Functional analysis of a novel SCN5A mutation G1712C identified in Brugada syndrome

• Published in: Nan fang yi ke da xue xue bao = Journal of Southern Medical University Vol. 37; no. 2; p. 256
• Main Authors: Chen, Yan-Yu, Liu, Shen-Rong, Xie, Liang-Zhen, Zhu, Ting-Yan, Chen, Yi-Zhen, Deng, Xiao-Jiang, Meng, Su-Rong, Peng, Jian
• Format: Journal Article
• Language: Chinese
• Published: China 20.02.2016
• Subjects: Brugada Syndrome - genetics; Genotype; HEK293 Cells; Humans; Mutagenesis, Site-Directed; Mutation; NAV1.5 Voltage-Gated Sodium Channel - genetics; Patch-Clamp Techniques; Polymerase Chain Reaction; Transfection
• ISSN: 1673-4254

To elucidate the molecular and electrophysiological mechanisms of Brugada syndrome through functional analysis of a novel SCN5A gene mutation G1712C. 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 Na channel 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 Na channel subunits (hH1 and mhH1, respectively), and the macroscopic Na currents 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. An HEK293 cell line that stably expressed Na channel β1-subunit was successfully established. After tra