The Regulation Of KCNQ1/KCNE1Potassium Channel Expression By Angiotensin II

Delayed rectifier K⁺current (IK) is the mainly repolarization outwardpotassium currents in human and mammalian ventricular myocytes, whichincludes the two components: slowly (I_Ks) and rapidly (I_Kr) activating delayedrectifier potassium current. At present, it generally considered that the alphasubunit encoded by KCNQ1and β subunit encoded by KCNE1together formthe IKschannel. KCNQ1is the member of KCNQ that belongs to Kvpotassium channels family (KCNQ1-5or Kv7.1-7.5). Heterologous expressionof KCNQ1produces the fast activation, slow deactivation of outward current.Five KCNE proteins found so far are able to play different regulation ofKCNQ1channel function, in which the KCNQ1/KCNE1heterologousexpression forms slow activation, slow deactivation and inactivation of current.The current kinetic characteristics and pharmacological characteristics arevery similar to the IKschannel of the heart.The gene mutations of KCNQ1and KCNE1are the molecular geneticfoundation causing channelopathies, including the long QT syndrome (LQT1,LQT5,) related to the downregulation of channel function or the short QTsyndrome (SQT2) caused by gain-of-function of the channel. Meanwhile,increasing evidence shows that many cardiovascular diseases, such as cardiachypertrophy and heart failure are associated with the down-regulation of IKschannel function and thus constitute one of the important causes of acquiredLQT. Both LQT and SQT can lead to arrhythmias, especially torsades depointes tachycardia with a high risk of sudden cardiac death. Therefore, studyon the regulation of IKschannel function is of great significance.Renin-angiotensin system (RAS) has an important role in regulating thecardiovascular system of normal physiological function and involves in thepathological process including high blood pressure, cardiac hypertrophy, and congestive heart failure. Angiotensin II (AngII) is the mainly effector of thissystem and recent studies suggest that AngII has a direct regulatory role oncardiac ion channel. In the previous study, we found that AngII showed acutedown regulation on IKsin guinea pig ventricular myocytes by activation of theAT1receptor. It is known that the stimulation of AT1receptor produces acutewithin minutes and chronic effects as gene transcription andpost-transcriptional regulation. However, the chronic regulation of IKschannelby AngII is not clear. In this study, the chronic regulation of the KCNQ1channel by AngII was investigated by using molecular biological techniques inhuman embryonic kidney cells (HEK293) co-expressed of KCNQ1, KCNE1,and human AT1receptor cDNA. The intracellular signal transduction pathwayrelated to the regulation by AngII was also analysised.1Effect of AngII on the expression of KCNQ1channel proteinsObjective: To observe the impact of different concentrations of AngII onthe KCNQ1channel protein expression and time course of the effect.Methods: HEK293cells were transiently co-transfected with taggedV5-KCNQ1or Myc-KCNQ1, KCNE1and human AT1receptor cDNA byusing lipofectin2000transfection reagents. After24hours, the abundance ofKCNQ1channel protein was determined with Western blot technique by usingspecific anti-tag antibody. The distribution of the protein on the cell membranewas observed by using immunofluorescence technique under the confocalmicroscope.Results:(1) The abundance of KCNQ1channel protein wassignificantly reduced after the cells were incubated with Ang II for24h andthe protein content of Ang II in10,100,1000nM is respectively80%,,68%,67%of control.(2) The time course of Ang II effect was observed at differentincubation time at2,6,12,24h. The result showed that the reduction of thechannel protein occurred at12h.(3) The immunofluorescence assay furtherconfirmed a decrease of fluorescence intensity of membrane channel proteinsafter incubation with Ang II (100nM) after24h.Conclusion: In the heterologous expression system, Ang II caused a decline of KCNQ1/KCNE1channel protein by prolonged incubation.2Effect of PKC signaling pathway on the role of Ang IIObjective: To investigate whether intracellular PKC signaling pathwayinvolved in the regulation of KCNQ1channels by AngII.Methods: HEK293cells were transiently co-transfected with taggedV5-KCNQ1or Myc-KCNQ1, KCNE1and human AT1receptor cDNA byusing lipofectin2000transfection reagents. After24hours, the abundance ofKCNQ1channel protein was determined with Western blot technique by usingspecific anti-tag antibody in the resence of Ang II without or with differentPKC inhibitors or agonists.Results:(1) Compared to the incubation of Ang II alone, the abundanceof KCNQ1channel protein was significantly increased in HEK cellsco-inculation with PKC inhibitor Bis-1(100nM)30min before adding Ang II(100nM) for24h, indicating that Bis-1antagonized the role of Ang II.(2) It isknown that long time PKC stimulation by potent PKC agonist PMA lead to thedepletion of PKC. We next examined the effect of PMA (100nM) on theregulation of Ang II on the channel abundance. Compared with the incubationof Ang II alone, PMA co-inculation with Ang II also significantly increasedthe abundance of KCNQ1channel protein.(3) To explore the regulation ofdirect PKC stimulation on the channel abundance, we observed the effects ofPKC activators PMA or OAG alone. It showed that protein content in thepresence of PMA at30min and24h is repectively49%and95%of the control;while the protein content after incubation with OAG at30min and24h isrepectively46%and40%of the control. The results indicated that directchronic stimulation of PKC led to the down-regulation of channel expressionand Ang II modulated the channel abundance through PKC signaling pathway.Conclusion: Ang II down regulates the KCNQ1channel abundancethrough intrcellular PKC signaling pathway.3Effect of KCNE1-S102mutation on the role of AngIIObjective: Recent studies have shown that the degradation of KCNQ1channel protein can be accelerated by PKC through the phosphorylation on the KCNE1S102. We investigated whether the mechanism underly thedown-regulation of the channel abundance by Ang II., while S102is on theKCNE1previously recognized PKC sites regulate channel function, in thisexperiment it compared the role of Ang II and KCNE1-S102A to confirmingwhether the two channels have different or not.Methods: The effects of Ang II on the KCNQ1abundance werecompared in HEK293cells transfected with wild type channelWT-KCNQ1/KCNE1or mutant KCNQ1/KCNE1-S102A.Results: There was no significant difference in the abundance of thechannel between WT-KCNQ1/KCNE1and mutant KCNQ1/KCNE1-S102Aafter incubation with Ang II or PKC activator OAG. The results suggestedthat KCNE1-S102phosphorylation was not involved in the down-regulationof the channel proteins of Ang II.Conclusion: KCNE1-S102mutation had no significant role on the effectof Ang II, suggesting that KCNE1-S102was not involved in thedown-regulation of the channel proteins of Ang II.