Assuntos
Odorantes , Neurônios Receptores Olfatórios/fisiologia , Trocador de Sódio e Cálcio/fisiologia , Animais , Cálcio/fisiologia , Cílios/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Reação em Cadeia da Polimerase , Olfato/fisiologia , Trocador de Sódio e Cálcio/genética , Transcrição GênicaAssuntos
Escherichia coli/metabolismo , Proteoma , Trocador de Sódio e Cálcio/química , Sequência de Aminoácidos , Animais , Transporte Biológico , Cálcio/metabolismo , Membrana Celular/metabolismo , Sequência Conservada , Feminino , Dados de Sequência Molecular , Oócitos/fisiologia , Células Procarióticas , XenopusRESUMO
Alternative splicing of RNA transcripts is a general characteristic for NCX genes in mammals, mollusks, and arthropods. Among the family of three NCX genes in mammals, the NCX1 gene contains six exons, namely, A, B, C, D, E, and F, that make up the alternatively spliced region. Studies of the NCX1 gene transcripts suggested that 16 distinct gene products can be produced from the NCX1 gene. The exons A and B are mutually exclusive when expressed. Generally, exon A-containing transcripts are predominantly found in excitable cells like cardiomyoctes and neurons, whereas exon B-containing transcripts are mostly found in nonexcitable cells like astrocytes and kidney cells. Other alternatively spliced exons (C-F) appear to be cassette-type exons and are found in various combinations. Interestingly, exon D is present in all characterized transcripts. The alternatively spliced isoforms of NCX1 show tissue-specific expression patterns, suggesting functional adaptation to tissues. To investigate functional differences among alternatively spliced isoforms of NCX1, we expressed an exon A-containing transcript present in cardiac tissue (NCX1.1) and an exon B-containing transcript found in the kidney (NCX1.3) in Xenopus oocytes. We demonstrated that the Na(+)/Ca(2+) exchangers expressed by exon A- and exon B-containing transcripts display differences in activation by PKA and by [Ca(2+)](i). We also observed that these two isoforms show differences in voltage dependence. Surprisingly, the alternatively spliced isoforms of NCX1 display greater functional differences among themselves than the products of different gene loci, NCX1, NCX2, and NCX3.
Assuntos
Processamento Alternativo , Regulação da Expressão Gênica , Proteínas de Membrana Transportadoras , Trocador de Sódio e Cálcio/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Cálcio/fisiologia , Éxons , Humanos , Mamíferos , Potenciais da Membrana , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Trocador de Sódio e Cálcio/fisiologiaAssuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Clonagem Molecular , Drosophila/fisiologia , Oócitos/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Recombinantes/metabolismo , Trocador de Sódio e Cálcio/genética , Transfecção , XenopusRESUMO
The transcript of the Na+-Ca2+ exchanger gene NCX1 undergoes alternative splicing to produce tissue-specific isoforms. The cloned NCX1 isoforms were expressed in Xenopus oocytes and studied using a two-electrode voltage clamp method to measure Na+-Ca2+ exchanger activity. The cardiac isoform (NCX1.1) expressed in oocytes was less sensitive to depolarizing voltages and to activation by [Ca2+]i than the renal isoform (NCX1.3). The cardiac isoform of NCX1 is more sensitive to activation by protein kinase A (PKA) than the renal isoform which may be explained by preferential phosphorylation. The cardiac isoform of NCX1 is phosphorylated to a greater extent than the renal isoform. The action of PKA phosphorylation which increases the activity of the cardiac isoform of the Na+-Ca2+ exchanger in oocytes was confirmed in adult rat ventricular cardiomyocytes by measuring Na+-dependent Ca2+ flux. We conclude that alternative splicing of NCX1 confers distinct functional characteristics to tissue-specific isoforms of the Na+-Ca2+ exchanger.
Assuntos
Rim/metabolismo , Miocárdio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Condutividade Elétrica , Ativação Enzimática/fisiologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fosforilação , Isoformas de Proteínas/metabolismo , Ratos , Trocador de Sódio e Cálcio/fisiologia , XenopusRESUMO
Phosphocholine (PC) is the immunodominant epitope found on the surface of Streptococcus pneumoniae (SPn). T15-idiotype Abs, whose heavy (H) chain variable region is encoded by the V1 gene, are dominant in the anti-PC response in adult mice and protect mice from lethal pneumococcal infection. The ability of anti-PC Abs using H chains other than the V1 H chain to protect against pneumococcal infection remains controversial. We generated V1(-/-) knockout mice to determine whether protective anti-PC Abs could be produced in the absence of the V1 gene. No anti-PC Abs were produced in V1(-/-) mice immunized with avirulent SPn; however, PC-BSA binding Abs were induced after immunization with PC-keyhole limpet hemocyanin but at significantly lower levels than those in wild-type mice. These Abs provided poor protection against virulent SPn; thus, <25% of V1(-/-) mice survived challenge with 10(4) bacteria as compared with 100% survival of V1(+/+) mice. The anti-PC Abs in V1(-/-) mice were heteroclitic, binding to nitrophenyl-PC better than to PC. None of nine hybridomas produced from V1(-/-) mice provided passive protection. However, the V1(-/-) mice produced normal amounts of Ab to SPn proteins that can partially protect mice against SPn. These data indicate that the V1 gene is critical for the production of anti-PC Abs providing optimum protection against infection with SPn, and the V1(-/-) mice could be useful in unmasking epitopes other than the immunodominant PC epitope on SPn capable of providing cross protection.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Deleção de Genes , Genes de Imunoglobulinas , Epitopos Imunodominantes/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Fosforilcolina/imunologia , Streptococcus pneumoniae/imunologia , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/genética , Anticorpos Monoclonais/genética , Vacinas Bacterianas/imunologia , Reações Cruzadas , Hemocianinas/imunologia , Hibridomas/imunologia , Imunidade Inata/genética , Camundongos , Camundongos Knockout , Vacinação , VirulênciaRESUMO
The mechanism of Ni2+ block of the Na+/Ca2+ exchanger was examined in Sf 9 cells expressing the human heart Na+/Ca2+ exchanger (NCX1-NACA1). As predicted from the reported actions of Ni2+, its application reduced extracellular Na+-dependent changes in intracellular Ca2+ concentration (measured by fluo 3 fluorescence changes). However, contrary to expectation, the reduced fluorescence was accompanied by measured 63Ni2+ entry. The 63Ni2+ entry was observed in Sf 9 cells expressing the Na+/Ca2+ exchanger but not in control cells. The established sequential transport mechanism of the Na+/Ca2+ exchanger could be compatible with these results if one of the two ion translocation steps is blocked by Ni2+ and the other permits Ni2+ translocation. We conclude that, because Ni2+ entry was inhibited by extracellular Ca2+ and enhanced by extracellular Na+, the Ca2+ translocation step moved Ni2+, whereas the Na+ translocation step was inhibited by Ni2+. A model is presented to discuss these findings.
Assuntos
Expressão Gênica , Níquel/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Spodoptera/metabolismo , Compostos de Anilina , Animais , Baculoviridae/genética , Transporte Biológico , Cálcio/farmacologia , Radioisótopos de Cálcio/metabolismo , Corantes Fluorescentes , Humanos , Níquel/farmacologia , Transfecção , XantenosRESUMO
Although inhibition of the Na+/Ca2+ exchanger normally increases [Ca2+]i in neonatal cardiac myocytes, application of the inhibitor Ni2+ appears to reduce [Ca2+] measured by fluo-3. To investigate how the apparent reduction in [Ca2+]i occurs we examined Ca2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells. Transport of Ca2+ by the Na+/Ca2+ exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca2+ indicator fluo-3, and the electrogenic function was determined by measuring the Na+/Ca2+ exchange current (INaCa) using patch clamp methods. INaCa was elicited with voltage-clamp steps or flash photolysis of caged Ca2+. We show significant expression of Na+/Ca2+ exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na+/Ca2+ exchanger. In addition to measurements of INaCa, characterization includes Ca2+ transport via the Na+/Ca2+ exchanger and the voltage dependence of Ca2+ transport. Application of Ni2+ blocked INaCa but, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni2+ was transported via the Na+/Ca2+ exchanger at a rate comparable to the Ca2+ transport. Once inside the cells, Ni2+ reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni2+ does indeed block INaCa, but is also rapidly translocated across the cell membrane by the Na+/Ca2+ exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.
Assuntos
Níquel/metabolismo , Trocador de Sódio e Cálcio/biossíntese , Animais , Transporte Biológico , Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Miocárdio/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Trocador de Sódio e Cálcio/genética , Spodoptera , Fatores de TempoRESUMO
Unique ATP-inhibitable K+ channels (KATP) in the kidney determine the rate of urinary K+ excretion and play an essential role in extracellular K+ balance. Here, we demonstrate that functionally similar low sulfonylurea affinity KATP channels are formed by two heterologous molecules, products of Kir1.1a and cystic fibrosis transmembrane conductance regulator (CFTR) genes. Co-injection of CFTR and Kir1.1a cRNA into Xenopus oocytes lead to the expression of K+ selective channels that retained the high open probability behavior of Kir1.1a but acquired sulfonylurea sensitivity and ATP-dependent gating properties. Similar to the KATP channels in the kidney but different from KATP channels in excitable tissues, the Kir1.1a/CFTR channel was inhibited by glibenclamide with micromolar affinity. Since the expression of Kir1.1a and CFTR overlap at sites in the kidney where the low sulfonylurea affinity KATP are expressed, our study offers evidence that these native KATP channels are comprised of Kir1.1a and CFTR. The implication that Kir subunits can interact with ABC proteins beyond the subfamily of sulfonylurea receptors provides an intriguing explanation for functional diversity in KATP channels.
Assuntos
Rim/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/química , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Eletrofisiologia , Expressão Gênica/genética , Microinjeções , Oócitos/metabolismo , Técnicas de Patch-Clamp , Potássio/urina , Canais de Potássio/genética , RNA Complementar/genética , Compostos de Sulfonilureia/farmacologia , XenopusRESUMO
The Na+/Ca2+ exchanger is a major transporter of Ca2+ in neurons and glial cells. The Na+/Ca2+ exchanger gene NCX1 expresses tissue-specific isoforms of the Na+/Ca2+ exchanger, and the isoforms have been examined here quantitatively using primary cultures of astrocytes and neurons. We present a PCR-based quantitative method, quantitative end-labeled reverse transcription-PCR (QERT-PCR), to determine the relative amounts of the NCX1 isoforms present in these cells. Six exons (A, B, C, D, E, and F) are alternatively spliced to produce the known NCX1 isoforms. Three exon B-containing isoforms (BDEF, BDF, and BD) are the predominant transcripts in primary rat cortical astrocytes and in C6 glioma cells. In contrast, exon A-containing isoforms (ADF and AD) are the predominant transcripts in primary rat hippocampal neurons. Functional differences between full-length constructs of NCX1 containing either the astrocyte isoform BD or the neuron isoform AD were examined in a Xenopus oocyte expression system. Although both isoforms function normally, the activity of the AD isoform can be increased 39% by activation of protein kinase A (PKA), whereas that of the BD isoform is not affected. We conclude that specific NCX1 isoforms are expressed in distinct patterns in astrocytes and neurons. Furthermore, the activity of a neuronal (but not glial) isoform of the Na+/Ca2+ exchanger can be altered by the activation of the PKA pathway.
Assuntos
Astrócitos/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Neurônios/enzimologia , Trocador de Sódio e Cálcio/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Córtex Cerebral/citologia , Ativação Enzimática/fisiologia , Regulação da Expressão Gênica/fisiologia , Glioma , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neurônios/citologia , Oócitos/fisiologia , Fosforilação , Reação em Cadeia da Polimerase/métodos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Trocador de Sódio e Cálcio/análise , Trocador de Sódio e Cálcio/genética , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/metabolismo , XenopusRESUMO
cDNAs for the Na+/Ca2+ exchanger from Drosophila melanogaster (Dmel/Nck) have been cloned by homology screening using the human heart Na+/Ca2+ exchanger cDNA. The overall deduced protein structure for Dmel/Nck is similar to that of mammalian Na+/Ca2+ exchanger genes NCX1 and NCX2, having six hydrophobic regions in the amino terminus separated from six at the carboxy-terminal end by a large intracellular loop. Sequence comparison of the Drosophila exchanger cDNAs with NCX1 and NCX2 Na+/Ca2+ exchangers are approximately 46% identical at the deduced amino acid level. Consensus phosphorylation sites for both protein kinase C and protein kinase A are present on the intracellular loop region of the Dmel/Nck. Alternative splicing for the Dmel/Nck gene is suggested in the same intracellular loop region as demonstrated for NCX1. Functionally, the Drosophila Na+/ Ca2+ exchanger expressed in oocytes differs from expressed mammalian NCX1 with regard to Ca2+ transport in Ca2+/ Ca2+ exchange and the effect of monovalent-dependent Ca2+/ Ca2+ exchange. The Dmel/Nck gene maps to chromosome 3 (93A-B) using in situ hybridization to polytene chromosomes, the same position as the Na(+)-K(+)-ATPase, a related transporter. We conclude that, although extracellular Na+ concentration-dependent Ca2+ transport is subserved by both human and Drosophila Na+/Ca2+ exchangers, there are clear and important differences in the transporters, which should be useful in deducing how the Na+/Ca2+ exchanger protein function depends on its structure.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Mapeamento Cromossômico , DNA Complementar/química , Drosophila/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/química , Membrana Celular/fisiologia , Clonagem Molecular , Sequência Consenso , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA Complementar/metabolismo , Drosophila/genética , Genes de Insetos , Biblioteca Genômica , Humanos , Hibridização In Situ , Mamíferos , Dados de Sequência Molecular , Oócitos/fisiologia , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Trocador de Sódio e Cálcio , XenopusRESUMO
The causes of observed deficiencies to the humoral immune response in aged humans are unknown. Since a major source of antibody diversity is generated at the VH-D-JH junctional regions of the immunoglobulin heavy chain, we determined whether differences in junctional diversity are manifested with aging. We compared the CDR3 regions of IgM heavy chain transcripts isolated from young adult and aged humans. A PCR assay that measures CDR3 length in the majority of mu-heavy chains showed the same average size and normal range of CDR3 length in aged individuals as observed in young adults. To characterize the features of junctional diversity of aged adults in more detail, we determined the CDR3 sequences of a subset of the mu-heavy chain repertoire that utilizes members of the VH 5 family. In general CDR3 length, D family usage, and JH gene usage were similar in aged compared to young adults. Thus, in contrast to dramatic changes in heavy chain junctional diversity associated with fetal to adult development, no major differences were found between young and aged adults. Since the CDR3 repertoire generated in aged individuals appears to be as diverse as that observed in younger adults, the decline in humoral immunocompetence with aging cannot be attributed to a restriction in heavy chain junctional diversification processes.
Assuntos
Idoso/fisiologia , Cadeias Pesadas de Imunoglobulinas/genética , Região de Junção de Imunoglobulinas/genética , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Região Variável de Imunoglobulina/genética , RNA/análise , Adulto , Humanos , Reação em Cadeia da Polimerase , Polimorfismo Genético , RNA/genética , Alinhamento de Sequência , Análise de Sequência de RNA , Deleção de Sequência , Transcrição GênicaRESUMO
We describe an analysis of the NCX1 gene and show that various tissues express different alternatively spliced forms of the gene. Alternative splicing has been confirmed by the genomic analysis of the Na(+)-Ca2+ exchanger gene. We also describe the Drosophila Na(+)-Ca2+ exchanger as having many of the same structural characteristics of the mammalian exchangers and this locus as possibly undergoing alternative splicing in the same region that has been described in the NCX1 gene. The general structure of the exchangers is similar to that of the alpha-subunit of the (Na(+)+ K+)-A Pase. Finally, sequence comparison of the various molecules demonstrates that structural characteristics of these molecules are more strongly conserved than the primary sequence of these products.
Assuntos
Processamento Alternativo , Proteínas de Transporte/genética , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Humanos , Dados de Sequência Molecular , Homologia de Sequência , Sódio/metabolismo , Trocador de Sódio e Cálcio , TransfecçãoAssuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Coração/fisiologia , Linfócitos/fisiologia , Músculo Liso/fisiologia , Fenômenos Fisiológicos do Sistema Nervoso , Animais , Cálcio/metabolismo , Humanos , Rim/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Sódio/metabolismo , Trocador de Sódio e CálcioRESUMO
A sensitive, highly resolvable, and quantitative method was designed to analyse the diversity of polymerase chain reaction (PCR)-amplified transcripts which possess length polymorphism. A reverse transcriptase-PCR technique was used to amplify rearranged T cell antigen receptor (TCR) transcripts isolated from human blood. Oligonucleotide primers specific for conserved TCR V and C region sequences were used in PCR, with one of the primers end-labeled with 32P. Amplified cDNA products were analysed by polyacrylamide sequencing gel electrophoresis with an M13mp18 sequencing ladder as a size marker. 32P-labeled products were detected by either autoradiography or PhosphorImager. The method allowed determination of the sizes of PCR products with the precision of one nucleotide. The resolution using this technique was much higher than by electrophoresis in agarose gel with ethidium bromide staining. The sizes of PCR products determined by sequencing gel electrophoresis were consistent with the lengths of nucleotide sequences obtained after subcloning PCR products in competent bacterial cells. Analysis of PCR products by sequencing gel electrophoresis was more rapid and as accurate as nucleotide sequence analysis in determining the relative ratios of TCR mRNA in mixtures of T cell clones. The method is applicable for analysis of both rearranged TCR and immunoglobulin genes.
Assuntos
Variação Genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Clonais , Eletroforese/métodos , Expressão Gênica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Receptores de Antígenos de Linfócitos T alfa-beta/análiseRESUMO
We have previously shown an increased percentage of gamma delta T cells expressing the TCR V delta 1 gene segment in the peripheral blood and bronchoalveolar lavage fluid of patients with systemic sclerosis (SSC). To estimate clonality of these V delta 1+ T cells, the diversity of V delta 1 junctional regions (V-D-J) was examined using a reverse transcriptase-PCR to amplify TCR delta-chain transcripts isolated from PBMC, lung, esophagus, stomach, or skin of patients and controls. Limited diversity of V delta 1-J delta junctional regions in SSC patients was demonstrated by comparing the size distribution of PCR-amplified junctional region cDNA from patients with that of controls. Sequence analyses confirmed that V delta 1-J delta junctional regions from the blood of SSc patients had less diversity than those from controls, in that a significantly higher proportion of sequences were repeated in patients (54.4 vs 19.4% in controls). Evidence for selection of the V delta 1+ T cells in the tissues of SSC patients came from the findings that the same V delta 1-J delta junctional sequences persisted in an individual patient over time and that identical junctional sequences were isolated from multiple sites. Analysis of deduced amino acid sequences revealed two clusters of similarities among the junctional regions from patients. These data suggest that expansion of V delta 1+ gamma delta T cells may be Ag driven in SSC patients.
Assuntos
Receptores de Antígenos de Linfócitos T gama-delta/genética , Escleroderma Sistêmico/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Sequência de Aminoácidos , Sequência de Bases , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Mensageiro/análise , Receptores de Antígenos de Linfócitos T gama-delta/químicaRESUMO
We have analyzed the gene structure that gives rise to tissue-specific isoforms of the Na/Ca exchanger. Five distinct isoforms of the Na/Ca exchanger from rabbit brain, kidney, and heart have been identified previously to which we now add a new brain isoform. Reverse-transcribed polymerase chain reaction, library screening, and sequence analysis of cDNA coding regions indicate that the only significant alteration of the Na/Ca exchanger cDNA in rabbit brain, kidney, and heart isoforms is located in the carboxyl end of the putative intracellular loop of the protein, a region recently linked to ionic and metabolic regulation of the Na/Ca exchanger. Additionally, we find that the Na/Ca exchanger isoforms found in lung and skeletal muscle may arise from among these same six isoforms. Examination of the gene structure of the Na/Ca exchanger in rabbit indicates how the single gene that encodes for the Na/Ca exchanger is alternatively spliced to give rise to the five rabbit isoforms. Specifically, sequence analysis of the intron-exon boundaries reveals the presence of two "mutually exclusive" exons in conjunction with four "cassette" exons in the region of the Na/Ca exchanger gene that codes for the carboxyl end of the predicted intracellular loop region. This unusual arrangement of exons in the Na/Ca exchanger gene could allow for the generation of up to 32 different Na/Ca exchanger mRNAs and accounts for the isoforms identified to date.
Assuntos
Processamento Alternativo , Encéfalo/metabolismo , Proteínas de Transporte/biossíntese , Éxons , RNA Mensageiro/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Proteínas de Transporte/genética , DNA/química , DNA/metabolismo , Primers do DNA , DNA Complementar/química , DNA Complementar/metabolismo , Variação Genética , Biblioteca Genômica , Íntrons , Rim/metabolismo , Dados de Sequência Molecular , Miocárdio/metabolismo , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Coelhos , Homologia de Sequência de Aminoácidos , Sódio/metabolismo , Trocador de Sódio e CálcioRESUMO
The cardiac (versus retinal rod) Na/Ca exchanger gene has been cloned, sequenced and shown by RNA analysis to be present in diverse tissues. Analysis of published sequences shows that a single isoform is found in heart tissue from many species (NACA1 isoform). We provide evidence here by ribonuclease (RNase) protection assays and by reverse transcriptase-polymerase chain reaction (PCR) amplification with sequence analysis that a new isoform encoding the Na/Ca exchanger is present in renal tissue. This isoform (NACA3) reveals a 7-amino acid deletion in the tested region compared with the NACA2 isoform described by Reilly and Shugrue [Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F1105-F1109, 1992] and is the dominant exchanger transcript in kidney. Analysis of the sequence of all isoforms indicates that the differences in the isoforms reside in the large intracellular loop region of the protein. Alternative splicing of a single Na/Ca exchanger message may be responsible for these tissue-specific transcripts.
Assuntos
Proteínas de Transporte/metabolismo , Rim/metabolismo , Animais , Sequência de Bases , Proteínas de Transporte/genética , Isomerismo , Sondas Moleculares/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , DNA Polimerase Dirigida por RNA , Coelhos , Ribonucleases , Trocador de Sódio e CálcioRESUMO
The cDNA that encodes the human Na+/Ca2+ exchanger (NCX1) involved in regulation of intracellular calcium levels has been isolated from a cardiac cDNA library. Using fluorescent in situ hybridization, the human cDNA was mapped to chromosome region 2p23-->p22 by co-hybridization with fluorescinated alu517-PCR amplified total human DNA to obtain an R-banding pattern.