Regulatory Effects Of Protein Kinase C Isoforms On KCNQ1 Protein Expression And Underlying Mechanism In Heterologous Expression System

Delayed rectifier potassium current IK is the main repolarized outward potassium current in mammalian ventricular myocytes,including slow activation of delayed rectifier potassium current(IKs)and fast activated delayed rectifier potassium current(IKr).The IKs channel consists of α subunit encoded by KCNQ1(KvLQT1)and β subunit encoded by KCNE1(Mink).The inherited KCNQ1 and KCNE1 gene mutations cause long QT syndrome(LQT)or short QT syndrome(SQT).Myocardial hypertrophy,chronic heart failure and myocardial infarction are often accompanied by the decrease of the IKs density and manifested as acquired LQT syndrome.Both LQTS and SQTS are characterized by high incidence of ventricular arrhythmias.Thus,it is of great importance to study the functional regulation of KCNQ1/KCNE1 channel.More and more evidence suggests that the protein phosphorylation kinase C(PKC)family involves in the regulation of IKs channel.But so far,the related results are still different,or even opposite.Based on their structure and activation requirements,PKCs are classified into three subgroups: traditional PKC(cPKC,including α,βI,βII,γ),the new PKC(nPKC,including δ,ε,π,θ)and atypical PKC(aPKC,including ζ,i/λ).It is well known that there are PKCα,βI,βII,e,θ,δ,γ and other isoforms in the myocardium of different animal species.It is possible that the different regulatory effect on IKs results from the activation of the distinct PKC isoforms.The ion channel current depends on the permeability of the channel pore,the open probability and the number of channels on the cell plasma membrane,which is regulated by gene transcription,protein synthesis,transport and degradation.At present,little is known how distinct PKC isoforms regulate the transport and degradation ofKCNQ1 protein on cell plasma membrane and thus affect the number of channels.Therefore,this study was designed to investigate the regulatory effect of distinct PKC isoforms on KCNQ1 protein expression and underlying mechanism in HEK cell stably expressing KCNQ1/KCNE1 channel by using nonselective PKC activator and selective membrane permeable PKC activator peptides.This is of great significance to understand the pathologic remodeling mechanism of myocardial ion channels and to provide a theoretical basis for the discovery of novel antiarrhythmic drugs which targeted PKC isoforms.Part1 Regulatory effects of distinct PKC isoforms on KCNQ1 protein expression.Objective: To investigate the effect of distinct PKC isoforms on expression of whole cell KCNQ1 protein and surface KCNQ1 protein on plasma membrane in heterologous expression system.Methods:1 Whole cell protein extraction: Cells were rinsed and gently scraped and centrifuged.Then collected cell and added in a ratio of 100: 1 in accordance with RIPA lysate and protease inhibitor(PMSF),and make sure the cell pellet was completely suspended.After ultrasonication and stewing on ice,the supernatant was collected by centrifugation.2 Extraction of cell surface protein: Biotinylation method was used to label cell membrane protein and cells were fully cracked.Then,the biotin-labeled membrane protein was separated with specific beads.Finally,the samples were eluted to get pure cell plasma membrane protein.3 Western blot: The extraction of total protein and plasma membrane protein were resolved by SDS-PAGE and transferred to PVDF membranes,blocked with 10% nonfatmilk,then incubated with the primary KCNQ1 antibody and fluorescently labeled secondary antibodies,scanned using Odyssey Infrared Imaging System.4 Transient transfection of siRNA: HEK293 cells or stably transfected KCNQ1/KCNE1-HEK293 cells were transiently transfected with siRNA usingLipofectin RNAmax and proceed to the next study after 48 h of transfection.Results:1 Effects of different PKC isoforms on KCNQ1 whole cell protein expression: The expression of KCNQ1 whole cell proteins were not significantly changed after incubation with non-selective PKC activator PMA(100 nM),OAG(10 μM)and selective cPKC activator peptides(200 n M),PKCε activator peptides(200 nM)for 24 h,respectively(P > 0.05).2 Effects of different PKC isoforms on KCNQ1 protein expression on plasma membrane: First,by incubating dexamethasone(50 nM)as a postitive control for 6 and 24 h,we tested the reliability of the biotinylation method used in this study.The expression of KCNQ1 proteins on plasma membrane were increased to 176%(P < 0.05)and 281%(P < 0.05),indicating the method is reliable.PMA,OAG and cPKC activator peptide,PKCε activator peptide were incubated with cells for 6 and 24 h,respectively.Western blot analysis showed that,compared to control,the KCNQ1 proteins expression on plasma membrane were significantly reduced to 36%(P < 0.01)and 24%(P < 0.01)after incubation with PMA.It was significantly reduced to 24%(P < 0.01)and30%(P < 0.01)after incubation with cPKC activator peptide.And it was significantly reduced to 34%(P < 0.01)and 34%(P < 0.01)after incubation with PKCε activator peptide.Whereas,the expression of KCNQ1 protein on plasma membrane was not significantly changed after incubation with OAG(P > 0.05).3 Effects of specific siRNA interference on PKC activator peptides:Using siRNA interference technique to knockdown the expression of different PKC isoforms,we further verified the specificity of PKC activator peptides.The results showed that knockdown of PKCα and β could reverse the effect of cPKC activator peptide on the down-regulation of KCNQ1 protein expression on plasma membrane(P < 0.05).Similarly,knockdown of PKCε could reverse the effect of PKCε activator peptide on the down-regulation of KCNQ1 protein expression on plasma membrane(P < 0.01).Thus,the experimentsfurther demonstrated the specific regulation of PKC isoforms.Conclusions: In the heterologous expression system,PMA,cPKC activator peptide and PKCε activator peptide had no significant influence on the expression of whole cell KCNQ1 protein,but those PKC activators significantly down-regulated KCNQ1 protein expression on plasma membrane,suggesting that PKC may modulate the process of channel protein transport or degradation.Part2 Mechanisms of different PKC isoforms on regulation of KCNQ1 channel protein expression on plasma membrane.Objective: To investigate the down-regulation mechanism of KCNQ1 protein expression on plasma membrane after activation of cPKC and PKCεisoforms.Methods: By interfering protein endocytosis and forward transport process,we analyzed the effect of activating different PKC isoforms on the above processes.The methods about extraction of surface protein on cell plasma membrane and Western blot were the same as first part.Results:1 Effects of cPKC and PKCε isoforms on endocytosis of KCNQ1 protein on plasma membrane: An endocytosis inhibitor Dynasore(80 μM)was used to observe the effect of activation of different PKC isoforms.The results showed that the down-regulation of KCNQ1 protein expression on plasma membrane was significantly reversed after incubation with PMA in the presence of Dynasore for 6 h(P < 0.01).The expression level is 34% in the absence and89% in the presence of Dynasore.Similarly,the down-regulation of KCNQ1 protein expression on plasma membrane by cPKC activator peptide was significantly blunted in the presence of Dynasore for 6 h(P < 0.01).The expression level is 24% in the absence and 89% in the presence of Dynasore.The above results showed that PMA and cPKC activator peptide enhanced the internalization and degradation of protein on plasma membrane.In contrast,the down-regulation of KCNQ1 protein expression on plasma membrane by PKCε activator peptide was not changed in the presence of Dynasore for 6 h(P > 0.05).The expression level is 29% in the absence and 39% in the presence of Dynasore.The finding suggested that PKCε activator peptide did not affect endocytosis.In addition,the incubation of Dynasore alone did not significantly alter the expression of KCNQ1 protein on plasma membrane and there may exist other compensatory pathways.2 Effects of cPKC and PKCε isoforms on degradation of KCNQ1 protein on plasma membrane: The expression of KCNQ1 protein on plasma membrane at different time in the presence of BFA(10 μM)could reflect the degradation rate of protein on plasma membrane since BFA blocked the forward trafficking of KCNQ1 protein.The expression level of KCNQ1 protein on plasma membrane at 6,24 h was normalized by the value at 0 h.The expression level in the control condition(BFA alone)at 6,24 h was 72%and 39%,repectively.The expression level of KCNQ1 surface protein in the presence of BFA and cPKC activator peptide was decreased to 30% and 14%,repectively.The degradation was significantly increased compared with control(P < 0.01).The expression level of KCNQ1 surface protein in the presence of BFA and PKCε activator peptide was 68% and 33%,respectively.There was no significant difference compared with control.The results further supported the notion that cPKC activator peptide accelerated the degradation of protein on plasma membrane,but PKCε did not affect this process and may inhibit protein forward transport or recycling process.In addition,we observed a similar effect of PMA as cPKC activator peptide on protein degradation.The expression level in the control condition(BFA alone)at 6,24 h was 82% and 32%,repectively.The expression level of KCNQ1 surface protein in the presence of BFA and PMA was 45% and 14%,respectively.The identical effect to cPKC activator peptide suggested that PMA may primarily activate cPKC and thus accelerate the degradation of KCNQ1 protein on plasma membrane.Conclusions: PMA,cPKC and PKCε activator peptides down-regulated KCNQ1 protein expression on plasma membrane were due to the fact that PMA and cPKC activator peptide accelerated the channel protein endocytosis,whereas PKCε activator peptide may inhibit protein forward transport or recycling process.Summary: In the heterologous expression system,both cPKC and PKCεisoforms significantly down-regulated the expression level of KCNQ1 protein on cell plasma membrane.The cPKC isoform accelerated the endocytosis and degradation,whereas the PKCε did not affect endocytosis and degradation and may inhibit protein forward transport or recycling process.