Pathogenesis Of LQT2 Syndrome Caused By HERG F68C Mutation

Long QT syndrome(long QT syndromes,LQTS)is a kind of malignant arrhythmia caused by prolonged repolarization of myocardium.It is a major cause of sudden death in adolescents and children.Currently,there are some limitations in the treatment of hereditary LQTS,and it can not be completely cured.The molecular pathological mechanism of long QT syndrome needs further study.In this study,we screened the mutations of HERG(KCNH2 gene)in LTQ2 family patients,analyzed the function of the identified new mutations of HERG,studied its molecular pathogenesis,and explored its rescue methods,hoping to provide the necessary basis for clinical individualized treatment of LQTS disease in the future.Result:1.We screend the HERG gene of patients with direct DNA sequence and identified a new heterozygous missense mutation F68 C.2.We transfected EGFP-HERG-WT plasmid or EGFP-HERG-F68 C plasmid into HEK293 T cells.The expression of HERG protein was detected by Western blot.The total protein of F68 C mutant(135KD+155KD)and 155 KD protein were significantly reduced.Confocal microscopy was used to observe the subcellular localization of HERG protein,F68 C mutant protein located on the membrane was significantly reduced.Patch-clamp recordings analysis found that the F68 C mutant channel could not detect the current.These results indicated that F68 C mutation not only affects the expression of HERG protein,but also affects the transport of 155 KD HERG protein.3.F68 C mutation is located in the N-terminal PAS(Per-Amt-Sim)region of the HERG potassium channel.To study the effect of mutation on PAS,HEK293 T cells was transfected with FLAG-tagged wild plasmid PAS-WT or mutant plasmid PAS-F68 C.Western blot analysis showed that F68 C mutant protein migrated slowly.Limited proteolysis hydrolysis after crude membranes extraction showed that the stability of F68 C mutant protein changed.Proteasome inhibitor MG132 could partially restore F68 C mutant protein after treatment.The results showed that F68 C mutation resulted in a significant change in the conformation of PAS domain,making it easily degraded by proteolysis.4.After transfection of WT or F68 C plasmids into HEK293 T cells,the cells were lysed with buffer containing 0.1% and 1% TritonX-100 respectively.After centrifugation,the expression of HERG in supernatant and sediment was detected.No insoluble aggregation of F68 C mutant protein was found.In addition,the expression of ER Stress(endoplasmic rediculum Stress)marker protein ATF4,ATF6,XBP1 and p-eIF2 a protein was detected by Western blot.It was found that F68 C mutation did not induce obvious endoplasmic reticulum stress response.5.We co-transfected the WT and F68 C plasmids into HEK293 T cells and found that the 155 KD HERG mature protein was significantly reduced compared with the single transfection of HERG-WT plasmid.In order to distinguish the expression and translocation into membrane of wild and mutant HERG proteins,we constructed wild and mutant HERG expression vectors with different labels and co-transfected the FLAG-WT/GFP-WT or FLAG-WT/GFP-F68 C plasmids into HEK293 T cells.Western blot analysis with GFP or FLAG antibody showed that when co-expressed wild-type and mutant plasmids,not only mutant HERG protein could not transfer into plasma membrane,but also wild-type HERG protein expressed on the membrane significantly reduced.Patch clamp analysis showed that HERG channel current dignificntly inhibited in HEK293 T cells co-transfected with WT and F68 C.The results showed that interfered with membrane transport of WT HERG protein,showing a negative dominant effect.6.We transfected F68 C plasmid into HEK293 T cells and treated them with low temperature,different concentrations of glycerol(5%-10%),DMSO(0.1%-2%)and drugs(E-4031,VX-809,cisapride,quinidine and pisthagargin),respectively.We found that the expression of HERG protein in 155 KD mature form did not increase significantly.It indicated that F68 C mutation could not be rescued by traditional methods.7.We co-transfected HEK293 T cells with WT and F68 C plasmids,and found that siRNA-13 could specifically silence the expression of F68 C mutant protein and effectively restore the current of HERG channel;moreover,siRNA-13 treatment did not significantly affect the proliferation and apoptosis of HEK293 T cells.It indicated that LQT2 syndrome caused by F68 C mutation can be salvaged by RNAi technology...