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

• DOI: 10.1074/jbc.m608300200
• Publication Date: 2006-09-30T00:23:18Z
• Authors: Alejandro Godoy; Valeska Ormazabal; Gustavo Moraga-Cid; Felipe A. Zúñiga; Paula Sotomayor; Valeria Barra; Osmán Vasquez; Viviana Montecinos; Lorena Mardones; Catherine Guzmán; Marcelo Villagrán; Luis G. Aguayo; Sergio A. Oñate; Alejandro M. Reyes; Juan G. Cárcamo; Coralia I. Rivas; Juan Carlos Vera
• Keywords: 0303 health sciences; Dose-Response Relationship, Drug; Sequence Homology, Amino Acid; Symporters; Molecular Sequence Data; Sodium; Organic Anion Transporters, Sodium-Dependent; Ascorbic Acid; Models, Biological; Kinetics; 03 medical and health sciences; Cations; Cell Line, Tumor; Humans; Calcium; Magnesium; Amino Acid Sequence; Melanoma; Sodium-Coupled Vitamin C Transporters

We characterized the human Na+-ascorbic acid transporter SVCT2 and developed a basic model for transport cycle that challenges current view it functions as Na+-dependent transporter. The properties of are modulated by Ca2+/Mg2+ reciprocal functional interaction between Na+ ascorbic defines substrate binding order stoichiometry. increased rate in cooperative manner, decreasing Km without affecting Vmax, thus converting low affinity form into high Inversely, affected bimodal concentration-dependent manner cooperativity, with absence cooperativity at concentrations. Our data consistent Na+:ascorbic stoichiometry 2:1 type acid:Na+. However, is not electrogenic. showed an absolute requirement function, both cations switching from inactive active conformation increasing Vmax or cooperativity. indicate may switch number states characteristic properties, including Ca2+/Mg2+. At least three can be envisioned, concentrations below 20 mm two conformations elevated whose acid. Thus, Ca2+/Mg2+-dependent Vitamin C exists chemically distinct forms: reduced ionizable (ascorbic acid) oxidized nonionic (dehydroascorbic (1Rose R.C. Biochim. Biophys. Acta. 1988; 947: 335-366Crossref PubMed Scopus (173) Google Scholar). Human cells acquire vitamin using different systems differ structural well terms. One system bidirectional, affinity, capacity includes several members facilitative glucose (GLUT) 4The abbreviations used are: GLUT, transporter; RT, reverse transcription; GFP, green fluorescent protein. family, shows specificity C, transports dehydroascorbic down concentration gradient. Fourteen isoforms (GLUT1-GLUT14) have been molecularly (2Joost H.G. Thorens B. Mol. Membr. Biol. 2001; 18: 247-256Crossref (573) Scholar, 3Joost Bell G.I. Best J.D. Birnbaum M.J. Charron Chen Y.T. Doege H. James D.E. Lodish H.F. Moley K.H. J.F. Mueckler M. Rogers S. Schurmann A. Seino Am. J. Physiol. 2002; 282: E974-E976Crossref (336) Scholar), there evidence obtained expression studies Xenopus laevis oocytes indicating GLUT1, GLUT3, GLUT4 (4Rumsey S.C. Kwon O. Xu G.W. Burant C.F. Simpson I. Levine Chem. 1997; 272: 18982-18989Abstract Full Text PDF (404) 5Rumsey Daruwala R. Al-Hasani Zarnowski I.A. 2000; 275: 28246-28253Abstract (162) 6Vera J.C. Rivas C.I. Fischbarg Golde D.W. Nature. 1993; 364: 79-82Crossref (450) 7Vera Zhang R.H. Farber C.M. Blood. 1994; 84: 1628-1634Crossref 8Vera Velasquez F.V. Concha II 1995; 270: 23706-23712Abstract (157) 9Maulen N.P. Henriquez E.A. Kempe Carcamo J.G. Schmid-Kotsas Bachem Grunert Bustamante M.E. Nualart F. Vera 2003; 278: 9035-9041Abstract (104) Scholar) transporters. regarding GLUT2 still controversial, no GLUT8, GLUT10, GLUT12-GLUT14 to GLUT5 fructose unable acid, which probably also situation GLUT7, GLUT9, GLUT11, although these last transporters less studied perspective. A second system, sodium-ascorbic co-transporters (SVCTs), composed show electrochemical sodium gradient (10Liang W.J. Johnson D. Jarvis S.M. 87-95Crossref (214) 11Tsukaguchi Tokui T. Mackenzie Berger U.V. X.Z. Wang Y. Brubaker R.F. Hediger M.A. 1999; 399: 70-75Crossref (731) SVCT1 expressed heterologous systems, but only has functionally detail. apparent range 50-200 μm, electrogenic, Hill coefficient (nH) near 2 (11Tsukaguchi difficult express efficiency, do degree reproducibility 12Rajan D.P. Huang W. Dutta Devoe L.D. Leibach F.H. Ganapathy V. Prasad P.D. Biochem. Res. Commun. 262: 762-768Crossref (150) 13Daruwala Song Koh W.S. Rumsey FEBS Lett. 460: 480-484Crossref (228) 14Wang Tsukaguchi Weremowicz Morton C.C. 267: 488-494Crossref (188) As result limitations, clear information values parameters such (from 6 200 μm), nH 1 2), stoichiometry, order, mechanism bivalent affect transport. present here detailed characterization melanoma propose cycle. conventional quantitative PCR immunocytochemistry immunoblotting, cloning HEK-293 cells, positive activity. Cell Culture—Human (SK-MEL-131) were grown RPMI 1640-25 Hepes (pH 7.4) 10% (v/v) fetal bovine serum penicillin/streptomycin (100 units/ml) (15Spielholz C. Houghton A.N. Cancer 57: 2529-2537PubMed RT-PCR—mRNA was prepared Micro poly(A) Pure kit (Ambion Inc.), transcription performed rapid RT-PCR (Clontech Inc.) (9Maulen Approximately 10 ng cDNA synthesis product template reaction mix containing 40 pmol primer pair (13Daruwala program: min 94 °C; 35 cycles each °C, 55 72 7 °C. products separated electrophoresis on 2% agarose gel visualized ethidium bromide. Immunolocalization—The fixed 4% p-formaldehyde, washed, blocked phosphate-buffered 7.6) saline, 1% albumin, incubated 18 h room temperature same buffer anti-SVCT goat preimmune (Santa Cruz Biotechnology, 1:1000), anti-goat IgG-horseradish peroxidase (DAKO, 1:100), revealed H2O2 3,3-diaminobenzidine (16Nualart Godoy Reinicke K. Brain 824: 97-104Crossref (83) 17Garcia Carrasco Montecinos V.P. Aguayo L.G. Tapia Cell. 80: 491-503Crossref (46) Uptake Assays—The uptake experiments carried out previously described 3-day-old cell monolayers 12-well tissue culture plates (8Vera 15Spielholz medium contained 15 7.6), 135 NaCl, 5 KCl, 1.8 CaCl2, 0.8 MgCl2, 0.1 dithiothreitol. For Na+-free buffer, NaCl replaced choline chloride. Monolayers washed medium, initiated replacing prewarmed μCi l-[14C]ascorbic (PerkinElmer Life Sciences) cold ascorbate indicated figure legends. terminated adding ice-cold stopping solution (15 pH 7.6, 0.2 HgCl2). lysed Tris/HCl 8.0), 0.2% SDS, incorporated radioactivity determined scintillation spectrometry. presented average standard deviation correspond minimum assays independently triplicate. Kinetic Michaelis-Menten equation linear transformation Eadie-Hofstee. Stoichiometry Determination—We parallel time course 22Na radiolabeled determination sodium/ascorbic ratio. mixture inhibitors (1 ouabain, amiloride, bumetanide) 0.25 22sodium Cloning Expression Cells—The full-length oligonucleotide pairs flank start stop codons SVCT2, respectively (12Rajan amplification done 0.4 μl template, 0.5 μm primer, following set reactions: 36 30 s 65 electrophoresis, stained bromide, extracted purified gels QIAX (Qiagen), cloned pEGFP vector (Invitrogen), sequenced, analyzed BLAST NCBI server www.ncbi.nlm.nih.gov/. SVCT1-GFP SVCT2-GFP constructed molecular biology techniques, vector, sequenced. Transfection Lipofectamine (Invitrogen) manufacturer's instructions. Patch Clamp—Patch clamp whole configuration 18-24 post-transfection Axopatch-1 (Axon Instrument) apparatus. Culture external 7.4), 150 5.4 1.0 2.0 glucose, stabilized min. Transfected selected under fluorescence microscopy established holding potential -60 mV. currents filtered kHz digitalized (Digidata 1200, Axotape Pcclamp 9.0; Axon Instruments) further nonlinear analysis. Statistics—The processed analysis program IgorPro (WaveMetrics). Origin (Microcal Inc.). Melanoma Cells Express Single Ascorbic Acid Transporter That Corresponds SVCT2—RT-PCR cDNAs RNA primers specific generated size expected (370 bp) (Fig. 1A, lanes 2). verified liver negative total brain 3 4, shown). These results agreement presence SVCT2-specific transcripts mammalian tissues protein level confirmed immunoblotting 1B) antibodies anti-SVCT1 -SVCT2 1, B-D). anti-SVCT2 membranes SK-MEL one reactive band estimated mass 50,000 daltons 1B, lane predicted coding region cDNA, immunoreactive 1). Control renal Madin-Darby canine kidney (data immunolocalization C-E). Proper reactivity known epithelial hepatocytes adrenal shown), previous differential 18Wang 1461: 1-9Crossref (123) Transport similar those 2, A-E). Time take up constant 130 pmol/min/million Dose-response approached saturation 100 2A), Eadie-Hofstee method straight line indicative single component, we calculated 17 ± (n = 8) 4 2B). rendered they depending cell, tissue, species where cloned. Rat X. 19-30 (SVCT1) 9.4 (SVCT2), whereas 75-250 22-69 (SVCT2) 19Castro Caprile Astuya Millan Vasquez Neurochem. 78: 815-823Crossref (106) In comparison, CaCo-2 113 reported differences attributable aspects experimental procedures. (cloned system) laboratories examined kinetic primary lines found all cases close this context, fully compatible its identification SVCT2. Cooperativity Na+:Ascorbic Stoichiometry—A fundamental property SVCT their potent activation ions. required ions, shown greater than 95% decrease cells) when incubation choline+, Cs+, K+, Li+ 2E). To analyze issue, saturation, measured graded extracellular (0-135 mm) Na+. increase function observed 2C), 20-fold went 0 mm, process Na50 (the 50% maximal effect) sigmoidal relationship concentration, suggesting effect 2C). Confirming interpretation, fitted equation, 1.9 2D), sites showing An interpreted first Na+-binding site increases does provide Na+: (20Cornish-Bowden Cornish-Bowden Fundamental Enzyme Kinetic. Butterworth & Co., Ltd., London1979: 147-176Google 21Smith-Maxwell Bennett E. Randles Kimmich G.A. 1990; 258: C234-C242Crossref erroneously directly addressed issue measuring comparing F-H). Under conditions, acid-coupled net 0.36 nmol/106 cells/min 2F), Na+-coupled 0.20 2G). molecule coupled molecules, resulting sodium:ascorbic 2H). Activates Decreasing Km—We Na+, curves hyperbolic, saturated 5mm capable transporting concentrations, millimolar 3, B). kinetics complex, nonlinear, dose-dependent 3C). almost Km, 120 followed second, slower phase decreased change Overall, more 100-fold value mm. contrast, appreciable remained ∼150 pmol/106 biphasic could occurrence successive conformational rearrangements occurring higher Moreover, changes supports acid-Na+. enough identify step overall Data Na+-glucose Na+-glutamate associated transitions expose (22Hirayama B.A. Loo D.D. Wright E.M. 2110-2115Abstract (71) 23Vayro Lo Silverman 1998; 332: 119-125Crossref (25) recent Lac permease, lactose activity driven H+ gradient, related (24Guan L. Kaback Proc. Natl. Acad. Sci. U. 2004; 101: 12148-12152Crossref our cannot Na+-driven site(s) leading opposed translocation steps (see Fig. 6A). Affects Na+50—Because co-transporter affects asked whether this, wide 5to500 moderate (5 μm) lost (>200 3D). curve sigmoidal, 1.4 D E), corresponding 500 clearly E). varied complex concentration; maximum 50-100 ∼1.0 >200 3F). On other hand, Na+50 unimodal, 80 50 reached lowest results, strongly influenced simultaneous co-transporter. rationalized if include demonstrating participate interpretation acid-Na+ above Km. It is, however, explain lack fact (which approaches (at caused μm). possibility that, site, increasin