Functional expression of a novel SCN5A mutation K317N identified in a Chinese Brugada syndrome family

Journal of Clinical and Experimental Medicine(2011)

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摘要
Objective After finding a case of novel SCN5A mutation associated with Brugada syndrome in a Chinese family,the primary functional analysis of novel mutation had been made in order to find out the molecular and cellular electrophysiological mechanism of Brugada syndrome.Methods Mammalian cell transfection and expression was carried out in No.293 cell line that stably expresses Na+channel β1-subunit,and it had been established by using LipofectamineTM2 000.Positive colonies were then selected by antibiotics screening with G418.Then,standard liposome method was used to transiently transfect HEK293 cells with Na+ channel subunit hH1 or mhH1.Macroscopic Na+ currents were recorded by using patch-clamp technique in whole cell mode.Data acquisition and analysis,generation of voltage commands and curve fitting were accomplished with pClamp 8.0 software(Axon Instruments).Results No.293 cell line that stably expresses Na+channel β1-subunit was established and positive colonies were selected with G418.Cells expressing the construct appeared green under UV epifluorescence.After transient transfection with WT subunit,large Na+current from stable β1-cell line was recorded.No detectable current was observed in the absence of subunit transfection.However,after transient transfection with K317N subunit,and Na+current had not been recorded anymore from stable β1-cell line.Conclusion Compared with normal Na+ channel,the wild-type channel exhibited a similar sodium current.The characteristic kinetics of sodium channel of WT-hH1 is same as normal cardiac muscle cell.The missense mutaion(K317N) in P-Loop region of domain I may be the cause responsible for failure of expression of sodium channel.
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关键词
Brugada syndrome,Whole cell patch clamp,Transfection,SCN5A,Sodium channel kinetics
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