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1.
J Mol Biol ; 358(2): 387-95, 2006 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-16527307

RESUMO

The Caenorhabditis elegans SLO-1 channel belongs to the family of calcium-activated large conductance BK potassium channels. SLO-1 has been shown to be involved in neurotransmitter release and ethanol response. Here, we report that SLO-1 also has a critical role in muscles. Inactivation of the slo-1 gene in muscles leads to phenotypes similar to those caused by mutations of the dystrophin homologue dys-1. Notably, slo-1 mutations result in a progressive muscle degeneration when put into a sensitized genetic background. slo-1 localization was observed by gfp reporter gene in both the M-line and the dense bodies (Z line) of the C.elegans body-wall muscles. Using the inside-out configuration of the patch clamp technique on body-wall muscle cells of acutely dissected wild-type worms, we characterized a Ca2+-activated K+ channel that was identified unambiguously as SLO-1. Since neither the abundance nor the conductance of SLO-1 was changed significantly in dys-1 mutants compared to wild-type animals, it is likely that the inactivation of dys-1 causes a misregulation of SLO-1. All in all, these results indicate that SLO-1 function in C.elegans muscles is related to the dystrophin homologue DYS-1.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/metabolismo , Distrofina/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Músculos/metabolismo , Distrofia Muscular Animal/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Caenorhabditis elegans/metabolismo , Músculos/patologia , Distrofia Muscular Animal/patologia , Mutação/genética , Fenótipo , Canais de Potássio Cálcio-Ativados/metabolismo
2.
J Mol Biol ; 332(5): 1037-46, 2003 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-14499607

RESUMO

Syntrophins are a family of PDZ domain-containing adaptor proteins required for receptor localization. Syntrophins are also associated with the dystrophin complex in muscles. We report here the molecular and functional characterization of the Caenorhabditis elegans gene stn-1 (F30A10.8), which encodes a syntrophin with homology to vertebrate alpha and beta-syntrophins. stn-1 is expressed in neurons and in muscles of C.elegans. stn-1 mutants resemble dystrophin (dys-1) and dystrobrevin (dyb-1) mutants: they are hyperactive, bend their heads when they move forward, tend to hypercontract, and are hypersensitive to the acetylcholinesterase inhibitor aldicarb. These phenotypes are suppressed when stn-1 is expressed under the control of a muscular promoter, indicating that they are caused by the absence of stn-1 in muscles. These results suggest that the role of syntrophin is linked to dystrophin function in C.elegans.


Assuntos
Proteínas de Caenorhabditis elegans , Proteínas Associadas à Distrofina , Distrofina/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Neuropeptídeos/fisiologia , Aldicarb/farmacologia , Animais , Caenorhabditis elegans , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio , Bases de Dados como Assunto , Genoma , Genótipo , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Músculos/patologia , Distrofia Muscular de Duchenne/metabolismo , Mutação , Fenótipo , Filogenia , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Sensibilidade e Especificidade , Técnicas do Sistema de Duplo-Híbrido
3.
Gene ; 294(1-2): 77-86, 2002 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-12234669

RESUMO

Dystrophin is the product of the gene mutated in Duchenne muscular dystrophy (DMD). Neither the function of dystrophin nor the physiopathology of the disease have been clearly established so far. In mammals, the dystrophin-glycoprotein complex (DGC) includes dystrophin, as well as transmembrane and cytoplasmic proteins. Since Caenorhabditis elegans possesses a dystrophin-like gene (dys-1), we investigated whether homologues of the DGC members could also be found in the C. elegans genome. Conserved homologues were found for dystroglycan, delta/gamma-sarcoglycan and syntrophin. Divergent but related proteins were found for alpha- and beta-sarcoglycans. No sarcospan counterpart was found. The expression of the conserved homologues was inactivated using the RNA interference technique. Phenotypes similar to that of dys-1 were obtained, both in the wild-type background and in combination with other mutations. These results strongly suggest that a protein complex comprising functional analogies with the DGC exists in C. elegans.


Assuntos
Caenorhabditis elegans/genética , Proteínas Associadas à Distrofina , Distrofina/genética , Glicoproteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Proteínas do Citoesqueleto/genética , Distroglicanas , Proteínas de Membrana/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas Musculares/genética , Músculos/anormalidades , Músculos/efeitos dos fármacos , Músculos/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/administração & dosagem , RNA de Cadeia Dupla/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
4.
Neuromuscul Disord ; 12(4): 371-7, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12062255

RESUMO

Duchenne muscular dystrophy is one of the most common neuromuscular diseases. It is caused by mutations in the dystrophin gene. Dystrobrevins are dystrophin-associated proteins potentially involved in signal transduction. The nematode Caenorhabditis elegans possesses one dystrophin-like (dys-1) and one dystrobrevin-like (dyb-1) gene. Mutations of dyb-1 and dys-1 lead to similar phenotypes, comprising hyperactivity and a tendency to hypercontract, which suggest that these proteins may participate in a common function. We show here that overexpression of the Dyb-1 protein delays the onset of the myopathy observed in the C. elegans double mutant (dys-1; hlh-1 mutations). This finding indicates that, in C. elegans, (1) the absence of dystrophin can be partly compensated for by extra doses of dystrobrevin, and (2) dystrobrevin is partly functional in absence of dystrophin.


Assuntos
Proteínas de Caenorhabditis elegans , Distrofina/genética , Locomoção/genética , Proteínas do Tecido Nervoso , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Animais , Caenorhabditis elegans , Modelos Animais de Doenças , Expressão Gênica/fisiologia , Músculos/metabolismo , Músculos/patologia , Distrofia Muscular de Duchenne/genética , Fenótipo
5.
Exp Cell Res ; 302(2): 170-9, 2005 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-15561099

RESUMO

In skeletal muscle, the localization of nNOS is destabilized in the absence of dystrophin, which impacts muscle function and satellite cell activation. In neurons, the adaptor protein, carboxy-terminal PDZ ligand of nNOS (CAPON), regulates the distribution of neuronal nitric oxide synthase (nNOS), which produces the key signaling molecule nitric oxide (NO). While a CAPON-like gene is known to compensate functionally for a dystrophic phenotype in muscle of Caenorhabditis elegans, CAPON expression has not been reported for mammalian muscle. Here, CAPON expression was identified in mouse muscle using Northern and Western blotting and in situ hybridization in combination with immunostaining for laminin. CAPON RNA was expressed in developing normal and dystrophic muscles near fiber junctions with tendons, and levels increased from 1 to 3 weeks. In regenerating normal muscle and also in dystrophic muscles in the mdx mouse, CAPON transcripts were prominent in satellite cells and new myotubes. Expression of CAPON RNA increased in diaphragm muscle of normal and mdx mice after treatment with L-arginine, the NOS substrate. Both CAPON and utrophin protein levels increased in dystrophic quadriceps muscle after treatment with the steroid deflazacort plus L-arginine, known to reduce the dystrophic phenotype. The identification of CAPON transcripts and protein in mammalian muscle and responses to L-arginine suggest CAPON may have a functional role in stabilizing neuronal NOS in skeletal muscle in the cytoskeletal complex associated with dystrophin/utrophin, with possible applications to therapy for human muscular dystrophy.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Óxido Nítrico Sintase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Arginina/farmacologia , Western Blotting , Diafragma/metabolismo , Distrofina/deficiência , Imuno-Histoquímica , Imunossupressores/farmacologia , Hibridização In Situ , Laminina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Pregnenodionas/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Fatores de Tempo , Utrofina/metabolismo
6.
Eur J Biochem ; 269(6): 1607-12, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11895430

RESUMO

Dystrobrevin is one of the intracellular components of the transmembrane dystrophin-glycoprotein complex (DGC). The functional role of this complex in normal and pathological situations has not yet been clearly established. Dystrobrevin disappears from the muscle membrane in Duchenne muscular dystrophy (DMD), which results from dystrophin mutations, as well as in limb girdle muscular dystrophies (LGMD), which results from mutations affecting other members of the DGC complex. These findings therefore suggest that dystrobrevin may play a pivotal role in the progression of these clinically related diseases. In this study, we used the Caenorhabditis elegans model to address the question of the relationship between dystrobrevin binding to dystrophin and dystrobrevin function. Deletions of the dystrobrevin protein were performed and the ability of the mutated forms to bind to dystrophin was tested both in vitro and in a two-hybrid assay, as well as their ability to rescue dystrobrevin (dyb-1) mutations in C. elegans. The deletions affecting the second helix of the Dyb-1 coiled-coil domain abolished the binding of dystrobrevin to dystrophin both in vitro and in the two-hybrid assay. These deletions also abolished the rescuing activity of a functional transgene in vivo. These results are consistent with a model according to which dystrobrevin must bind to dystrophin to be able to function properly.


Assuntos
Caenorhabditis elegans/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Associadas à Distrofina , Distrofina/metabolismo , Proteínas de Membrana/metabolismo , Animais , Sítios de Ligação , Caenorhabditis elegans/genética , Proteínas do Citoesqueleto/química , Teste de Complementação Genética , Proteínas de Membrana/química , Mutagênese , Técnicas do Sistema de Duplo-Híbrido
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