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Article ; Online: Functional characterization of the human facilitative glucose transporter 12 (GLUT12) by electrophysiological methods.

Pujol-Giménez, Jonai / Pérez, Alejandra / Reyes, Alejandro M / Loo, Donald D F / Lostao, Maria Pilar

American journal of physiology. Cell physiology

2015 Volume 308, Issue 12, Page(s) C1008–22

Abstract: GLUT12 is a member of the facilitative family of glucose transporters. The goal of this study was to characterize the functional properties of GLUT12, expressed in Xenopus laevis oocytes, using radiotracer and electrophysiological methods. Our results ... ...

Abstract	GLUT12 is a member of the facilitative family of glucose transporters. The goal of this study was to characterize the functional properties of GLUT12, expressed in Xenopus laevis oocytes, using radiotracer and electrophysiological methods. Our results showed that GLUT12 is a facilitative sugar transporter with substrate selectivity: d-glucose ≥ α-methyl-d-glucopyranoside (α-MG) > 2-deoxy-d-glucose(2-DOG) > d-fructose = d-galactose. α-MG is a characteristic substrate of the Na(+)/glucose (SGLT) family and has not been shown to be a substrate of any of the GLUTs. In the absence of sugar, (22)Na(+) was transported through GLUT12 at a higher rate (40%) than noninjected oocytes, indicating that there is a Na(+) leak through GLUT12. Genistein, an inhibitor of GLUT1, also inhibited sugar uptake by GLUT12. Glucose uptake was increased by the PKA activator 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) but not by the PKC activator phorbol-12-myristate-13-acetate (PMA). In high K(+) concentrations, glucose uptake was blocked. Addition of glucose to the external solution induced an inward current with a reversal potential of approximately -15 mV and was blocked by Cl(-) channel blockers, indicating the current was carried by Cl(-) ions. The sugar-activated Cl(-) currents were unaffected by genistein. In high external K(+) concentrations, sugar-activated Cl(-) currents were also blocked, indicating that GLUT12 activity is voltage dependent. Furthermore, glucose-induced current was increased by the PKA activator 8-Br-cAMP but not by the PKC activator PMA. These new features of GLUT12 are very different from those described for other GLUTs, indicating that GLUT12 must have a specific physiological role within glucose homeostasis, still to be discovered.
MeSH term(s)	8-Bromo Cyclic Adenosine Monophosphate/pharmacology ; Animals ; Biological Transport ; Chloride Channels/antagonists & inhibitors ; Chloride Channels/metabolism ; Chlorides/metabolism ; Cyclic AMP-Dependent Protein Kinases/metabolism ; Enzyme Activation ; Enzyme Activators/pharmacology ; Genistein/pharmacology ; Glucose/analogs & derivatives ; Glucose/metabolism ; Glucose Transport Proteins, Facilitative/antagonists & inhibitors ; Glucose Transport Proteins, Facilitative/metabolism ; Humans ; Hydrogen-Ion Concentration ; Kinetics ; Membrane Potentials ; Oocytes ; Patch-Clamp Techniques ; Sodium/metabolism ; Xenopus laevis
Chemical Substances	Chloride Channels ; Chlorides ; Enzyme Activators ; Glucose Transport Proteins, Facilitative ; SLC2A12 protein, human ; 8-Bromo Cyclic Adenosine Monophosphate (23583-48-4) ; Sodium (9NEZ333N27) ; Genistein (DH2M523P0H) ; Cyclic AMP-Dependent Protein Kinases (EC 2.7.11.11) ; Glucose (IY9XDZ35W2)
Language	English
Publishing date	2015-04-08
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	392098-7
ISSN	1522-1563 ; 0363-6143
ISSN (online)	1522-1563
ISSN	0363-6143
DOI	10.1152/ajpcell.00343.2014
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Article: Effect of nordihydroguaiaretic acid on cell viability and glucose transport in human leukemic cell lines.

Leon, David / Parada, Daniela / Vargas-Uribe, Mauricio / Perez, Alejandra A / Ojeda, Lorena / Zambrano, Angara / Reyes, Alejandro M / Salas, Mónica

FEBS open bio

2016 Volume 6, Issue 10, Page(s) 1000–1007

Abstract: The polyphenol nordihydroguaiaretic acid (NDGA) has antineoplastic properties, hence it is critical to understand its action at the molecular level. Here, we establish that NDGA inhibits glucose uptake and cell viability in leukemic HL-60 and U-937 cell ... ...

Abstract	The polyphenol nordihydroguaiaretic acid (NDGA) has antineoplastic properties, hence it is critical to understand its action at the molecular level. Here, we establish that NDGA inhibits glucose uptake and cell viability in leukemic HL-60 and U-937 cell lines. We monitored hexose uptake using radio-labeled 2-deoxyglucose (2DG) and found that the inhibition by NDGA followed a noncompetitive mechanism. In addition, NDGA blocked hexose transport in human red blood cells and displaced prebound cytochalasin B from erythrocyte ghosts, suggesting a direct interaction with the glucose transporter GLUT1. We propose a model for the mechanism of action of NDGA on glucose uptake. Our study shows for the first time that NDGA can act as inhibitor of the glucose transporter GLUT1.
Language	English
Publishing date	2016-10
Publishing country	England
Document type	Journal Article
ISSN	2211-5463
ISSN	2211-5463
DOI	10.1002/2211-5463.12106
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Article: Data on SVCT2 transporter expression and localization in cancer cell lines and tissues

Roa, Francisco J. / Peña, Eduardo / Inostroza, Eveling / Sotomayor, Kirsty / González, Mauricio / Gutierrez-Castro, Francisco A. / Maurin, Michelle / Sweet, Karen / Labrousse, Claire / Gatica, Marcell / Aylwin, Carlos F. / Mendoza, Pamela / Maldonado, Mafalda / Delgado, Carolina / Madariaga, Jaime / Panes, Jessica / Silva-Grecchi, Tiare / Concha, Ilona I. / Moraga-Cid, Gustavo /

Reyes, Alejandro M. / Muñoz-Montesino, Carola / Vera, Juan Carlos / Rivas, Coralia I.

Data in Brief. 2019 Aug., v. 25

2019

Abstract: The data presented in this article are related to the research paper entitled “Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer”, available in Free Radical Biology and ... ...

Abstract	The data presented in this article are related to the research paper entitled “Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer”, available in Free Radical Biology and Medicine Journal [1]. In this article, we examined the SVCT2 transporter expression in various breast cancer cell lines using RT-PCR and Western blot assays. In addition, we analyzed the subcellular localization of SVCT2 by immunofluorescence colocalization assays and cellular fractionation experiments. Finally, an analysis of different cancer tissue microarrays immunostained for SVCT2 and imaged by The Human Protein Atlas (https://www.proteinatlas.org) is presented.
Keywords	Western blotting ; ascorbic acid ; breast neoplasms ; fluorescent antibody technique ; fractionation ; mitochondria ; neoplasm cells
Language	English
Dates of publication	2019-08
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	2786545-9
ISSN	2352-3409
ISSN	2352-3409
DOI	10.1016/j.dib.2019.103972
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Article ; Online: Data on SVCT2 transporter expression and localization in cancer cell lines and tissues.

Roa, Francisco J / Peña, Eduardo / Inostroza, Eveling / Sotomayor, Kirsty / González, Mauricio / Gutierrez-Castro, Francisco A / Maurin, Michelle / Sweet, Karen / Labrousse, Claire / Gatica, Marcell / Aylwin, Carlos F / Mendoza, Pamela / Maldonado, Mafalda / Delgado, Carolina / Madariaga, Jaime / Panes, Jessica / Silva-Grecchi, Tiare / Concha, Ilona I / Moraga-Cid, Gustavo /

Reyes, Alejandro M / Muñoz-Montesino, Carola / Vera, Juan Carlos / Rivas, Coralia I

Data in brief

2019 Volume 25, Page(s) 103972

Abstract: The data presented in this article are related to the research paper entitled "Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer", available in Free Radical Biology and ... ...

Abstract	The data presented in this article are related to the research paper entitled "Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer", available in Free Radical Biology and Medicine Journal [1]. In this article, we examined the SVCT2 transporter expression in various breast cancer cell lines using RT-PCR and Western blot assays. In addition, we analyzed the subcellular localization of SVCT2 by immunofluorescence colocalization assays and cellular fractionation experiments. Finally, an analysis of different cancer tissue microarrays immunostained for SVCT2 and imaged by The Human Protein Atlas (https://www.proteinatlas.org) is presented.
Language	English
Publishing date	2019-05-06
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2786545-9
ISSN	2352-3409 ; 2352-3409
ISSN (online)	2352-3409
ISSN	2352-3409
DOI	10.1016/j.dib.2019.103972
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Article ; Online: Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer.

Peña, Eduardo / Roa, Francisco J / Inostroza, Eveling / Sotomayor, Kirsty / González, Mauricio / Gutierrez-Castro, Francisco A / Maurin, Michelle / Sweet, Karen / Labrousse, Claire / Gatica, Marcell / Aylwin, Carlos F / Mendoza, Pamela / Maldonado, Mafalda / Delgado, Carolina / Madariaga, Jaime / Panes, Jessica / Silva-Grecchi, Tiare / Concha, Ilona I / Moraga-Cid, Gustavo /

Reyes, Alejandro M / Muñoz-Montesino, Carola / Vera, Juan Carlos / Rivas, Coralia I

Free radical biology & medicine

2019 Volume 135, Page(s) 283–292

Abstract: The potential role of vitamin C in cancer prevention and treatment remains controversial. While normal human cells obtain vitamin C as ascorbic acid, the prevalent form of vitamin C in vivo, the uptake mechanisms by which cancer cells acquire vitamin C ... ...

Abstract	The potential role of vitamin C in cancer prevention and treatment remains controversial. While normal human cells obtain vitamin C as ascorbic acid, the prevalent form of vitamin C in vivo, the uptake mechanisms by which cancer cells acquire vitamin C has remained unclear. The aim of this study is to characterize how breast cancer cells acquire vitamin C. For this, we determined the expression of vitamin C transporters in normal and breast cancer tissue samples, and in ZR-75, MCF-7, MDA-231 and MDA-468 breast cancer cell lines. At the same time, reduced (AA) and oxidized (DHA) forms of vitamin C uptake experiments were performed in all cell lines. We show here that human breast cancer tissues differentially express a form of SVCT2 transporter, that is systematically absent in normal breast tissues and it is increased in breast tumors. In fact, estrogen receptor negative breast cancer tissue, exhibit the most elevated SVCT2 expression levels. Despite this, our analysis in breast cancer cell lines showed that these cells are not able to uptake ascorbic acid and depend on glucose transporter for the acquisition of vitamin C by a bystander effect. This is consistent with our observations that this form of SVCT2 is completely absent from the plasma membrane and is overexpressed in mitochondria of breast cancer cells, where it mediates ascorbic acid transport. This work shows that breast cancer cells acquire vitamin C in its oxidized form and are capable of accumulated high concentrations of the reduced form. Augmented expression of an SVCT2 mitochondrial form appears to be a common hallmark across all human cancers and might have implications in cancer cells survival capacity against pro-oxidant environments.
MeSH term(s)	Ascorbic Acid/metabolism ; Breast Neoplasms/genetics ; Breast Neoplasms/pathology ; Bystander Effect ; Female ; Gene Expression Regulation, Neoplastic/genetics ; Humans ; MCF-7 Cells ; Mitochondria/genetics ; Mitochondria/pathology ; Mitochondrial Membrane Transport Proteins/genetics ; Oxidation-Reduction ; Reactive Oxygen Species/metabolism ; Sodium/metabolism ; Sodium-Coupled Vitamin C Transporters/genetics
Chemical Substances	Mitochondrial Membrane Transport Proteins ; Reactive Oxygen Species ; SLC23A2 protein, human ; Sodium-Coupled Vitamin C Transporters ; Sodium (9NEZ333N27) ; Ascorbic Acid (PQ6CK8PD0R)
Language	English
Publishing date	2019-03-19
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	807032-5
ISSN	1873-4596 ; 0891-5849
ISSN (online)	1873-4596
ISSN	0891-5849
DOI	10.1016/j.freeradbiomed.2019.03.015
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Article ; Online: Noncompetitive blocking of human GLUT1 hexose transporter by methylxanthines reveals an exofacial regulatory binding site.

Ojeda, Paola / Pérez, Alejandra / Ojeda, Lorena / Vargas-Uribe, Mauricio / Rivas, Coralia I / Salas, Monica / Vera, Juan Carlos / Reyes, Alejandro M

American journal of physiology. Cell physiology

2012 Volume 303, Issue 5, Page(s) C530–9

Abstract: Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; ... ...

Abstract	Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes. The displacement of previously bound cytochalasin B revealed a direct interaction between the methylxanthines and GLUT1. Methylxanthines behave as noncompetitive blockers (inhibition constant values of 2-3 mM) in exchange and zero-trans efflux assays, whereas mixed inhibition with a notable uncompetitive component is observed in zero-trans influx assays (inhibition constant values of 5-12 mM). These results indicate that methylxanthines do not bind to either exofacial or endofacial d-glucose-binding sites but instead interact at a different site accessible by the external face of the transporter. Additionally, infinite-cis exit assays (Sen-Widdas assays) showed that only pentoxifylline disturbed d-glucose for binding to the exofacial substrate site. Interestingly, coinhibition assays showed that methylxanthines bind to a common site on the transporter. We concluded that there is a methylxanthine regulatory site on the external surface of the transporter, which is close but distinguishable from the d-glucose external site. Therefore, the methylxanthine moiety may become an attractive framework for the design of novel specific noncompetitive facilitative GLUT inhibitors.
MeSH term(s)	Binding Sites ; Biological Transport ; Cell Membrane ; Cytochalasin B/metabolism ; Deoxyglucose/metabolism ; Erythrocytes/metabolism ; Glucose/metabolism ; Glucose Transporter Type 1/antagonists & inhibitors ; Glucose Transporter Type 1/metabolism ; Humans ; Protein Conformation ; Xanthines/classification ; Xanthines/pharmacology
Chemical Substances	Glucose Transporter Type 1 ; SLC2A1 protein, human ; Xanthines ; Cytochalasin B (3CHI920QS7) ; Deoxyglucose (9G2MP84A8W) ; Glucose (IY9XDZ35W2)
Language	English
Publishing date	2012-06-06
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	392098-7
ISSN	1522-1563 ; 0363-6143
ISSN (online)	1522-1563
ISSN	0363-6143
DOI	10.1152/ajpcell.00145.2012
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Article ; Online: Hexose transporter GLUT1 harbors several distinct regulatory binding sites for flavones and tyrphostins.

Pérez, Alejandra / Ojeda, Paola / Ojeda, Lorena / Salas, Mónica / Rivas, Coralia I / Vera, Juan C / Reyes, Alejandro M

Biochemistry

2011 Volume 50, Issue 41, Page(s) 8834–8845

Abstract: The facilitative hexose transporter GLUT1 activity is blocked by tyrosine kinase inhibitors that include natural products such as flavones and isoflavones and synthetic compounds such as tyrphostins, molecules that are structurally unrelated to the ... ...

Abstract	The facilitative hexose transporter GLUT1 activity is blocked by tyrosine kinase inhibitors that include natural products such as flavones and isoflavones and synthetic compounds such as tyrphostins, molecules that are structurally unrelated to the transported substrates [Vera, et al. (2001) Biochemistry, 40, 777-790]. Here we analyzed the interaction of GLUT1 with quercetin (a flavone), genistein (an isoflavone), and tyrphostin A47 and B46 to evaluate if they share one common or have several binding sites on the protein. Kinetic assays showed that genistein, quercetin, and tyrphostin B46 behave as competitive inhibitors of equilibrium exchange and zero-trans uptake transport and noncompetitive inhibitors of net sugar exit out of human red cells, suggesting that they interact with the external surface of the GLUT1 molecule. In contrast, tyrphostin A47 was a competitive inhibitor of equilibrium exchange and zero-trans exit transport and a noncompetitive inhibitor of net sugar entry into red cells, suggesting that it interacts with the cytoplasmic surface of the transporter. Genistein protected GLUT1 against iodide-elicited fluorescence quenching and also decreased the affinity of d-glucose for its external binding site, while quercetin and tyrphostins B46 and A47 promoted fluorescence quenching and did not affect the external d-glucose binding site. These findings are explained by a carrier that presents at least three binding sites for tyrosine kinase inhibitors, in which (i) genistein interacts with the transporter in a conformation that binds glucose on the external surface (outward-facing conformation), in a site which overlaps with the external binding site for d-glucose, (ii) quercetin and tyrphostin B46 interact with the GLUT1 conformation which binds glucose by the internal side of the membrane (inward-facing conformation), but to a site accessible from the external surface of the protein, and (iii) the binding site for tyrphostin A47 is accessible from the inner surface of GLUT1 by binding to the inward-facing conformation of the transporter. These data provide groundwork for a molecular understanding of how the tyrosine kinase inhibitors directly affect glucose transport in animal cells.
MeSH term(s)	Allosteric Site ; Binding Sites ; Binding, Competitive ; Erythrocytes/metabolism ; Flavones/chemistry ; Genistein/pharmacology ; Glucose/chemistry ; Glucose Transporter Type 1/chemistry ; Humans ; Kinetics ; Monosaccharide Transport Proteins/metabolism ; Protein Conformation ; Proteolipids/chemistry ; Spectrometry, Fluorescence/methods ; Tyrphostins/chemistry
Chemical Substances	Flavones ; Glucose Transporter Type 1 ; Monosaccharide Transport Proteins ; Proteolipids ; SLC2A1 protein, human ; Tyrphostins ; proteoliposomes ; Genistein (DH2M523P0H) ; Glucose (IY9XDZ35W2)
Language	English
Publishing date	2011-10-18
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/bi200748b
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Article ; Online: Mitochondrial ascorbic acid transport is mediated by a low-affinity form of the sodium-coupled ascorbic acid transporter-2.

Muñoz-Montesino, Carola / Roa, Francisco J / Peña, Eduardo / González, Mauricio / Sotomayor, Kirsty / Inostroza, Eveling / Muñoz, Carolina A / González, Iván / Maldonado, Mafalda / Soliz, Carlos / Reyes, Alejandro M / Vera, Juan Carlos / Rivas, Coralia I

Free radical biology & medicine

2014 Volume 70, Page(s) 241–254

Abstract: Despite the fundamental importance of the redox metabolism of mitochondria under normal and pathological conditions, our knowledge regarding the transport of vitamin C across mitochondrial membranes remains far from complete. We report here that human ... ...

Abstract	Despite the fundamental importance of the redox metabolism of mitochondria under normal and pathological conditions, our knowledge regarding the transport of vitamin C across mitochondrial membranes remains far from complete. We report here that human HEK-293 cells express a mitochondrial low-affinity ascorbic acid transporter that molecularly corresponds to SVCT2, a member of the sodium-coupled ascorbic acid transporter family 2. The transporter SVCT1 is absent from HEK-293 cells. Confocal colocalization experiments with anti-SVCT2 and anti-organelle protein markers revealed that most of the SVCT2 immunoreactivity was associated with mitochondria, with minor colocalization at the endoplasmic reticulum and very low immunoreactivity at the plasma membrane. Immunoblotting of proteins extracted from highly purified mitochondrial fractions confirmed that SVCT2 protein was associated with mitochondria, and transport analysis revealed a sigmoidal ascorbic acid concentration curve with an apparent ascorbic acid transport Km of 0.6mM. Use of SVCT2 siRNA for silencing SVCT2 expression produced a major decrease in mitochondrial SVCT2 immunoreactivity, and immunoblotting revealed decreased SVCT2 protein expression by approximately 75%. Most importantly, the decreased protein expression was accompanied by a concomitant decrease in the mitochondrial ascorbic acid transport rate. Further studies using HEK-293 cells overexpressing SVCT2 at the plasma membrane revealed that the altered kinetic properties of mitochondrial SVCT2 are due to the ionic intracellular microenvironment (low in sodium and high in potassium), with potassium acting as a concentration-dependent inhibitor of SVCT2. We discarded the participation of two glucose transporters previously described as mitochondrial dehydroascorbic acid transporters; GLUT1 is absent from mitochondria and GLUT10 is not expressed in HEK-293 cells. Overall, our data indicate that intracellular SVCT2 is localized in mitochondria, is sensitive to an intracellular microenvironment low in sodium and high in potassium, and functions as a low-affinity ascorbic acid transporter. We propose that the mitochondrial localization of SVCT2 is a property shared across cells, tissues, and species.
MeSH term(s)	Ascorbic Acid/metabolism ; Biological Transport/genetics ; Free Radicals/metabolism ; Gene Expression Regulation ; HEK293 Cells ; Humans ; Mitochondria/metabolism ; Oxidation-Reduction ; RNA, Small Interfering ; Sodium-Coupled Vitamin C Transporters/genetics ; Sodium-Coupled Vitamin C Transporters/metabolism
Chemical Substances	Free Radicals ; RNA, Small Interfering ; SLC23A1 protein, human ; Sodium-Coupled Vitamin C Transporters ; Ascorbic Acid (PQ6CK8PD0R)
Language	English
Publishing date	2014-05
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	807032-5
ISSN	1873-4596 ; 0891-5849
ISSN (online)	1873-4596
ISSN	0891-5849
DOI	10.1016/j.freeradbiomed.2014.02.021
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Article: Histidine Residues in the Na⁺-coupled Ascorbic Acid Transporter-2 (SVCT2) Are Central Regulators of SVCT2 Function, Modulating pH Sensitivity, Transporter Kinetics, Na⁺ Cooperativity, Conformational Stability, and Subcellular Localization

Ormazabal, Valeska / Zuñiga, Felipe A / Escobar, Elizabeth / Aylwin, Carlos / Salas-Burgos, Alexis / Godoy, Alejandro / Reyes, Alejandro M / Vera, Juan Carlos / Rivas, Coralia I

Journal of biological chemistry. 2010 Nov. 19, v. 285, no. 47

2010

Abstract: Na⁺-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of ... ...

Abstract	Na⁺-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His¹⁰⁹, His²⁰³, His²⁰⁶, His²⁶⁹, and His⁴¹³, are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na⁺ cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His⁴¹³, localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na⁺ and loss of Na⁺ cooperativity, which leads to a decreased Vmax without altering the transport Km; (ii) exofacial histidine residues His²⁰³, His²⁰⁶, and His⁴¹³ may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport Km; and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function.
Language	English
Dates of publication	2010-1119
Size	p. 36471-36485.
Publishing place	American Society for Biochemistry and Molecular Biology
Document type	Article
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
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Article ; Online: Cis-regulatory elements involved in species-specific transcriptional regulation of the SVCT1 gene in rat and human hepatoma cells.

Muñoz, Alejandra / Villagrán, Marcelo / Guzmán, Paula / Solíz, Carlos / Gatica, Marcell / Aylwin, Carlos / Sweet, Karen / Maldonado, Mafalda / Escobar, Elizabeth / Reyes, Alejandro M / Toledo, Jorge R / Sánchez, Oliberto / Oñate, Sergio A / Carlos Vera, Juan / Rivas, Coralia I

Free radical biology & medicine

2015 Volume 85, Page(s) 183–196

Abstract: Ascorbic acid is transported into cells by the sodium-coupled vitamin C transporters (SVCTs). Recently, we obtained evidence of differential regulation of SVCT expression in response to acute oxidative stress in cells from species that differ in their ... ...

Abstract	Ascorbic acid is transported into cells by the sodium-coupled vitamin C transporters (SVCTs). Recently, we obtained evidence of differential regulation of SVCT expression in response to acute oxidative stress in cells from species that differ in their capacity to synthesize vitamin C, with a marked decrease in SVCT1 mRNA and protein levels in rat hepatoma cells that was not observed in human hepatoma cells. To better understand the regulatory aspects involved, we performed a structural and functional analysis of the proximal promoter of the SVCT1 rat gene. We cloned a 1476-bp segment containing the proximal promoter of the rat SVCT1 gene and generated deletion-derived truncated promoters of decreasing sizes and mutant promoters by modification of consensus binding sites for transcription factors by site-directed mutagenesis. We next analyzed their capacity to direct the transcription of a reporter gene after transfection into rat H4IIE and human HepG2 hepatoma cells, in experiments involving the coexpression of transcription factors whose consensus binding sequences are present in the SVCT1 promoter. This analysis revealed the presence of two critical cis-regulatory elements of the transcriptional activity of the rat SVCT1 gene promoter, sites containing consensus sequences for the binding of the transcription factors Bach1 and HNF4 that are not present in equivalent locations in the human SVCT1 gene promoter. Moreover, a consensus site for HNF1 that is crucial for the regulation of the human SVCT1 promoter is present in the SVCT1 rat promoter but has no effect on its transcriptional activity. These findings imply that regulation of vitamin C metabolism in the rat, a species with the capacity to synthesize large amounts of ascorbic acid, may differ from that of humans, a species that must obtain ascorbic acid from the diet through a transport mechanism that depends on proper SVCT1 expression.
MeSH term(s)	Animals ; Cell Line, Tumor ; Humans ; Promoter Regions, Genetic ; Rats ; Regulatory Sequences, Nucleic Acid ; Sodium-Coupled Vitamin C Transporters/genetics ; Species Specificity
Chemical Substances	SLC23A1 protein, human ; Slc23a1 protein, rat ; Sodium-Coupled Vitamin C Transporters
Language	English
Publishing date	2015-08
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	807032-5
ISSN	1873-4596 ; 0891-5849
ISSN (online)	1873-4596
ISSN	0891-5849
DOI	10.1016/j.freeradbiomed.2015.04.024
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