Chronic Regulation Of HERG Potassium Channel By AKAP5 Mediated PKA

Sudden cardiac death(SCD)is a kind of natural death caused by cardiac causes characterized by sudden loss of consciousness within 1 hour after the onset of acute symptoms.Severe ventricular arrhythmia(VA)is the main cause of sudden cardiac death.A large number of ion channels are distributed on the myocardial cell membrane,the rapidly activating delayed rectifier K current(IKr)plays an important role in maintaining myocardial action potential plateau phase and phase 3 repolarization phase.HERG gene(human ether-a-go-go-related gene)encodes the alpha subunit of IKr,and a mutation of the HERG gene causes a decrease in cardiac IKr current,resulting in congenital long QT syndrome(LQT)associated with autosomal 7.LQT often leads to various ventricular arrhythmias,especially torsades de piontes(TDp),with a high risk of sudden cardiac death.It has been shown that the activation of protein kinase A(PKA)can acutely regulate HERG channel by inhibiting IKr current.However,unlike acute regulation,chronic regulation occurs in hours or later,often involving transcription and post-transcriptional regulation of genes.It has been reported that the sustained activation of PKA can significantly increase the expression of HERG channel in heterologous expression systems or cardiomyocytes,and PKA plays a role in the phosphorylation of HERG channel.AKAP are a class of functionally related and structurally different proteins that bind to PKA.AKAP can bind to a variety of signaling molecules to form a signal complex that targets a specific region to achieve the corresponding function.More than ten kinds of AKAP have been found in myocardial hypertrophy.The expression of AKAP was significantly changed in cardiac diseases such as cardiac hypertrophy,arrhythmia,myocardial ischemia and heart failure,suggesting that AKAP play an important role in the pathological process of the heart.AKAP5 is a widely expressed anchoring protein that can interact with PKA,protein kinase C(PKC),calcineurin(Ca N),calmodulin(Ca M)and other signaling molecules.However,it is not clear whether AKAP5 can chronically regulate HERG channel by binding PKA.Therefore,this study intends to investigate the chronic regulation of AKAP5 on HERG channels by molecular biology and gene intervention in heterologous expression systems and to explore its mechanism.The completion of this study provides a new target for the prevention and treatment of LQTS in the future,which can effectively prevent sudden cardiac death and has important practical significance.Part I: Establishment and identification of HEK293 cell lines expressing HERG potassium channel and AKAP5 anchoring proteinObjective: to establish a HEK293 cell line which can express HERG potassium channel and AKAP5 anchoring protein separately and simultaneously express these two proteins.Methods: HERG gene and AKAP5 gene were transfected and co-transfected into HEK293 cell line with Lipofectamine2000 transfection reagent.The expression of two genes was identified by agarose gel PCR,and the expression of HERG protein and AKAP5 protein were identified by Western blot.Results: Agarose gel PCR results showed that AKAP5 and HERG plasmids were normally expressed in HEK293 cells,and Western blot results showed that AKAP5 anchoring protein and HERG potassium channel could be expressed normally.Conclusion: HEK293 cell lines expressing HERG and AKAP5 separately and simultaneously expressing these two proteins were successfully established.Part ?:The chronic regulatory effect of AKAP5 on HERG potassium channelObjective: to observe whether the presence of AKAP5 anchoring protein affects the expression of HERG potassium channel in transfected cells with PKA agonist forskolin at different concentrations or at different treatment times.Methods: the transfected cells were divided into two groups: HEK293-HERG group which only expressed HERG and HEK293-HERG+AKAP5 group which expressed both HERG and AKAP5.The expression of HERG protein was detected by Western blot after the cells were incubated with PKA agonist forskolin at different concentrations or different treatment times.Results: The expression of HERG was significantly increased in both groups of HEK293-HERG and HEK293-HERG+AKAP5 cells with PKA agonist forskolin 1?m,10?m,1000?m for 24 hours or forskolin 10?m for 6h,12 h and 24 h.The increase in the expression of HERG was more pronounced in the HEK293-HERG+AKAP5 group than in the HEK293-HERG group.Conclusion: AKAP5 is involved in the chronic regulation of HERG potassium channel by PKA.Part III: Molecular mechanism of AKAP5 on chronic regulation of HERG potassium channelObjective: To elucidate the molecular mechanism of AKAP5 on chronic regulation of HERG potassium channel at the cellular level,that is,AKAP5 can chronically regulate HERG potassium channel by specifically anchoring and activating PKA.Methods: Immunofluorescence and co-immunoprecipitation were used to identify the binding and colocalization of HERG,AKAP5 and PKA.Results: immunofluorescence and immunoprecipitation showed that HERG,PKA and AKAP5 combined and co-located on the cell membrane.Conclusion: AKAP5 may modulate HERG potassium channel by specifically anchoring and activating PKA to directly phosphorylate HERG potassium channel.