Test The Function Of HERG New Mutation Q738X

Inherited LQTS is caused by mutations in coding myocardial cell ion channel gene.Up to now, genetic studies have discovered at least thirteen forms of LQTS related gene. The LQTS patient of our country mainly acquire LQT2. The cause of LQT2is Mutation in the hERG gene. The human ether-a-go-go-related genen codes the rapidly activating delay rectifier potassium channel. The IKr is essential to the repolarization phase of cardiac action potential. Recently, Clinical tested a novel hERG Q738X mutation. Before we made use of whole cell patchclamp technique and confocal laser technology to identify the function of mutant Q738X.The experimental results showed that singlely transfecting Q738X-hERG in to HEK-293did not record current. Co-transfecting Q738X-hERG and WT-hEG could record current, While the current scope comparing with the same amount of WT-hERG HEK-293cell reduced the half. The confocal laser technology showed that the Q738X-hERG proteins were detained in the endoplasmic reticulum. This conclusion:Q738X mutants are not the dominant negative LQT2mutation, the defective trafficking Q738QX maybe cause LQT2. So we will confirm that Q738X lead to the specific mechanism LQT2.Cells were transfected transiently with lipofectamine method.Transfected plasmid contained:①Wild type WT-hERG②mutantn type Q738X-hERG③control group untransfected HEK-293cell. The expression of hERG protein were studied with western blot.In addition, We constructed Flag-WT-hERG and Myc-Q738X-hERG plasmid.We aslo transfected transiently cells with lipofectamine method. Transfected plasmid contained:①cotranfect Flag-WT-hERG and WT-hERG;②contranfect Myc-Q738X-hERG and Q738X-hERG;③contranfect Myc-Q738X-hERG and Flag-WT-hERG. Using immunoprecipitation decected whether Q738X subunits associated with wild-type hERG subunit or not.Western blot analysis revealed that only80KDa immature hERG protein was expressed in Q738X-transfected cell,whereas both mature and immature forms of hERG protein were observed in WT-transfeced cells. Immunoprecipitation study showed that Q738X mutant proteins were not physically associated in the endoplasmic reticulum as heteromeric, which suggested that the mutant channels were retained in the endoplasmic reticulum.