RESUMO
Carnitine is essential for beta-oxidation of fatty acids, and a defect of cell membrane transport of carnitine leads to fatal systemic carnitine deficiency. We have already shown that a defect of the organic cation/carnitine transporter OCTN2 is a primary cause of systemic carnitine deficiency. In the present study, we further isolated and characterized new members of the OCTN family, OCTN1 and -3, in mice. All three members were expressed commonly in kidney, and OCTN1 and -2 were also expressed in various tissues, whereas OCTN3 was characterized by predominant expression in testis. When their cDNAs were transfected into HEK293 cells, the cells exhibited transport activity for carnitine and/or the organic cation tetraethylammonium (TEA). Carnitine transport by OCTN1 and OCTN2 was Na(+)-dependent, whereas that by OCTN3 was Na(+)-independent. TEA was transported by OCTN1 and OCTN2 but not by OCTN3. The relative uptake activity ratios of carnitine to TEA were 1.78, 11.3, and 746 for OCTN1, -2, and -3, respectively, suggesting high specificity of OCTN3 for carnitine and significantly lower carnitine transport activity of OCTN1. Thus, OCTN3 is unique in its limited tissue distribution and Na(+)-independent carnitine transport, whereas OCTN1 efficiently transported TEA with minimal expression of carnitine transport activity and may have a different role from other members of the OCTN family.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , DNA Complementar , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Proteínas de Transporte de Cátions Orgânicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , SimportadoresRESUMO
The bromodomain is a structural motif characteristic of proteins involved in chromatin-dependent regulation of transcription. Bromodomain proteins have been identified as integral components of chromatin remodeling complexes and frequently possess histone acetyltransferase activity. Their encoding genes have been identified at translocation breakpoints, and at least one, CBP, is a tumor suppressor gene. We have identified a series of novel bromodomain genes by EST database and cDNA library screening. Comparison of sequences for four clones indicated that they represent genes belonging to a novel bromodomain family. Full-length sequences for these genes, which are widely expressed, predict encoded proteins of between 1527 and 1972 amino acids. In addition to a carboxy-terminal bromodomain, an adjacent PHD finger, and a WACZ motif, at least four other conserved novel motifs are present in each protein. The genes contain regions conserved with Drosophila Acf1 and Caenorhabditis elegans ZK783.4. The novel genes, termed BAZ1A, BAZ1B, BAZ2A, and BAZ2B, localize to chromosomes 14q12-q13, 7q11-q21, 12q24.3-qter, and 2q23-q24, respectively. Conservation of multiple domains throughout these genes with Acf1 indicates that they are likely to be components of chromatin remodeling complexes.
Assuntos
Cromatina/metabolismo , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 2 , Cromossomos Humanos Par 7 , Etiquetas de Sequências Expressas , Feminino , Biblioteca Gênica , Humanos , Células Híbridas , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , RNA Mensageiro/análise , Alinhamento de Sequência , Fatores de Transcrição/metabolismoRESUMO
Carnitine deficiency, either primary or drug-induced, causes critical symptoms and is thought to involve alteration of active transport of carnitine across the plasma membrane of tissues as the underlying mechanism. Recently, we showed that human organic cation transporter, hOCTN2, cloned as a member of the organic cation transporter family, is a physiologically important Na(+)-dependent high-affinity carnitine transporter in humans. In this study, we further characterized the functional properties of hOCTN2 and examined the interaction between hOCTN2-mediated carnitine transport and clinically used drugs to assess possible toxicological effects. When expressed in human embryonic kidney (HEK)293 cells, hOCTN2 showed low but significant stereospecific transport activity: D-carnitine was transported with lower affinity (K(m) = 10.9 microM) than the L-isomer (K(m) = 4.3 microM). One Na(+) appeared to be associated with the transport of one carnitine molecule. hOCTN2-mediated transport of acetyl-L-carnitine was also Na(+)-dependent and of high affinity, with a K(m) value of 8.5 microM. To examine the transport activity for organic cations other than carnitine and the possible relationship of drug-induced carnitine deficiency with hOCTN2, the inhibitory effect of several drugs on hOCTN2-mediated L-carnitine transport was examined. Many zwitterionic drugs, such as cephaloridine, and many cationic drugs, such as quinidine and verapamil, exhibited significant inhibitory effects. Among these inhibitors, tetraethylammonium, pyrilamine, quinidine, verapamil, and valproate were found to be transported by hOCTN2. The results suggest that the carnitine deficiency-related toxicological effects by long-term treatment with such drugs might be ascribed to a functional alteration of hOCTN2-mediated carnitine transport.
