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

doi:10.3969/j.issn.1673-4254.2017.02.19

Abstract：Objective To elucidate the molecular and electrophysiological mechanisms of Brugada syndrome through functional analysis of a novel SCN5A gene mutation G1712C.Methods 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.Results An HEK293 cell line that stably expressed Na + channel β1-subunit was successfully established.After transient transfection with the WT subunit,large Na+ currents were recorded from the stable β1-cell line.Transient transfection with the G1712C subunit,however,did not elicit a Na + current in the cells.Conclusion Compared with normal Na + channel,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.