Mechanistic Insights and Functional Determinants of the Transport Cycle of the Ascorbic Acid Transporter SVCT2
2006; Elsevier BV; Volume: 282; Issue: 1 Linguagem: Inglês
10.1074/jbc.m608300200
ISSN1083-351X
AutoresAlejandro Godoy, Valeska Ormazábal, Gustavo Moraga‐Cid, Felipe Zúñiga, Paula Sotomayor, Valeria Barra, Osmán Vásquez, Viviana P. Montecinos, Lorena Mardones, Catherine Guzmán, Marcelo Villagrán, Luis G. Aguayo, Sergio A. Oñate, Alejandro Reyes, Juan G. Cárcamo, Coralia I. Rivas, Juan Carlos Vera,
Tópico(s)Vitamin K Research Studies
ResumoWe 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 Hill coefficient (nH) for Na+ near 2 (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). SVCT2 has been difficult to express in heterologous systems at high efficiency, and the data from these studies do not show a high degree of reproducibility (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, 12Rajan D.P. Huang W. Dutta B. Devoe L.D. Leibach F.H. Ganapathy V. Prasad P.D. Biochem. Biophys. Res. Commun. 1999; 262: 762-768Crossref PubMed Scopus (150) Google Scholar, 13Daruwala R. Song J. Koh W.S. Rumsey S.C. Levine M. FEBS Lett. 1999; 460: 480-484Crossref PubMed Scopus (228) Google Scholar, 14Wang Y. Mackenzie B. Tsukaguchi H. Weremowicz S. Morton C.C. Hediger M.A. Biochem. Biophys. Res. Commun. 2000; 267: 488-494Crossref PubMed Scopus (188) Google Scholar). As a result of these limitations, there is no clear information regarding the values of basic functional parameters such as the transport Km (from 6 to 200 μm), the nH (from 1 to 2), the Na+:ascorbic acid stoichiometry, the substrate binding order, or the mechanism by which bivalent cations affect transport. We present here a detailed characterization of the ascorbic acid transporter SVCT2 of human melanoma cells and propose a basic model for the transport cycle. Our data from conventional and quantitative PCR immunocytochemistry and immunoblotting, cloning and expression in HEK-293 cells, and transport and electrochemical studies indicate that SVCT2 is a high affinity transporter of Na+ and ascorbic acid that shows positive cooperativity for Na+ and has an absolute requirement for Ca2+/Mg2+ for activity. 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+. Cell Culture—Human melanoma cells (SK-MEL-131) were grown in RPMI 1640-25 mm Hepes (pH 7.4) with 10% (v/v) fetal bovine serum and penicillin/streptomycin (100 units/ml) (15Spielholz C. Golde D.W. Houghton A.N. Nualart F. Vera J.C. Cancer Res. 1997; 57: 2529-2537PubMed Google Scholar). RT-PCR—mRNA was prepared using the Micro poly(A) Pure kit (Ambion Inc.), and reverse transcription was performed using the rapid RT-PCR kit (Clontech Inc.) (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). Approximately 10 ng of cDNA synthesis product was used as template in a reaction mix containing 40 pmol of the primer pair (13Daruwala R. Song J. Koh W.S. Rumsey S.C. Levine M. FEBS Lett. 1999; 460: 480-484Crossref PubMed Scopus (228) Google Scholar), with the program: 2 min at 94 °C; 35 cycles of 2 min each at 94 °C, 1 min at 55 °C, and 2 min at 72 °C; and 7 min at 72 °C. The PCR products were separated by electrophoresis on 2% agarose gel and visualized with ethidium bromide. Immunolocalization—The cells were fixed in 4% p-formaldehyde, washed, and blocked in phosphate-buffered (pH 7.6) saline, 1% bovine serum albumin, incubated for 18 h at room temperature in the same buffer containing anti-SVCT or goat preimmune serum (Santa Cruz Biotechnology, 1:1000), washed, and incubated with anti-goat IgG-horseradish peroxidase (DAKO, 1:100), and the reaction revealed with H2O2 and 3,3-diaminobenzidine (16Nualart F. Godoy A. Reinicke K. Brain Res. 1999; 824: 97-104Crossref PubMed Scopus (83) Google Scholar, 17Garcia M.A. Carrasco M. Godoy A. Reinicke K. Montecinos V.P. Aguayo L.G. Tapia J.C. Vera J.C. Nualart F. J. Cell. Biochem. 2001; 80: 491-503Crossref PubMed Scopus (46) Google Scholar). Uptake Assays—The uptake experiments were carried out as previously described using 3-day-old cell monolayers in 12-well tissue culture plates (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, 15Spielholz C. Golde D.W. Houghton A.N. Nualart F. Vera J.C. Cancer Res. 1997; 57: 2529-2537PubMed Google Scholar). The transport medium contained 15 mm Hepes buffer (pH 7.6), 135 mm NaCl, 5 mm KCl, 1.8 mm CaCl2, 0.8 mm MgCl2, and 0.1 mm dithiothreitol. For experiments in Na+-free buffer, NaCl was replaced with choline chloride. Monolayers were washed with transport medium, and uptake experiments were initiated by replacing the medium with prewarmed transport medium containing 0.1 μCi of l-[14C]ascorbic acid (PerkinElmer Life Sciences) and cold ascorbate at the concentrations indicated in the figure legends. Uptake was terminated by adding ice-cold stopping solution (15 mm Hepes buffer, pH 7.6, 135 mm NaCl, 5 mm KCl, 0.8 mm MgCl2, 1.8 mm CaCl2, 0.2 mm HgCl2). The monolayers were washed and lysed in 10 mm Tris/HCl (pH 8.0), 0.2% SDS, and the incorporated radioactivity was determined by scintillation spectrometry. The data are presented as the average standard deviation and correspond to a minimum of three assays performed independently in triplicate. Kinetic parameters were determined using the Michaelis-Menten equation and by using the linear transformation of Eadie-Hofstee. Stoichiometry Determination—We performed parallel time course uptake assays of 22Na and radiolabeled ascorbic acid for determination of the sodium/ascorbic acid uptake ratio. The transport medium contained a mixture of sodium transport inhibitors (1 mm ouabain, 0.1 mm amiloride, and 0.1 mm bumetanide) and 0.25 μCi of 22sodium (PerkinElmer Life Sciences) or radiolabeled ascorbic acid. Cloning of SVCT2 and Expression in HEK-293 Cells—The full-length SVCT1 and SVCT2 cDNA were obtained by RT-PCR using oligonucleotide primer pairs that flank the start and stop codons of SVCT1 and SVCT2, respectively (12Rajan D.P. Huang W. Dutta B. Devoe L.D. Leibach F.H. Ganapathy V. Prasad P.D. Biochem. Biophys. Res. Commun. 1999; 262: 762-768Crossref PubMed Scopus (150) Google Scholar). PCR amplification was done using 0.4 μl of cDNA template, 0.5 μm of each primer, and the following set of reactions: 2 min at 94 °C; 36 cycles of 30 s at 94 °C, 30 s at 65 °C, and 2 min at 72 °C; and 7 min at 72 °C. The PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide, extracted and purified from agarose gels with the QIAX kit (Qiagen), cloned in the pEGFP vector (Invitrogen), sequenced, and analyzed by BLAST at the NCBI server at www.ncbi.nlm.nih.gov/. SVCT1-GFP and SVCT2-GFP were constructed using standard molecular biology techniques, cloned in the pEGFP vector, and sequenced. Transfection experiments in HEK-293 cells were performed using Lipofectamine (Invitrogen) following the manufacturer's instructions. Patch Clamp—Patch clamp in the whole cell configuration was performed 18-24 h post-transfection using an Axopatch-1 (Axon Instrument) apparatus. Culture medium was replaced with an external solution containing 10 mm Hepes (pH 7.4), 150 mm NaCl, 5.4 mm KCl, 1.0 mm MgCl2, 2.0 mm CaCl2, and 10 mm glucose, and the cells were stabilized at room temperature for 30 min. Transfected cells were selected under fluorescence microscopy and the whole cell configuration was established with a holding potential of -60 mV. The currents were filtered at 2 kHz and digitalized (Digidata 1200, Axotape and Pcclamp 9.0; Axon Instruments) for further nonlinear analysis. Statistics—The transport data were processed for nonlinear analysis with the program IgorPro (WaveMetrics). Patch clamp data were analyzed with the program Origin (Microcal Inc.). Melanoma Cells Express a Single Ascorbic Acid Transporter That Corresponds to SVCT2—RT-PCR experiments using cDNAs prepared from RNA obtained from melanoma cells and primers specific for SVCT1 or SVCT2 generated amplification products of the size expected for SVCT2 (370 bp) but not for SVCT1 (Fig. 1A, lanes 1 and 2). The specificity of the amplification reaction was verified by using human liver RNA that was positive for SVCT1 and negative for SVCT2 and total human brain RNA that was positive for SVCT2 and negative for SVCT1 (Fig. 1A, lanes 3 and 4, and data not shown). These results are in agreement with studies of the presence of SVCT1 and SVCT2-specific transcripts in mammalian tissues and cells (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, 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, 12Rajan D.P. Huang W. Dutta B. Devoe L.D. Leibach F.H. Ganapathy V. Prasad P.D. Biochem. Biophys. Res. Commun. 1999; 262: 762-768Crossref PubMed Scopus (150) Google Scholar). Expression of SVCT2 at the protein level was confirmed by immunoblotting (Fig. 1B) and immunocytochemistry with antibodies anti-SVCT1 or -SVCT2 (Fig. 1, B-D). The immunoblotting experiments using anti-SVCT2 and total membranes prepared from SK-MEL cells showed one reactive band with an estimated molecular mass of 50,000 daltons (Fig. 1B, lane 2), which is consistent with the size of SVCT2 predicted from the coding region of the cDNA, but there was no immunoreactive band with anti-SVCT1 (Fig. 1B, lane 1). Control experiments revealed an immunoreactive band in membranes prepared from renal Madin-Darby canine kidney cells that express SVCT1 (data not shown). The immunolocalization experiments confirmed expression of SVCT2 in the melanoma cells, but were negative for SVCT1 (Fig. 1, C-E). Proper reactivity of the antibodies was confirmed by using cells and tissues known to express the different transporters. Human renal epithelial cells were positive for SVCT1 and SVCT2, hepatocytes were positive for SVCT1 and negative for SVCT2, and adrenal cells were positive for SVCT2 and negative for SVCT1 (data not shown), which is consistent with previous data on the differential expression of SVCT1 and SVCT2 in different tissues and cells (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, 18Wang H. Dutta B. Huang W. Devoe L.D. Leibach F.H. Ganapathy V. Prasad P.D. Biochim. Biophys. Acta. 1999; 1461: 1-9Crossref PubMed Scopus (123) Google Scholar). Transport studies confirmed that the melanoma cells expressed an ascorbic acid transporter with functional properties similar to those of SVCT2 (Fig. 2, A-E). Time course analysis of ascorbic acid uptake revealed that the melanoma cells take up ascorbic acid at a constant rate of 130 pmol/min/million cells for at least 10 min (data not shown). Dose-response experiments showed that transport approached saturation at 100 μm ascorbic acid (Fig. 2A), and analysis of the transport data with the Eadie-Hofstee method generated a straight line that is indicative of the presence of a single functional component, for which we calculated an apparent Km of 17 ± 2 μm (n = 8) and a Vmax of 150 ± 4 pmol/min/million cells (Fig. 2B). Expression studies of cloned SVCT1 and SVCT2 have rendered results indicating that they may show different Km values depending on the expression system used and also on the cell, tissue, or species from where the transporters were cloned. Rat SVCT1 and SVCT2 expressed in X. laevis oocytes showed transport Km values of 19-30 μm (SVCT1) and 9.4 μm (SVCT2), whereas for the human isoforms the Km values were 75-250 μm (SVCT1) and 22-69 μm (SVCT2) (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, 12Rajan D.P. Huang W. Dutta B. Devoe L.D. Leibach F.H. Ganapathy V. Prasad P.D. Biochem. Biophys. Res. Commun. 1999; 262: 762-768Crossref PubMed Scopus (150) Google Scholar, 13Daruwala R. Song J. Koh W.S. Rumsey S.C. Levine M. FEBS Lett. 1999; 460: 480-484Crossref PubMed Scopus (228) Google Scholar, 14Wang Y. Mackenzie B. Tsukaguchi H. Weremowicz S. Morton C.C. Hediger M.A. Biochem. Biophys. Res. Commun. 2000; 267: 488-494Crossref PubMed Scopus (188) Google Scholar, 18Wang H. Dutta B. Huang W. Devoe L.D. Leibach F.H. Ganapathy V. Prasad P.D. Biochim. Biophys. Acta. 1999; 1461: 1-9Crossref PubMed Scopus (123) Google Scholar, 19Castro M. Caprile T. Astuya A. Millan C. Reinicke K. Vera J.C. Vasquez O. Aguayo L.G. Nualart F. J. Neurochem. 2001; 78: 815-823Crossref PubMed Scopus (106) Google Scholar). In comparison, in human CaCo-2 cells, SVCT1 and SVCT2 have transport Km values of 113 and 15 μm, respectively (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). The reported differences may be attributable to aspects of the experimental procedures. Thus, two different transport Km values have been determined for the same transporter (cloned from the same tissue and expressed in the same system) by two different laboratories (13Daruwala R. Song J. Koh W.S. Rumsey S.C. Levine M. FEBS Lett. 1999; 460: 480-484Crossref PubMed Scopus (228) Google Scholar, 14Wang Y. Mackenzie B. Tsukaguchi H. Weremowicz S. Morton C.C. Hediger M.A. Biochem. Biophys. Res. Commun. 2000; 267: 488-494Crossref PubMed Scopus (188) Google Scholar). We have examined the kinetic properties of SVCT2 expressed in 10 different primary cells and cell lines and found that in all cases the transport Km is close to 20 μm (data not shown). In this context, the functional properties of the ascorbic acid transporter expressed in the melanoma cells are fully compatible with its molecular identification as SVCT2. Na+ Cooperativity and the Na+:Ascorbic Acid Stoichiometry—A fundamental property of the ascorbic acid transporters of the SVCT type is their potent activation by sodium ions. Uptake of ascorbic acid by the melanoma cells required the presence of sodium ions, as shown by a greater than 95% decrease in the rate of uptake (from 130 to less than 7 pmol/min/million cells) when the Na+ in the incubation buffer was replaced with choline+, Cs+, K+, or Li+ (Fig. 2E). To further analyze this issue, transport of 100 μm ascorbic acid, a concentration at which SVCT2 is near saturation, was measured in the presence of graded extracellular concentrations (0-135 mm) of Na+. A clear increase in the ascorbic acid transport rate as a function of increasing Na+ concentration was observed (Fig. 2C), with the rate increasing at least 20-fold when the concentration of extracellular Na+ went from 0 to 135 mm, a process that was characterized by a Na50 (the Na+ concentration that increased the transport rate to 50% of the maximal effect) of 35 mm and a sigmoidal relationship between uptake rate and Na+ concentration, suggesting that the effect of Na+ on the transport rate was cooperative (Fig. 2C). Confirming this interpretation, when the data were fitted to the Hill equation, the line obtained showed an nH of 1.9 (Fig. 2D), which is consistent with the presence of at least two sodium sites showing positive cooperativity. An nH of 1.9 can be interpreted as indicating that the binding of Na+ to the first Na+-binding site increases the affinity of the second Na+ site and does not provide information on the Na+: ascorbic acid stoichiometry (20Cornish-Bowden A. Cornish-Bowden A. Fundamental of Enzyme Kinetic. Butterworth & Co., Ltd., London1979: 147-176Google Scholar, 21Smith-Maxwell C. Bennett E. Randles J. Kimmich G.A. Am. J. Physiol. 1990; 258: C234-C242Crossref PubMed Google Scholar). However, previous results obtained in expression studies of cloned SVCT2, which indicated a cooperative Na+ effect with an nH near 2, have been erroneously interpreted as indicating a 2:1 Na+:ascorbic acid stoichiometry (12Rajan D.P. Huang W. Dutta B. Devoe L.D. Leibach F.H. Ganapathy V. Prasad P.D. Biochem. Biophys. Res. Commun. 1999; 262: 762-768Crossref PubMed Scopus (150) Google Scholar, 18Wang H. Dutta B. Huang W. Devoe L.D. Leibach F.H. Ganapathy V. Prasad P.D. Biochim. Biophys. Acta. 1999; 1461: 1-9Crossref PubMed Scopus (123) Google Scholar). We directly addressed this issue by measuring the transport of 22Na in the presence and in the absence of 100 μm ascorbic acid and comparing it with the transport of ascorbic acid in the presence and in the absence of 135 mm NaCl (Fig. 2, F-H). Under these conditions, the ascorbic acid-coupled net 22Na transport rate was 0.36 nmol/106 cells/min (Fig. 2F), and the Na+-coupled net ascorbic acid transport rate was 0.20 nmol/106 cells/min (Fig. 2G). Thus, at 100 μm ascorbic acid and 135 mm NaCl, the transport of one molecule of ascorbic acid was coupled to the transport of two Na+ molecules, resulting in a 2:1 sodium:ascorbic acid stoichiometry (Fig. 2H). Na+ Activates Transport by Decreasing the Ascorbic Acid Transport Km—We examined the mechanism by which Na+ increases the rate of ascorbic acid transport. At 5 and 15 mm Na+, the saturation curves were hyperbolic, and the transporter was still not saturated at 5mm ascorbic acid, indicating that SVCT2 was fully capable of transporting ascorbic acid at low Na+ concentrations, but with a transport Km in the millimolar range (Fig. 3, A and B). A detailed analysis of the transport kinetics using graded extracellular Na+ concentrations (from 5 to 35 mm) revealed that Na+ increased the transport rate by decreasing the transport Km for ascorbic acid in a complex, nonlinear, and dose-dependent manner (Fig. 3C). Thus, there was an almost linear decrease in the ascorbic acid transport Km, from 2 mm to 120 μm as Na+ increased from 5 to 20 mm, followed by a second, slower phase in which the transport Km decreased from 120 to 17 μm with a change in Na+ from 20 to 135 mm (Fig. 3C). Overall, there was more than a 100-fold decrease in the value of the Km for ascorbic acid when Na+ was increased from 5 to 135 mm. In contrast, there was no appreciable change in the transport Vmax, which remained at ∼150 pmol/106 cells/min when Na+ was increased from 5 to 135 mm (Fig. 3C). Our data showing a biphasic effect of Na+ on the Km of SVCT2 for ascorbic acid could be interpreted as indicating the occurrence of two successive conformational rearrangements in SVCT2, the first occurring at low concentrations of Na+ followed by a second at higher Na+ concentrations. Moreover, the kinetic data indicating that the ascorbic acid transport Km changes as a function of the Na+ concentration supports a substrate binding order of the type Na+-ascorbic acid-Na+. The kinetic analysis does not provide enough data to identify the specific step in the overall transport cycle that is affected by the presence of Na+. Data obtained with the Na+-glucose and the Na+-glutamate co-transporters was interpreted as indicating that the Na+ effect on the transport Km is associated with conformational transitions that expose the substrate binding site (22Hirayama B.A. Loo D.D. Wright E.M. J. Biol. Chem. 1997; 272: 2110-2115Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 23Vayro S. Lo B. Silverman M. Biochem. J. 1998; 332: 119-125Crossref PubMed Scopus (25) Google Scholar). However, recent data with the Lac permease, a transporter of lactose whose transport activity is driven by the electrochemical H+ gradient, indicated that the H+ effect is not related to changes in the transporter affinity for the substrate (24Guan L. Kaback R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12148-12152Crossref PubMed Scopus (46) Google Scholar). Thus, our present data cannot be interpreted as indicating the occurrence of Na+-driven changes in the substrate binding site(s) leading to increased affinity for ascorbic acid, as opposed to conformational changes that directly affect the substrate translocation steps (see Fig. 6A). Ascorbic Acid Affects the Na+ Cooperativity and the Na+50—Because SVCT2 is a co-transporter of Na+ and ascorbic acid, and Na+ affects the transport of ascorbic acid, we asked whether ascorbic acid affects the Na+ cooperativity. For this, we selected a wide range of ascorbic acid concentrations, from 5to500 μm, and measured the rate of transport of ascorbic acid at increasing Na+ concentrations. These experiments revealed that the Na+ cooperativity was moderate at low ascorbic acid concentrations (5 μm) and was lost at high (>200 μm) ascorbic acid (Fig. 3D). Thus, although at 5 μm ascorbic acid the ascorbic acid uptake curve was sigmoidal, with an nH of less than 1.4 (Fig. 3, D and E), the corresponding curve at 500 μm ascorbic acid was clearly hyperbolic, with an nH of 1.0 (Fig. 3, D and E). A detailed analysis revealed that the nH for Na+ varied in a complex and bimodal manner as a function of the ascorbic acid concentration; it increased from 1.4 at low ascorbic acid concentrations to a maximum value of 2 at 50-100 μm ascorbic acid and decreased to ∼1.0 at ascorbic acid concentrations >200 μm (Fig. 3F). On the other hand, the Na+50 decreased in a unimodal, nonlinear manner with increasing concentrations of ascorbic acid, from 80 mm at 5 μm ascorbic acid to 35 mm at 50 μm ascorbic acid, and reached a lowest value of 20 mm at 500 μm ascorbic acid (Fig. 3F). These results, indicating that the transport of ascorbic acid is strongly influenced by the simultaneous presence of Na+ and that the interaction of Na+ with the transporter is affected by the presence of ascorbic acid, are fully consistent with the function of SVCT2 as a Na+-ascorbic acid co-transporter. The effect of ascorbic acid on the Na+ cooperativity can be rationalized if we include in the analysis the evidence indicating that the Na+:ascorbic acid stoichiometry is 2:1 and that the substrate binding order is Na+-ascorbic acid-Na+. Thus, the data demonstrating low Na+ cooperativity at low ascorbic acid concentrations can be interpreted as evidence indicating that ascorbic acid does not participate in the binding of the first Na+ but affects the binding of the second Na+, an interpretation that is consistent with a binding order of the type Na+-ascorbic acid-Na+ and the Na+ cooperativity observed at concentrations of ascorbic acid near or above the transport Km. It is, however, difficult to explain the lack of cooperativity observed at ascorbic acid concentrations greater than 200 μm and the fact that these high concentrations of ascorbic acid still affect the Na+50 (which approaches 20 mm at 500 μm ascorbic acid) without evidence of further ascorbic acid transport (at these concentrations, SVCT2 approaches saturation caused by a transport Km of less than 20 μm). One possibility is that, at high concentrations, ascorbic acid may affect the functional properties of the first Na+-binding site, increasin
Referência(s)