Functional expression of congenital long QT syndrome related HERG mutation A561V in vitro / 中华心血管病杂志
Chinese Journal of Cardiology
; (12): 143-146, 2007.
Article
em Zh
| WPRIM
| ID: wpr-304952
Biblioteca responsável:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the functional expression of HERG mutation A561V detected in a Chinese congenital long QT syndrome family.</p><p><b>METHODS</b>The mutation gene A561V was cloned into eukaryotic expressive vector pcDNA3 by quick site-directed mutagenesis PCR and restriction enzymes. The wild-type HERG, heterozygous type HERG and HERG mutation A561V were respectively cotransfected with pRK5-GFP into HEK293 cells by Suprefact transfection regent. The protein expression was measured by immunofluorescence method and Western blot. The electrophysiological characteristics of transfected cells were determined by whole cell patch-clamp technique.</p><p><b>RESULTS</b>Direct sequence analyses revealed a C to T transition at position 1682. A561V mutation was correctly combined to eukaryotic expressive vector pcDNA3 and expressed in HEK293 cells. The protein expression of mutation and heterozygosis were located in cytoplasm and cellular membrane. 155 kDa and 135 kDa protein bands were detected in wild type HERG channel while only 135 kDa protein band was shown in heterozygous and mutational channels. Significant HERG tail-current was recorded in wild type HERG channel but not in mutation and heterozygosis channels.</p><p><b>CONCLUSION</b>This study evidenced a functional dominant-negative current suppression in HEK293 cells transfected with HERG mutation A561V.</p>
Texto completo:
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Índice:
WPRIM
Assunto principal:
Síndrome do QT Longo
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Análise Mutacional de DNA
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Transfecção
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Expressão Gênica
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Linhagem Celular
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Técnicas de Patch-Clamp
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Canais de Potássio Éter-A-Go-Go
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Canal de Potássio ERG1
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Genética
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Mutação
Limite:
Humans
Idioma:
Zh
Revista:
Chinese Journal of Cardiology
Ano de publicação:
2007
Tipo de documento:
Article