RESUMEN
We have cloned a new subtype of the amino acid transport system N2 (SN2 or second subtype of system N) from rat brain. Rat SN2 consists of 471 amino acids and belongs to the recently identified glutamine transporter gene family that consists of system N and system A. Rat SN2 exhibits 63% identity with rat SN1. It also shows considerable sequence identity (50-56%) with the members of the amino acid transporter A subfamily. In the rat, SN2 mRNA is most abundant in the liver but is detectable in the brain, lung, stomach, kidney, testis, and spleen. When expressed in Xenopus laevis oocytes and in mammalian cells, rat SN2 mediates Na(+)-dependent transport of several neutral amino acids, including glycine, asparagine, alanine, serine, glutamine, and histidine. The transport process is electrogenic, Li(+) tolerant, and pH sensitive. The transport mechanism involves the influx of Na(+) and amino acids coupled to the efflux of H(+), resulting in intracellular alkalization. Proline, alpha-(methylamino)isobutyric acid, and anionic and cationic amino acids are not recognized by rat SN2.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros , Aminoácidos/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Membrana , Simportadores , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos/genética , Aminoácidos/farmacología , Animales , Química Encefálica , Proteínas Portadoras/química , Proteínas Portadoras/genética , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Transportador de Péptidos 1 , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/metabolismo , Isoformas de Proteínas , Ratas , Alineación de Secuencia , Cloruro de Sodio/farmacología , Especificidad por Sustrato , Distribución Tisular , Xenopus laevisRESUMEN
Caveolae are flask-shaped membrane invaginations present in most mammalian cells. They are distinguished by the presence of a striated coat composed of the protein, caveolin. Caveolae have been implicated in numerous cellular processes, including potocytosis in which caveolae are hypothesized to co-localize with folate receptor alpha and participate in folate uptake. Our laboratory has recently localized folate receptor alpha to the basolateral surface of the retinal pigment epithelium (RPE). It is present also in many other cells of the retina. In the present study, we asked whether caveolae were present in the RPE, and if so, whether their pattern of distribution was similar to folate receptor alpha. We also examined the distribution pattern of caveolin-1, which can be a marker of caveolae. Extensive electron microscopical analysis revealed caveolae associated with endothelial cells. However, none were detected in intact or cultured RPE. Laser scanning confocal microscopical analysis of intact RPE localized caveolin-1 to the apical and basal surfaces, a distribution unlike folate receptor alpha. Western analysis confirmed the presence of caveolin-1 in cultured RPE cells and laser scanning confocal microscopy localized the protein to the basal plasma membrane of the RPE, a distribution like that of folate receptor alpha. This distribution was confirmed by electron microscopic immunolocalization. The lack of caveolae in the RPE suggests that these structures may not be essential for folate internalization in the RPE.
Asunto(s)
Proteínas Portadoras/metabolismo , Caveolas/ultraestructura , Caveolinas/metabolismo , Ácido Fólico/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Epitelio Pigmentado Ocular/ultraestructura , Receptores de Superficie Celular , Retina/metabolismo , Retina/ultraestructura , Animales , Western Blotting , Caveolina 1 , Línea Celular , Técnicas de Cultivo , Receptores de Folato Anclados a GPI , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos ICR , Microscopía Confocal , Microscopía ElectrónicaRESUMEN
PURPOSE: The purpose of this investigation was to provide evidence for the expression of the cystine/glutamate transporter (x(c)(-)) in the human retinal pigment epithelial cell line ARPE-19, clone the light chain of the transporter from an ARPE-19 cell cDNA library and study its function, and investigate the regulation of this transporter by nitric oxide (NO) in ARPE-19 cells. METHODS: Uptake of radiolabeled cystine and glutamate was measured in ARPE-19 cells. The functional identity of x(c)(-) in these cells was established by substrate specificity and Na(+)-independence of the uptake process. The human x(c)(-) light chain (human xCT) was cloned from an ARPE-19 cell cDNA library. The functional identity of the cloned human xCT was investigated by heterologous coexpression of the light chain with the heavy chain (human 4F2hc) in HeLa cells. ARPE-19 cells were treated with or without the NO donor 3-nitroso-N:-acetylpenicillamine (SNAP) and the expression of x(c)(-) was studied at the functional and molecular levels. RESULTS: ARPE-19 cells take up cystine as well as glutamate in the absence of Na(+). Substrate specificity studies indicate that although the uptake of cystine in the absence of Na(+) is mediated by multiple amino acid transport systems including x(c)(-), the uptake of glutamate in the absence of Na(+) occurs exclusively via x(c)(-). The human xCT cloned from ARPE-19 cells is a protein of 501 amino acids. These cells express the heavy chain 4F2hc as evidenced from RT-PCR analysis. Coexpression of human xCT with 4F2hc in HeLa cells leads to the induction of cystine and glutamate uptake with characteristics similar to that of x(c)(-). The activity of x(c)(-) in ARPE-19 cells is upregulated by SNAP, and the process is associated with an increase in the expression of xCT with no detectable change in the expression of 4F2hc. CONCLUSIONS: ARPE-19 cells express the cystine/glutamate transporter x(c)(-) (the light chain xCT and the heavy chain 4F2hc) as is evident from functional and molecular studies. NO upregulates this transport system and the process is associated with an increase in xCT mRNA but with no change in 4F2hc mRNA.
Asunto(s)
Sistema de Transporte de Aminoácidos y+ , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Cistina/metabolismo , Proteínas del Ojo/química , Proteínas del Ojo/fisiología , Ácido Glutámico/metabolismo , Penicilamina/análogos & derivados , Epitelio Pigmentado Ocular/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Clonación Molecular , Cartilla de ADN/química , Proteínas del Ojo/genética , Expresión Génica , Biblioteca de Genes , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Datos de Secuencia Molecular , Óxido Nítrico/metabolismo , Penicilamina/farmacología , Epitelio Pigmentado Ocular/citología , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
N-Acetylaspartate is a highly specific marker for neurons and is present at high concentrations in the central nervous system. It is not present at detectable levels anywhere else in the body other than brain. Glial cells express a high-affinity transporter for N-acetylaspartate, but the molecular identity of the transporter has not been established. The transport of N-acetylaspartate into glial cells is obligatory for its intracellular hydrolysis, a process intimately involved in myelination. N-Acetylaspartate is a dicarboxylate structurally related to succinate. We investigated in the present study the ability of NaDC3, a Na(+)-coupled high-affinity dicarboxylate transporter, to transport N-acetylaspartate. The cloned rat and human NaDC3s were found to transport N-acetylaspartate in a Na(+)-coupled manner in two different heterologous expression systems. The Michaelis-Menten constant for N-acetylaspartate was approximately 60 microM for rat NaDC3 and approximately 250 microM for human NaDC3. The transport process was electrogenic and the Na(+):N-acetylaspartate stoichiometry was 3:1. The functional expression of NaDC3 in the brain was demonstrated by in situ hybridization and reverse transcription-polymerase chain reaction as well as by isolation of a full-length functional NaDC3 from a rat brain cDNA library. In addition, the expression of a Na(+)-coupled high-affinity dicarboxylate transporter and the interaction of the transporter with N-acetylaspartate were demonstrable in rat primary astrocyte cultures. These studies establish NaDC3 as the transporter responsible for the Na(+)-coupled transport of N-acetylaspartate in the brain. This transporter is likely to be an essential component in the metabolic role of N-acetylaspartate in the process of myelination.
Asunto(s)
Ácido Aspártico/análogos & derivados , Encéfalo/metabolismo , Proteínas Portadoras/fisiología , Sodio/metabolismo , Animales , Ácido Aspártico/farmacocinética , Astrocitos/metabolismo , Transporte Biológico , Proteínas Portadoras/genética , Línea Celular , Cloruros/metabolismo , Transportadores de Ácidos Dicarboxílicos , Ácidos Dicarboxílicos/farmacocinética , Humanos , Masculino , Pirrolidinas/farmacocinética , RatasRESUMEN
We examined in this study the expression of the potential-sensitive organic cation transporter OCT3 in the kidney. A functionally active OCT3 was cloned from a mouse kidney cDNA library. The cloned transporter was found to be capable of mediating potential-dependent transport of a variety of organic cations including tetraethylammonium. This function was confirmed in two different heterologous expression systems involving mammalian cells and Xenopus laevis oocytes. We have also isolated the mouse OCT3 gene and deduced its structure and organization. The OCT3 gene consists of 11 exons and 10 introns. In situ hybridization studies in the mouse kidney have shown that OCT3 mRNA is expressed primarily in the cortex. The expression is evident in the proximal and distal convoluted tubules. The expression of OCT3 in human kidney was confirmed by RT-PCR. We have also cloned OCT3 from human placenta and human kidney. Human OCT3 exhibits 86% identity with mouse OCT3 in amino acid sequence. Human OCT3 was found to transport tetraethylammonium and a variety of other organic cations. The transport process was electrogenic. We conclude that OCT3 is expressed in mammalian kidney and that it plays an important role in the renal clearance of cationic drugs.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Riñón/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , 1-Metil-4-fenilpiridinio/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Unión Competitiva/fisiología , Radioisótopos de Carbono , Cationes/metabolismo , Clonación Molecular , ADN Complementario , Proteínas de Unión al ADN/análisis , Dopaminérgicos/farmacología , Exones , Regulación de la Expresión Génica/fisiología , Células HeLa , Herbicidas/farmacología , Humanos , Hibridación in Situ , Intrones , Ratones , Niacinamida/análogos & derivados , Niacinamida/farmacología , Factor 3 de Transcripción de Unión a Octámeros , Oocitos/fisiología , Epitelio Pigmentado Ocular/citología , ARN Mensajero/análisis , Tetraetilamonio/farmacocinética , Factores de Transcripción/análisis , Tritio , Xenopus laevisRESUMEN
We have cloned and functionally characterized the human Na(+)-dependent high-affinity dicarboxylate transporter (hNaDC3) from placenta. The hNaDC3 cDNA codes for a protein of 602 amino acids with 12 transmembrane domains. When expressed in mammalian cells, the cloned transporter mediates the transport of succinate in the presence of Na(+) [concentration of substrate necessary for half-maximal transport (K(t)) for succinate = 20+/-1 microM]. Dimethylsuccinate also interacts with hNaDC3. The Na(+)-to-succinate stoichiometry is 3:1 and concentration of Na(+) necessary for half-maximal transport (K(Na(+))(0.5)) is 49+/-1 mM as determined by uptake studies with radiolabeled succinate. When expressed in Xenopus laevis oocytes, hNaDC3 induces Na(+)-dependent inward currents in the presence of succinate and dimethylsuccinate. At a membrane potential of -50 mV, K(Suc)(0.5) is 102+/-20 microM and K(Na(+))(0.5) is 22+/-4 mM as determined by the electrophysiological approach. Simultaneous measurements of succinate-evoked charge transfer and radiolabeled succinate uptake in hNaDC3-expressing oocytes indicate a charge-to-succinate ratio of 1:1 for the transport process, suggesting a Na(+)-to-succinate stoichiometry of 3:1. pH titration of citrate-induced currents shows that hNaDC3 accepts preferentially the divalent anionic form of citrate as a substrate. Li(+) inhibits succinate-induced currents in the presence of Na(+). Functional analysis of rat-human and human-rat NaDC3 chimeric transporters indicates that the catalytic domain of the transporter lies in the carboxy-terminal half of the protein. The human NaDC3 gene is located on chromosome 20q12-13.1, as evidenced by fluorescent in situ hybridization. The gene is >80 kbp long and consists of 13 exons and 12 introns.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Transportadores de Ácidos Dicarboxílicos , Ácidos Dicarboxílicos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente , Sodio/metabolismo , Simportadores , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 20/genética , ADN Complementario/genética , Femenino , Humanos , Cinética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Oocitos/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
We provide evidence here that b(0,+) amino acid transporter (b(0, +)AT) interacts with 4F2 heavy chain (4F2hc) as well as with the protein related to b(0,+) amino acid transporter (rBAT) to constitute functionally competent b(0,+)-like amino acid transport systems. This evidence has been obtained by co-expression of b(0, +)AT and 4F2hc or b(0,+)AT and rBAT in human retinal pigment epithelial cells and in COS-1 cells. The ability to interact with 4F2hc and rBAT is demonstrable with mouse b(0,+)AT as well as with human b(0,+)AT. Even though both the 4F2hc x b(0,+)AT complex and the rBAT x b(0,+)AT complex exhibit substrate specificity that is characteristic of system b(0,+), these two complexes differ significantly in substrate affinity. The 4F2hc x b(0,+)AT complex has higher substrate affinity than the rBAT x b(0,+)AT complex. In situ hybridization studies demonstrate that the regional distribution pattern of mRNA in the kidney is identical for b(0,+)AT and 4F2hc. The pattern of rBAT mRNA expression is different from that of b(0,+)AT mRNA and 4F2hc mRNA, but there are regions in the kidney where b(0,+)AT mRNA expression overlaps with rBAT mRNA expression as well as with 4F2hc mRNA expression.
Asunto(s)
Aminoácidos/metabolismo , Proteínas Portadoras/metabolismo , Sistemas de Transporte de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , ADN Complementario , Humanos , Hibridación in Situ , Ratones , Conformación Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por SustratoRESUMEN
The differential polarized distribution of the reduced- folate transporter (RFT-1) and folate receptor alpha (FRalpha), the two proteins involved in the transport of folate, has been characterized in normal mouse retinal pigment epithelium (RPE) and in cultured human RPE cells. RPE cells mediate the vectorial transfer of nutrients from choroidal blood to neural retina. Whereas FRalpha is known to be present in many cell types of the neural retina, in situ hybridization analysis in the present study demonstrated that RFT-1 is present only in RPE. Laser-scanning confocal microscopy using antibodies specific for RFT-1 demonstrated an apical distribution of this protein in cultured human and intact mouse RPE, which contrasts with the basolateral distribution of FRalpha in these cells. The expression of RFT-1 in the RPE cell apical membrane was confirmed by functional studies with purified apical membrane vesicles from bovine RPE. These studies, done with N(5)-methyltetrahydrofolate (the predominant folate derivative in blood) and folate as substrates, have shown that RFT-1 functions in a Na(+)- and C1(-)-independent manner. The transporter is specific for folate and its analogs. A transmembrane H(+) gradient influences the transport function of this protein markedly; the transport mechanism is likely to be either folate/H(+) co-transport or folate/OH(-) exchange. Based on the differential polarization of FRalpha and RFT-1 in RPE, we suggest that these two proteins work in a concerted manner to bring about the vectorial transfer of folate across the RPE cell layer from the choroidal blood to the neural retina. This constitutes the first report of the differential polarization of the two folate transport proteins in any polarized epithelium.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Epitelio Pigmentado Ocular/metabolismo , Receptores de Superficie Celular , Animales , Transporte Biológico , Proteínas Portadoras/genética , Bovinos , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Receptores de Folato Anclados a GPI , Humanos , Concentración de Iones de Hidrógeno , Hibridación in Situ , Cinética , Proteínas de la Membrana/genética , Ratones , Microscopía Confocal , Placenta/metabolismo , ARN Mensajero/metabolismo , Especificidad por Sustrato , Tetrahidrofolatos/metabolismoRESUMEN
PURPOSE: Dopamine has several important functions in the retina including a possible role in controlling photoreceptor disk shedding to the RPE. While some cells express a transporter for dopamine, the RPE cell does not, leading us to ask whether the newly described catecholamine transport system, the extraneuronal monoamine transporter (uptake(2)) (also known as organic cation transporter 3 (OCT3), is present in RPE and might function as a transporter for dopamine. OCT3 also accepts histamine as a transportable substrate and so we investigated the interaction of this retinal neurotransmitter with OCT3. METHODS: OCT3 expression in the mouse eye was analyzed by in situ hybridization, Northern blot analysis and RT-PCR. OCT3 function was analyzed in cultured human ARPE-19 cells by monitoring the uptake of 1-methyl-4-phenyl pyridinium (MPP(+)), a neurotoxin, which is a known substrate for OCT3. RESULTS: In situ hybridization analysis showed that OCT3 is expressed in mouse RPE and in several cell types of the neural retina, including photoreceptor, ganglion, amacrine, and horizontal cells. The expression of OCT3 in RPE was confirmed by Northern blot analysis and RT-PCR. The characteristics of MPP( +) uptake in cultured ARPE-19 cells included the stimulation of transport by alkaline pH, high affinity (K(t) = 28 +/- 4 microM), competition with several cationic drugs and monoamine neurotransmitters and sensitivity to steroids. In addition, the uptake of MPP(+) in RPE cells was inhibited by dopamine and histamine with IC(50) values (concentration needed for 50% inhibition) of 637 +/- 84 microM and 150 +/- 20 microM, respectively. CONCLUSIONS. This study provides the first report on the expression and function of an organic cation transporter, OCT3, in the eye and in particular the RPE. The data have physiological and pharmacological relevance as it is likely that OCT3 participates in the clearance of dopamine and histamine from the subretinal space and may also play a key role in the disposition of the retinal neurotoxin MPP(+).
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Transporte de Catión Orgánico , Epitelio Pigmentado Ocular/metabolismo , Retina/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , 1-Metil-4-fenilpiridinio/farmacocinética , Anfetamina/farmacología , Animales , Monoaminas Biogénicas/farmacología , Northern Blotting , Células Cultivadas , Ojo/metabolismo , Femenino , Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Hibridación in Situ , Cinética , Metanfetamina/farmacología , Ratones , Ratones Endogámicos ICR , Nicotina/farmacología , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/efectos de los fármacos , Procainamida/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Esteroides/farmacología , Tetraetilamonio/farmacología , Factores de TiempoRESUMEN
We investigated the expression of interleukin-1 (IL-1) receptors and their involvement in the regulation of the serotonin transporter gene expression in human placenta. IL-1beta is an activator of the serotonin transporter gene expression in JAR human placental choriocarcinoma cells as demonstrated by an increase in the steady-state levels of the transporter mRNA and in serotonin transport activity. This activation is blocked by IL-1 receptor antagonist. Genistein also blocks the effect of IL-1beta, indicating involvement of tyrosine phosphorylation in the process. Treatment of JAR cells with IL-1beta activates mitogen-activated protein kinases and nuclear factor-kappaB. The nuclear factor-kappaB that is responsive to IL-1beta in these cells is the p65 homodimer. Northern blot analysis and reverse transcription-polymerase chain reaction revealed that JAR cells and human placenta express type I and type II IL-1 receptors. The binding sites for (125)I-IL-1beta are localized predominantly in the maternal-facing brush border membrane of the syncytiotrophoblast. These results show that IL-1 in the maternal circulation is likely to play a critical role in the regulation of the serotonin transporter gene expression in the placenta.
Asunto(s)
Proteínas Portadoras/genética , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Placenta/metabolismo , Receptores de Interleucina-1/fisiología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1/metabolismo , Interleucina-1/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Embarazo , Receptores de Interleucina-1/análisis , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Células Tumorales CultivadasRESUMEN
We have isolated a cDNA from a rabbit intestinal cDNA library which, when co-expressed with the heavy chain of the human 4F2 antigen (4F2hc) in mammalian cells, induces system L-like amino acid transport activity. This protein, called LAT2, consists of 535 amino acids and is distinct from LAT1 which also interacts with 4F2hc to induce system L-like amino acid transport activity. LAT2 does not interact with rBAT, a protein with a significant structural similarity to 4F2hc. The 4F2hc/LAT2-mediated transport process differs from the 4F2hc/LAT1-mediated transport in substrate specificity, substrate affinity, tissue distribution, interaction with D-amino acids, and pH-dependence. The 4F2hc/LAT2-associated transport process has a broad specificity towards neutral amino acids with K(t) values in the range of 100-1000 microM, does not interact with D-amino acids to any significant extent, and is stimulated by acidic pH. In contrast, the 4F2hc/LAT1-associated transport process has a narrower specificity towards neutral amino acids, but with comparatively higher affinity (K(t) values in the range of 10-20 microM), interacts with some D-amino acids with high affinity, and is not influenced by pH. LAT2 is expressed primarily in the small intestine and kidney, whereas LAT1 exhibits a much broader tissue distribution.
Asunto(s)
Aminoácidos/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Mucosa Intestinal/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos , Animales , Transporte Biológico Activo , Proteínas Portadoras/química , Clonación Molecular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Glutamina/metabolismo , Humanos , Cinética , Datos de Secuencia Molecular , Conejos , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
We have isolated a cDNA from human placenta, which, when expressed heterologously in mammalian cells, mediates the transport of the water-soluble vitamin thiamine. The cDNA codes for a protein of 497 amino acids containing 12 putative transmembrane domains. Northern blot analysis indicates that this transporter is widely expressed in human tissues. When expressed in HeLa cells, the cDNA induces the transport of thiamine (K(t) = 2.5 +/- 0.6 microM) in a Na(+)-independent manner. The cDNA-mediated transport of thiamine is stimulated by an outwardly directed H(+) gradient. Substrate specificity assays indicate that the transporter is specific to thiamine. Even though thiamine is an organic cation, the cDNA-induced thiamine transport is not inhibited by other organic cations. Similarly, thiamine is not a substrate for the known members of mammalian organic cation transporter family. The thiamine transporter gene, located on human chromosome 1q24, consists of 6 exons and is most likely the gene defective in the metabolic disorder, thiamine-responsive megaloblastic anemia. At the level of amino acid sequence, the thiamine transporter is most closely related to the reduced-folate transporter and thus represents the second member of the folate transporter family.
Asunto(s)
Proteínas Portadoras/metabolismo , Cromosomas Humanos Par 1 , Ácido Fólico/metabolismo , Proteínas de Transporte de Membrana , Secuencia de Aminoácidos , Transporte Biológico , Northern Blotting , Proteínas Portadoras/genética , Clonación Molecular , Exones , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Intrones , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
We have cloned a transporter protein from rabbit small intestine, which, when coexpressed with the 4F2 heavy chain (4F2hc) in mammalian cells, induces a b(0,+)-like amino acid transport activity. This protein (4F2-lc6 for the sixth member of the 4F2 light chain family) consists of 487 amino acids and has 12 putative transmembrane domains. At the level of amino acid sequence, 4F2-lc6 shows significant homology (44% identity) to the other five known members of the 4F2 light chain family, namely LAT1 (4F2-lc1), y(+)LAT1 (4F2-lc2), y(+)LAT2 (4F2-lc3), xCT (4F2-lc4), and LAT2 (4F2-lc5). The 4F2hc/4F2-lc6 complex-mediated transport process is Na(+)-independent and exhibits high affinity for neutral and cationic amino acids and cystine. These characteristics are similar to those of the b(0,+)-like amino acid transport activity previously shown to be associated with rBAT (protein related to b(0,+) amino acid transport system). However, the newly cloned 4F2-lc6 does not interact with rBAT. This is the first report of the existence of a b(0,+)-like amino acid transport process that is independent of rBAT. 4F2-lc6 is expressed predominantly in the small intestine and kidney. Based on the characteristics of the transport process mediated by the 4F2hc/4F2-lc6 complex and the expression pattern of 4F2-lc6 in mammalian tissues, we suggest that 4F2-lc6 is a new candidate gene for cystinuria.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos , Sistemas de Transporte de Aminoácidos , Aminoácidos/metabolismo , Antígenos CD/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cistinuria/genética , Glicoproteínas de Membrana/metabolismo , Alanina/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Proteínas Portadoras/química , Clonación Molecular , Cistina/metabolismo , Dimerización , Proteína-1 Reguladora de Fusión , Intestino Delgado/metabolismo , Cinética , Datos de Secuencia Molecular , Oocitos , ARN Mensajero/metabolismo , Conejos , Especificidad por Sustrato , Xenopus laevisRESUMEN
OBJECTIVE: We sought to investigate the expression of cannabinoid receptors in human placenta and BeWo choriocarcinoma cells and study their role in the regulation of the serotonin transporter. STUDY DESIGN: Expression of the 2 types of cannabinoid receptors (CB1 and CB2) in human placenta and BeWo cells was investigated by reverse transcriptase-polymerase chain reaction and Northern blot analysis. The involvement of the receptors in the regulation of the serotonin transporter expression was studied by using a cannabinoid receptor agonist (WIN 55212-2). BeWo cells were treated with the agonist in the presence or absence of forskolin, and the serotonin transporter activity was measured by assessing paroxetine-sensitive serotonin transport. Serotonin transporter density in cell membranes was monitored by measuring paroxetine-sensitive binding of RTI-55, a specific high-affinity ligand for the transporter. Agonist-induced changes in intracellular levels of cyclic adenosine monophosphate were also monitored. RESULTS: Reverse transcriptase-polymerase chain reaction and Northern blot analysis demonstrated unequivocally that human placenta and BeWo cells express both types of cannabinoid receptors. Treatment of BeWo cells with the receptor agonist blocked the activity of the constitutive, as well as the forskolin-induced, serotonin transporter without affecting the serotonin transporter density. This effect is not mediated by alterations in intracellular cyclic adenosine monophosphate levels. CONCLUSION: The results show that cannabinoid receptors are expressed in human placenta and BeWo cells and play a role in the regulation of the serotonin transporter activity. Human placenta is therefore a direct target for cannabinoids, and marijuana use during pregnancy is likely to affect the placental clearance of serotonin through the serotonin transporter.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Placenta/metabolismo , Receptores de Droga/fisiología , Benzoxazinas , Northern Blotting , Membrana Celular/metabolismo , Coriocarcinoma/metabolismo , AMP Cíclico/metabolismo , Femenino , Expresión Génica , Humanos , Morfolinas/farmacología , Naftalenos/farmacología , Embarazo , Receptores de Cannabinoides , Receptores de Droga/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Células Tumorales CultivadasRESUMEN
PURPOSE: Folic acid is essential for DNA, RNA, and protein synthesis, and deficiencies in folate can lead to nutritional amblyopia and optic neuropathy. The transport of folate from the choroidal blood supply to the retina is only now beginning to be understood. The reduced-folate transporter was reported recently to be present in cultured human retinal pigment epithelial (RPE) cells and is thought to be localized to the apical region of these cells. The authors hypothesize that the RPE plays a role in the vectorial transport of folate from the choroidal blood to the neural retina and uses not only the reduced-folate transporter but also the folate receptor alpha in mediating this transport. The purpose of the present study was to determine whether the folate receptor alpha was present in the RPE and, if so, whether it was distributed along the basolateral membrane of the RPE, supporting a role for the protein in the initial steps of folate transport into the RPE. METHODS: The expression of the folate receptor alpha in mouse RPE was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), functional assays, in situ hybridization, immunohistochemistry, and laser scanning confocal microscopy. RESULTS: RT-PCR analysis, cloning of the RT-PCR product, and subsequent sequencing established that folate receptor alpha mRNA transcripts are expressed in the mouse RPE/choroid and are expressed also in the neural retina. A heterologous functional expression assay using MTX(R)-ZR-75-1 cells showed that the folate receptor alpha cDNA obtained by RT-PCR from the RPE/choroid complex and the neural retina was functional as assessed by the binding of folic acid and by the uptake of N5-methyltetrahydrofolate. In situ hybridization localized the folate receptor alpha mRNA to the mouse RPE cells and to cells of the neural retina. The folate receptor alpha was detected immunohistochemically in the mouse and rat RPE and in several layers of the neural retina. Laser scanning confocal microscopy revealed the distribution of the folate receptor alpha along the basolateral region of the RPE and not the apical region. CONCLUSIONS: The present work represents the first analysis of the folate receptor alpha expression in intact mammalian retina. The receptor is present and functional in mouse RPE. It is distributed specifically along the basolateral surface of the RPE and is proposed to work in a coordinated manner with the reduced-folate transporter in the vectorial transport of folate from the choroidal blood to the neural retina.
Asunto(s)
Proteínas Portadoras/biosíntesis , Epitelio Pigmentado Ocular/metabolismo , Receptores de Superficie Celular/biosíntesis , Retina/metabolismo , Animales , Northern Blotting , Proteínas Portadoras/genética , Cartilla de ADN/química , Receptores de Folato Anclados a GPI , Técnicas para Inmunoenzimas , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Microscopía Confocal , ARN Mensajero/metabolismo , Ratas , Receptores de Superficie Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We report here on the cloning and functional characterization of human LAT1, a subunit of the amino acid transport system L. The hLAT1 cDNA, obtained from a human placental cDNA library, codes for a protein of 507 amino acids. When functionally expressed in mammalian cells together with the heavy chain of the rat 4F2 antigen (r4F2hc), hLAT1 induces the transport of neutral amino acids. When expressed independently, neither hLAT1 nor r4F2hc was capable of amino acid transport to any significant extent. Thus, the hLAT1-r4F2hc heterodimeric complex is responsible for the observed amino acid transport. The transport process induced by the heterodimer is Na+ independent and is not influenced by pH. It recognizes exclusively neutral amino acids with high affinity. LAT1-specific mRNA is expressed in most human tissues with the notable exception of the intestine.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos , Animales , Antígenos CD/biosíntesis , Antígenos CD/fisiología , Transporte Biológico , Northern Blotting , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Línea Celular , Clonación Molecular , ADN Complementario/aislamiento & purificación , Proteína-1 Reguladora de Fusión , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Placenta/química , RatasRESUMEN
We have cloned a Na+-dependent, high affinity dicarboxylate transporter (NaDC3) from rat placenta. NaDC3 exhibits 48% identity in amino acid sequence with rat NaDC1, a Na+-dependent, low affinity dicarboxylate transporter. NaDC3-specific mRNA is detectable in kidney, brain, liver, and placenta. When expressed in mammalian cells, NaDC3 mediates Na+-dependent transport of succinate with a Kt of 2 microM. The transport function of NaDC3 shows a sigmoidal relationship with regard to Na+ concentration, with a Hill coefficient of 2.7. NaDC3 accepts a number of dicarboxylates including dimethylsuccinate as substrates and excludes monocarboxylates. Li+ inhibits NaDC3 in the presence of Na+. Transport of succinate by NaDC3 is markedly influenced by pH, the transport function gradually decreasing when pH is acidified from 8. 0 to 5.5. In contrast, the influence of pH on NaDC3-mediated transport of citrate is biphasic in which a pH change from 8.0 to 6. 5 stimulates the transport and any further acidification inhibits the transport. In addition, the potency of citrate to compete with NaDC3-mediated transport of succinate increases 25-fold when pH is changed from 7.5 to 5.5. These data show that NaDC3 interacts preferentially with the divalent anionic species of citrate. This represents the first report on the cloning and functional characterization of a mammalian Na+-dependent, high affinity dicarboxylate transporter.
Asunto(s)
Proteínas Portadoras/química , Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Cartilla de ADN , ADN Complementario , Transportadores de Ácidos Dicarboxílicos , Ácidos Dicarboxílicos/metabolismo , Cinética , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de AminoácidoRESUMEN
We investigated the transport of cationic neurotoxins and neurotransmitters by the potential-sensitive organic transporter OCT3 and its steroid sensitivity using heterologous expression systems and also analyzed the expression of OCT3 in the brain. When expressed in mammalian cells, OCT3 mediates the uptake of the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and the neurotransmitter dopamine. Competition experiments show that several cationic neuroactive agents including amphetamines interact with OCT3. When expressed in Xenopus laevis oocytes, OCT3-mediated MPP+ uptake is associated with inward currents under voltage-clamp conditions. The MPP+-induced currents are saturable with respect to MPP+ concentration, and half-maximal saturation (K0.5) occurs at about 25 microM MPP+ with membrane potential clamped at -50 mV. The K0.5 for MPP+ is markedly influenced by membrane potential. OCT3 is inhibited by several steroids, and beta-estradiol is the most potent inhibitor (Ki approximately 1 microM). The pattern of steroid sensitivity of OCT3 is different from that of OCT1 and OCT2 but correlates significantly with that of the extraneuronal monoamine transporter (uptake2). The transport characteristics and steroid sensitivity provide strong evidence for the molecular identity of OCT3 as uptake2. OCT3 is expressed in the brain as evidenced from Northern blot analysis, reverse transcription-polymerase chain reaction, and in situ hybridization using OCT3-specific probes. The molecular identity of the transcript hybridizing to the probe has been established by sequencing the reverse transcription-polymerase chain reaction product and also by the isolation of the OCT3 cDNA from a brain cDNA library. Regional distribution studies with in situ hybridization show that OCT3 is expressed widely in different brain regions, especially in the hippocampus, cerebellum, and cerebral cortex. OCT3 is likely to play a significant role in the disposition of cationic neurotoxins and neurotransmitters in the brain.
Asunto(s)
Monoaminas Biogénicas/metabolismo , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , 1-Metil-4-fenilpiridinio/metabolismo , Animales , Línea Celular , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Cinética , Neurotoxinas/metabolismo , Neurotransmisores/metabolismo , Oocitos/metabolismo , Ratas , Xenopus laevisRESUMEN
We have isolated a cDNA from rat placenta which, when expressed heterologously, mediates the transport of a wide spectrum of organic cations. The cDNA codes for a protein of 551 amino acids containing 12 putative transmembrane domains. Northern blot analysis indicates that this transporter is expressed most abundantly in the placenta and moderately in the intestine, heart, and brain. The expression is comparatively low in the kidney and lung and is undetectable in the liver. This transporter is distinct from the previously cloned organic cation transporters (OCT1, OCT2, NKT, NLT, RST, and OCTN1). When expressed in HeLa cells, the cDNA induces the transport of tetraethylammonium and guanidine. Competition experiments indicate that this transport process recognizes a large number of organic cations, including the neurotoxin 1-methyl-4-phenylpyridinium, as substrates. The cDNA-induced transport is markedly influenced by extracellular pH. However, when expressed in Xenopus laevis oocytes, the cDNA-induced transport is electrogenic, associated with the transfer of positive charge into the oocytes. Under voltage clamp conditions, tetraethylammonium evokes inward currents that are concentration- and potential-dependent. This potential-sensitive organic cation transporter, designated as OCT3, represents a new member of the OCT gene family.
Asunto(s)
Proteínas de Unión al ADN/genética , Placenta/metabolismo , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Proteínas de Unión al ADN/química , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Factor 3 de Transcripción de Unión a Octámeros , Oocitos/metabolismo , Conformación Proteica , Ratas , Factores de Transcripción/química , Xenopus laevisRESUMEN
Previous studies have shown that a Na+-dependent transport system is responsible for the transplacental transfer of the vitamins pantothenate and biotin and the essential metabolite lipoate. We now report the isolation of a rat placental cDNA encoding a transport protein responsible for this function. The cloned cDNA, when expressed in HeLa cells, induces Na+-dependent pantothenate and biotin transport activities. The transporter is specific for pantothenate, biotin, and lipoate. The Michaelis-Menten constant (Kt) for the transport of pantothenate and biotin in cDNA-transfected cells is 4.9 +/- 1.1 and 15.1 +/- 1.2 microM, respectively. The transport of both vitamins in cDNA-transfected cells is inhibited by lipoate with an inhibition constant (Ki) of approximately 5 microM. The nucleotide sequence of the cDNA (sodium-dependent multivitamin transporter (SMVT)) predicts a protein of 68.6 kDa with 634 amino acids and 12 potential transmembrane domains. Protein data base search indicates significant sequence similarity between SMVT and known members of the Na+-dependent glucose transporter family. Northern blot analysis shows that SMVT transcripts are present in all of the tissues that were tested. The size of the principal transcript is 3.2 kilobases. SMVT represents the first Na+-dependent vitamin transporter to be cloned from a mammalian tissue.