Km from to with a in Na+ from 20 to mm there a in the of the Km for ascorbic acid Na+ increased from to there no in the transport Vmax, which at Na+ increased from to mm Our data a of Na+ the Km of SVCT2 for ascorbic acid be as indicating the of two in the at low concentrations of Na+ by a second at Na+ concentrations. the data indicating that the ascorbic acid transport Km as a of the Na+ concentration a substrate binding order of the type Na+-ascorbic The not data to the in the transport cycle that is affected by the of obtained with the and the co-transporters as indicating that the Na+ the transport Km is with that the substrate binding J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. M. J. PubMed Scopus Google Scholar). However, data with the a transporter of whose transport is by the electrochemical that the is not to in the transporter affinity for the substrate R. S. A. PubMed Scopus Google Scholar). Thus, data be as indicating the of in the substrate binding to increased affinity for ascorbic acid, as to that the substrate the Na+ and the SVCT2 is a of Na+ and ascorbic acid, and Na+ the transport of ascorbic acid, ascorbic acid the Na+ cooperativity. a range of ascorbic acid from μm, and the rate of transport of ascorbic acid at increasing Na+ concentrations. that the Na+ cooperativity at low ascorbic acid concentrations and at high ascorbic acid Thus, although at ascorbic acid the ascorbic acid with an of less and the at ascorbic acid with an of and A that the for Na+ in a and bimodal manner as a of the ascorbic acid it increased from at low ascorbic acid concentrations to a of at ascorbic acid and to at ascorbic acid concentrations the the in a manner with increasing concentrations of ascorbic acid, from mm at ascorbic acid to mm at ascorbic acid, and a of 20 mm at ascorbic acid indicating that the transport of ascorbic acid is by the of Na+ and that the interaction of Na+ with the transporter is affected by the of ascorbic acid, are consistent with the of SVCT2 as a Na+-ascorbic acid The of ascorbic acid the Na+ cooperativity can be in the the evidence indicating that the Na+:ascorbic acid stoichiometry is 2:1 and that the substrate binding order is Na+-ascorbic Thus, the data low Na+ cooperativity at low ascorbic acid concentrations can be as evidence indicating that ascorbic acid not in the binding of the Na+ but the binding of the second an that is consistent with a binding order of the type Na+-ascorbic and the Na+ cooperativity at concentrations of ascorbic acid or the transport to the of cooperativity at ascorbic acid concentrations and the that these high concentrations of ascorbic acid still the 20 mm at ascorbic acid) without evidence of ascorbic acid transport these SVCT2 by a transport Km of less 20 One is at high ascorbic acid may the functional properties of the increasing affinity to a to the affinity of the second with the that the Na+ cooperative is high ascorbic acid concentrations may the affinity of the second decreasing it to a to the affinity of the also in a of the Na+ cooperativity. The data between these two and for SVCT2 the of and ascorbic acid in the of the transporter functional in the absence of or but there no transport in the absence of both cations the absence of there no transport of the absence or the of or showed that the rate of ascorbic acid transport increased as a of increasing concentrations of and at mm with the of a functional with an apparent Km of and a Vmax of and to the of the rate of ascorbic acid transport increased as a of increasing concentrations and at mm with the of a functional with an apparent Km of and a Vmax of and using a in the Vmax of transport without in the transport Km using concentrations from that the transport rate at low concentrations is to a in the transport Vmax without in the transport Km the Na+ cooperativity or the affected at low concentrations and the transport rate at low concentrations also to a transport Vmax, without in the transport the Na+ cooperativity, or the not The of and transport can be as indicating that SVCT2 binding for and and that the binding of the cations a in the transporter from a to a active Thus, Ca2+/Mg2+ are for the of which can be as a transporter. the the binding of Na+ a in the transporter from a low to a high affinity which that SVCT2 is by but it is not a Na+-dependent transporter. SVCT2 of the for a 2:1 Na+:ascorbic acid transport stoichiometry and SVCT2 is electrogenic. SVCT2 is expressed at low in the of cells of the in cells or of the in laevis the of with the transport of ascorbic acid H. Tokui T. Mackenzie B. Berger U.V. Chen X.Z. Wang Y. Brubaker R.F. Hediger M.A. Nature. 1999; 399: 70-75Crossref PubMed Scopus (731) Google Scholar, B. Biophys. 1999; PubMed Scopus Google Scholar, R. J. S.C. Levine M. 1999; PubMed Scopus Google Scholar, Y. Mackenzie B. H. S. Hediger M.A. Biophys. 2000; PubMed Scopus Google Scholar). We in several to with the transport of ascorbic acid in the an probably by the low of SVCT2 expression in these cells unable to expression in laevis oocytes with in SVCT2 not are of in cells low of the or the which is in with in the cells J. Am. J. Physiol. PubMed Google Scholar). a to and to evidence that SVCT2 is the ascorbic acid transporter expressed in the a SVCT2 from cells by using for the and of human SVCT2 The to and the data by at the at The that the from the cells with the human SVCT2 that the ascorbic acid transporter expressed by the cells is SVCT2 not We the functional properties of SVCT2 in cells with the of the cells by expression of at the of the cells showed an of without of the of ascorbic acid a in the rate of transport of ascorbic acid in the with cells showed in transport at ascorbic acid and of the transport data with the a from which an apparent Km of The Vmax of transport in the cells with a of cells in cells studies that transport of ascorbic acid by cells to the of Na+ and Ca2+/Mg2+. We a in transport in the absence of Na+ in the and no transport in the of in the absence of and not At ascorbic acid, the of sodium transport as by an of and a of to the for SVCT2 in the the Na+ cooperativity at high ascorbic acid concentrations not The data that expressed at high as the in the properties of SVCT2 from including an ascorbic acid transport Km μm, by Na+ in a cooperative manner, of Na+ cooperativity at high ascorbic acid and absolute Ca2+/Mg2+ for the high of expression in the the properties of SVCT2 by Na+ with the transport of ascorbic acid in the cells and fluorescent and to with concentrations of ascorbic acid from 20 to showed no evidence of ascorbic Na+ and are consistent with indicating no ascorbic acid in laevis oocytes SVCT2 H. Tokui T. Mackenzie B. Berger U.V. Chen X.Z. Wang Y. Brubaker R.F. Hediger M.A. Nature. 1999; 399: 70-75Crossref PubMed Scopus (731) Google Scholar). We the that is not to a current of low with these to these a number of ascorbic Na+ in cells with the expression studies in laevis oocytes have that SVCT1 is H. Tokui T. Mackenzie B. Berger U.V. Chen X.Z. Wang Y. Brubaker R.F. Hediger M.A. Nature. 1999; 399: 70-75Crossref PubMed Scopus (731) Google Scholar), these as for both the of the and a of in the of the cells by expression of at the of the which is to the in cells of ascorbic acid by cells a in the ascorbic acid transport rate with transport that by Na+ and with an apparent Km of and a Vmax that to that in cells not fluorescent and to with concentrations of ascorbic acid from 20 showed a ascorbic current and Thus, SVCT1 expressed at high as the in cells the functional properties for including the obtained in cells the indicating that SVCT2 is not electrogenic. can the apparent that a transporter that transports Na+ and ascorbic acid with a 2:1 stoichiometry is not SVCT2 is a of the of transporters that the from the of Na+ in to an electrochemical Na+ gradient to the of ascorbic acid. is the transport of the transporters J. I. S. 2003; PubMed Scopus Google Scholar, M. and Scholar) and the of Na+-ascorbic acid can be by a of at least binding of the Na+ to the conformation of binding of ascorbic acid, binding of the second a that in the of and to the We in a manner only the of the transport in the of ascorbic acid and and of the transport At least three can be at to the of the are low to the and be there is of a in the transporters to the or SVCT2 functions as an that the of a with the of two Na+ and ascorbic acid. A of the absence of of SVCT2 the of the transport cycle from to and from to SVCT2 can three different of the by two that as a of the of and an inactive form that the capacity of ascorbic acid and only in the absence of an low affinity form with cations that transports ascorbic acid with low affinity in the of Na+ concentrations below 20 and a high affinity form with that with Na+ in a cooperative manner, is of both Na+ and ascorbic acid, and transports ascorbic acid with a Km of Our data indicate in SVCT2 is at the of the as well as We have a for SVCT2 in a number of cells and in the and also in in of human not is no current data indicating SVCT2 is of ascorbic acid and be in the of ascorbic acid PubMed Scopus Google Scholar, Mol. Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). The concentration of Na+ is a concentration at which SVCT2 is active but shows a transport Km for ascorbic acid the although is at the concentration of can from low the to concentrations the type and the functional of the cells Am. J. Physiol. 1993; PubMed Google Scholar). We SVCT2 may in two functional states by The and and to an mm as high affinity transporters of ascorbic acid the the and to a low in as low affinity ascorbic acid transporters whose may be modulated by in the concentrations. At the these properties a transport cycle that the binding and of ascorbic acid at the and of the with the that ascorbic acid is into the down the electrochemical Na+ gradient. developed a model for the transport cycle of SVCT2 that challenges the current view indicating that SVCT2 is a Na+-dependent transporter Our data that Na+ the rate of ascorbic acid transport in a cooperative manner, decreasing the transport Km without affecting the transport Thus, the of Na+ is to a low affinity form of SVCT2 into a high affinity transporter. ascorbic acid affected in a bimodal and concentration-dependent manner the Na+ cooperativity, with an absence of cooperativity at high ascorbic acid indicating a reciprocal functional interaction between the two ascorbic acid and interaction defines the substrate binding order and the transport stoichiometry. data are consistent with a cycle characterized by a 2:1 Na+:ascorbic acid stoichiometry 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 SVCT2 from an inactive into an active conformation by increasing the transport Vmax without affecting the transport the Na+ cooperativity, or the We that SVCT2 is a Ca2+/Mg2+-dependent of Na+ and ascorbic acid. active states can be envisioned, including a low affinity conformation at Na+ concentrations below 20 mm and a high affinity conformation at elevated Na+ concentrations whose Na+ cooperativity is modulated by ascorbic acid. We that the functional properties of SVCT2 from cells are to SVCT2 from cells and We used cells to studies and functional data that SVCT2 and show no evidence of SVCT1 has been that SVCT1 is expressed in SVCT2 is expressed in a number of cells and (10Liang W.J. Johnson D. Jarvis S.M. Mol. Membr. Biol. 2001; 18: 87-95Crossref PubMed Scopus (214) Google Scholar, 11Tsukaguchi H. Tokui T. Mackenzie B. Berger U.V. Chen X.Z. Wang Y. Brubaker R.F. Hediger M.A. Nature. 1999; 399: 70-75Crossref PubMed Scopus (731) Google Scholar). However, SVCT1 and SVCT2 expression in a number of and that both transporters are expressed in including cells and cells human cells cells and human and human cells and from and and of cells from human and human the only expressed SVCT1 in and cells showed expression of SVCT2 have two cells that SVCT2 in and show no evidence of SVCT1 the and the human and in both SVCT2 shows sodium and absolute Ca2+/Mg2+ for not We and expressed in SVCT2 from cells human and human cells and that show by sodium and Ca2+/Mg2+ not We that the of SVCT2 function, indicating by sodium and is not and to SVCT2 expressed different