RESUMO
The ICln protein is expressed ubiquitously in mammals. Experiments designed to knock down the ICln protein in NIH 3T3 fibroblasts as well as in epithelial cells led to the conclusion that this protein is crucially involved in volume regulation after cytoplasmic swelling. Reconstitution of the ICln protein in lipid bilayers revealed the ion channel nature of ICln. Here we describe a new human promoter sequence, composed of 89 nucleotides, which is responsible for a highly constitutive expression of the ICln protein. The promoter sequence lacks a TATA box, and the transcription can be effected at multiple sites. In addition to the starting sites, upstream sequence elements are mandatory for an efficient transcription of the ICln gene (CLNS1A). These new nucleotide elements were defined by site-directed mutagenesis.
Assuntos
Canais Iônicos/genética , Regiões Promotoras Genéticas/genética , Proteínas/genética , Animais , Sítios de Ligação , Linhagem Celular , Tamanho Celular/genética , Clonagem Molecular , Regulação da Expressão Gênica , Genes Reporter , Haplorrinos , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , TransfecçãoRESUMO
The SGK protein kinase is a novel member of the serine/threonine protein kinase family. Its corresponding gene belongs to the group of immediate-early genes. SGK transcription is controlled by cell volume alterations in different cell lines. To analyze the genomic structure and chromosomal location of the SGK gene, a human P1 clone was isolated by screening a human genomic library with a SGK cDNA probe. This clone was confirmed to encode the authentic SGK gene by the detection of exon-intron structures and the correspondence between the nucleotide sequences of exons and human cDNA. Using this P1 clone as a probe for fluorescence in situ hybridization, a single chromosomal locus for SGK was assigned to band 6q23, a region frequently affected by deletion in various human neoplasms.
Assuntos
Cromossomos Humanos Par 6/genética , Genes/genética , Proteínas Nucleares , Mapeamento Físico do Cromossomo , Proteínas Serina-Treonina Quinases/genética , Linhagem Celular , Clonagem Molecular , Dosagem de Genes , Humanos , Proteínas Imediatamente Precoces , Masculino , Dados de Sequência Molecular , Mapeamento por Restrição , Análise de Sequência de DNARESUMO
Expression cloning revealed a chloride channel (ICln) that we found to be fundamental for the regulatory volume decrease in a variety of cells. The chromosomal localization of the human ICln-gene showed two loci, one at chromosome 11 in position q13.5-q14.1, termed CLNS1A, and a second one at chromosome 6 at position p12.1-q13, termed CLNS1B. In this study, we offer a detailed characterization of the CLNS1A gene and provide the exact position (6p12) and sequence data of CLNS1B, an intronless gene 91.3% homologous to the coding region of CLNS1A.
Assuntos
Canais de Cloreto/genética , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 6 , Canais Iônicos , Sequência de Aminoácidos , Sequência de Bases , Canais de Cloreto/biossíntese , Canais de Cloreto/química , Mapeamento Cromossômico , Primers do DNA , Éxons , Biblioteca Genômica , Humanos , Hibridização in Situ Fluorescente , Íntrons , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
ICln is a cloned chloride channel paramount for regulatory volume decrease. Two different loci that carry the coding region for ICln were identified in the human genome. By PCR strategies an intronless copy of the gene was located on chromosome 6 at position 6p12.1-6q13 (CLNS1B). By fluorescence in situ hybridization a copy carrying introns with a putative length of 19 kb was located at chromosome 11 on position 11q13.5-q14.1 (CLNS1A). The characterization and chromosomal localization of the ICln gene offer the opportunity to study the regulatory sites of this gene in greater detail and could be helpful in establishing linkages between ICln and potential human diseases.
Assuntos
Canais de Cloreto/genética , Cromossomos Humanos Par 11/genética , Genes , Canais Iônicos , Mapeamento Cromossômico , DNA Complementar/genética , Biblioteca Gênica , Humanos , Hibridização in Situ Fluorescente , Reação em Cadeia da PolimeraseRESUMO
Cell volume regulation is a ubiquitous cell regulatory mechanism based on meticulously controlled ion transport mechanisms. Keeping the absolute volume constant seems to be of the highest priority for most cells and is achieved at the expense of altered intracellular ion concentrations. We have been able to demonstrate that ICln, a chloride channel cloned from epithelial cells, is paramount for the ability of swollen cells to regulate their volume back to that under resting conditions. A unique feature of ICln is the distinct sensitivity of these channels for nucleotides and nucleoside analogues added to the extracellular fluid. In addition, cromolyn sodium and nedocromil sodium, drugs used by patients with asthma, are able to impede the function of these channels.
Assuntos
Tamanho Celular/efeitos dos fármacos , Canais de Cloreto/farmacologia , AnimaisRESUMO
Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.
Assuntos
Canais de Cloreto/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Animais , Sequência de Bases , Biotransformação/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Canais de Cloreto/efeitos dos fármacos , Células Epiteliais , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Fibroblastos/metabolismo , Soluções Hipotônicas , Cinética , Camundongos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
BACKGROUND: The antiviral drugs AZT and acyclovir are generally used in the treatment of infections with human immunodeficiency virus (HIV) and herpes simplex virus (HSV). These substances are known to impede virus replication by premature nucleic acid chain termination. It is not yet clear, however, if this is the sole mechanism responsible for the antiviral and/or the numerous side effects observed in patients treated with these agents. We investigated the swelling-induced chloride current in fibroblasts, which we demonstrated is closely related or identical to a cloned epithelial chloride channel, ICln: This chloride channel can be blocked by nucleotides. MATERIALS AND METHODS: Electrophysiological, fluorescence optical, and volume measurements were made to determine the effect of nucleoside analogs on the swelling-dependent chloride current (ICl) in NIH 3T3 fibroblasts and in human T cell lymphoma (H9) cells and the cAMP-dependent chloride current in CaCo cells. RESULTS: AZT and acyclovir block the swelling-dependent chloride current and the chloride flux in fibroblasts, and the regulatory volume decrease (RVD) and ICl in H9 cells. This immediate effect can be substantially reduced by the simultaneous incubation of the cells with thymidine-5'-diphosphate (TDP) or uridine, both of which are by themselves unable to affect ICl. CONCLUSIONS: We show here a novel molecular mechanism by which antiviral drugs of the nucleoside analog family could lead to impairments of the kidney, bone marrow, gastrointestinal, and neuronal functions, and how these side effects could possibly be restricted by the presence of TDP or uridine.
