Regulation Of DPP6 On The Expression Of Cardiac Kv4 Voltage-gated K~+ Channel

Dipeptidyl peptidase-like protein 6(DPP6)can serve as an auxiliary subunit to form cardiac voltage gated potassium channel 4(Kv4)channel together with K⁺channel-interacting protein 2(KCh IP2),which is the molecular basis of transient outward potassium current(I_to).And I_to is the main component of ventricular repolarization phase 1 in large mammals,including human.In recent years,it has been found that mutations in DPP6 gene can increase I_to and further trigger familial idiopathic ventricular fibrillation(IVF).Therefore,it is important to investigate the regulation and mechanism of DPP6on cardiac Kv4 channel.Previous studies have shown that DPP6 can directly regulate channel gating by binding with Kv4 subunit,and initial experiments have shown that DPP6 may effect the expression level of Kv4 membrane protein,but the mechanism of this action remains unclear.The expression of membrane protein depends on gene transcription,protein synthesis,and protein trafficking.In addition,cardiac Kv4 channel is a ternary complex,in which KCh IP2 acts as a transcription factor to regulate the transcription of Kv4.3 and promote the forward trafficking of Kv4 channel.However,we have no idea whether the effect of DPP6 on the expression of cardiac Kv4 protein depends on KCh IP2.And studies still need to be done to understand how DPP6 works.Different isoforms of DPP6 have been discovered in the brain tissue and the most common types are DPP6-L and DPP6-S.Moreover,our previous study has found that NS5806,a small molecule of sulfonic ureas,may regulate the function of cardiac Kv4 channel by binding to the intracellular site of DPP6.And NS5806 may have opposite effects on cardiac Kv4 channel because of the difference between DPP6-L and DPP6-S isoforms.However,cardiac DPP6isoforms are still unclear.Therefore,this study was designed to clarify the isoforms of DPP6 in the rat left ventricle by Liquid Chromatograph Mass Spectrometer/Mass Spectrometer(LC-MS/MS),and detect the effect of DPP6 on the expression of Kv4.3 membrane protein.And in this way,it can provide an experimental basis for exploring the physiological and pathophysiological contribution of DPP6 in heart.Objective:This study aimed to clearly define the DPP6 isoform in rat heart,and analyze the functional regulation of DPP6 on the Kv4 channel.Methods:1.The identification of DPP6 isoform in rat left ventricular myocardium:We detected the majority content of DPP6 in rat left ventricular tissue by LC-MS/MS and q RT-PCR.2.Regulation and the mechanism of DPP6 on the expression of Kv4membrane protein:In the heterologous expression line,different auxiliary subunits were expressed to form channel complex with Kv4.3.The m RNA,total protein and membrane protein expression levels of Kv4.3 were respectively analyzed by q RT-PCR and Western Blot.The two-color fluorescent labeled Kv4.3 plasmid was constructed,and the effect of DPP6 on Kv4.3 protein trafficking was observed by flow cytometry.In addition,the effects of two DPP6 mutants(L747P and Q526H)on the expression of Kv4.3 protein were observed by Western Blot..Results:1.The isoforms of DPP6 in rat left ventricular myocardium:At the protein level,four unique peptides of DPP6-L and two unique peptides of DPP6-S were found by LC-MS/MS,and the intensity-based Absolute Quantification(i BAQ)value of DPP6-L was much higher than DPP6-S.It is indicated that DPP6-L is the mainly isoform in left ventricular myocardium of rat.At the m RNA level,DPP6-L was much higher than DPP6-S in rat brain tissue.Both DPP6-L and DPP6-S in the left ventricular myocardium of rat were less than rat brain tissue.However,there was no statistical difference between DPP6-L and DPP6-S in the left ventricular myocardium of rat.2.The regulation effect of DPP6 on Kv4 channel:At the m RNA level,DPP6 or KCh IP2 co-expressed with Kv4.3 did not affect the expression of Kv4.3 m RNA compared with Kv4.3 expressed alone(P>0.05).At the protein level,compared with Kv4.3 expressed alone,DPP6co-expression with Kv4.3 did not affect the expression of total Kv4.3 protein(P>0.05),but increased the expression of Kv4.3 membrane protein(P<0.05).Compared with Kv4.3 expressed alone,the expression of Kv4.3 were increased when KCh IP2 was co-expressed with Kv4.3(P<0.05).However,when DPP6 was co-expressed with KCh IP2 and Kv4.3,the expression of total Kv4.3 protein was lower than the condition KCh IP2 was co-expressed with Kv4.3(P<0.05),and the expression of Kv4.3 membrane protein when DPP6was co-expressed with KCh IP2 and Kv4.3 was not different from the condition that KCh IP2 was co-expressed with Kv4.3(P>0.05).The flow cytometry results showed that,when DPP6 and Kv4.3 were co-expressed with incubation of Dynasore(Motor Protein inhibitor with the effect of endocytosis),the degree of Kv4.3 protein on the membrane increased more with time dependent compared with Kv4.3 expressed alone(P<0.05).It is further suggested that DPP6 could promote the forward trafficking of Kv4.3.3.The effect of DPP6 mutants on Kv4.3 protein expression.Compared with DPP6-WT,DPP6-L747P and DPP6-Q526H had no significant effect on the expression of total Kv4.3 protein(P>0.05).However,the expression of Kv4.3 membrane protein was significantly increased(P<0.05).Conclusion:1.In rat left ventricular myocardium,it is found that DPP6-L is the main isoform of cardiac DPP6.2.The effects of DPP6 and KCh IP2 are different.Firstly,the mode of action is different:DPP6 is involved in the regulation of post-transcriptional process of Kv4.3,which is independent of KCh IP2 and promotes the forward trafficking of Kv4.3.And DPP6 can weaken the effect of increasement of total Kv4.3 protein by KCh IP2.Secondly,the efficiency is different:the effect of DPP6 is far less than the regulation effect of KCh IP2 on the Kv4.3 channel,and can be covered up by the strong effect of KCh IP2.3.Two DPP6 mutants,DPP6-L747P and DPP6-Q526H,which may cause IVF,significantly increased the expression of Kv4.3 membrane protein compared with DPP6-WT.This may be the main mechanism of gain of function mutation...