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Functional analysis of a novel SCN5A mutation G1712C identified in Brugada syndrome / 南方医科大学学报
Journal of Southern Medical University ; (12): 256-260, 2016.
Artigo em Chinês | WPRIM | ID: wpr-273778
ABSTRACT
<p><b>OBJECTIVE</b>To elucidate the molecular and electrophysiological mechanisms of Brugada syndrome through functional analysis of a novel SCN5A gene mutation G1712C.</p><p><b>METHODS</b>A recombinant plasmid pRc<CMV-hH1 containing the mutant human cardiac sodium channel α subunit (hH1) cDNA was constructed using in vitro PCR-based site-directed mutagenesis technique. LipofectamineTM 3000 was used to transfect the plasmid DNA into HEK293 cell line to induce stable expression of Nachannel β1-subunit, and the positive colonies were selected by screening with G418.The standard liposome method was used to transiently transfect HEK293 cells with either the wild-type or mutant Nachannel subunits (hH1 and mhH1, respectively), and the macroscopic Nacurrents were recorded using whole-cell patch-clamp technique. Data acquisition and analysis, generation of voltage commands and curve fitting were accomplished with EPC-10, PatchMaster and IGOR Pro 6.0.</p><p><b>RESULTS</b>An HEK293 cell line that stably expressed Nachannel β1-subunit was successfully established. After transient transfection with the WT subunit, large Nacurrents were recorded from the stable β1-cell line. Transient transfection with the G1712C subunit, however, did not elicit a Nacurrent in the cells.</p><p><b>CONCLUSION</b>Compared with normal Nachannel, the wild-type channel exhibits a similar sodium current. The characteristic kinetics of sodium channel of WT-hH1 was identical to that in normal cardiac muscle cell, and the missense mutation (G1712C) in the P-loop region of the domain IV may have caused the failure of sodium channel expression.</p>
Assuntos
Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Assunto principal: Transfecção / Reação em Cadeia da Polimerase / Mutagênese Sítio-Dirigida / Técnicas de Patch-Clamp / Síndrome de Brugada / Células HEK293 / Genética / Genótipo / Mutação Tipo de estudo: Estudo prognóstico Limite: Humanos Idioma: Chinês Revista: Journal of Southern Medical University Ano de publicação: 2016 Tipo de documento: Artigo

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Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Assunto principal: Transfecção / Reação em Cadeia da Polimerase / Mutagênese Sítio-Dirigida / Técnicas de Patch-Clamp / Síndrome de Brugada / Células HEK293 / Genética / Genótipo / Mutação Tipo de estudo: Estudo prognóstico Limite: Humanos Idioma: Chinês Revista: Journal of Southern Medical University Ano de publicação: 2016 Tipo de documento: Artigo