The Mechanisms Of Ubiquitin-like Protein FAT10 Regulates Potassium Channel Kv7.1

Background:Kv7.1 is a type of voltage-dependent potassium channel,encoded by the KCNQ1gene,assembles with KCNE1 to form a channel complex which constitutes the slow component of the delayed rectifier current I_Ks(slow delayed rectifier potassium current,I_Ks).I_Kss plays an important role in the repolarization of action potential?AP?.Studies have reported that the enhancement and reduction of Kv7.1 channel are both to induce arrhythmias.Therefore,the stability of Kv7.1 is crucial.FAT10?F-AdjacentTranscript-10,FAT10?is a newly discovered ubiquitin-like protein which involved in the translation and modification of many proteins.For the first time,our team demonstrated that FAT10 stabilizes the expression of substrate proteins by antagonizing the level of ubiquitination.However,the effection of FAT10 on regulation of Kv7.1 remains largely unknown.Objective:The objective of this study is to investigate whether FAT10 is involved in regulation of Kv7.1 function,and to further clarify the specific molecular mechanisms.Methods:1.The FAT10 overexpression and interference adenovirus were used as the gain-of-functional and loss-of-functional approaches to study the effects of FAT10 in NRCMs?neonatal rat cardiomyocytes,NRCMs?.2.To determine the effection of FAT10 on regulation of Kv7.1:The protein synthesis inhibitor CHX and the proteasome inhibitor MG132 were used to determine whether Kv7.1 was degraded by ubiquitination.The effects of FAT10 on the ubiquitination of Kv7.1 in cardiomyocytes were detected by WB and Co-IP experiments.Results:1.Overexpression of FAT10 increased Kv7.1 expression,while interference with FAT10 decreased Kv7.1 expression.Overexpression and interference with FAT10,Western Blot method detected Kv7.1 protein level was positively correlated with FAT10.2.FAT10 interacted with Kv7.1To further clarify the regulation of Kv7.1 by FAT10,co-immunoprecipitation?co-IP?and immunofluorescence was used to confirm the endogenous interaction of FAT10 and Kv7.1.3.Kv7.1 was degraded by the ubiquitin–proteasome system?UPS?In NRCMs,we found after the treatment of cycloheximide CHX,western blot results showed a decrease in Kv7.1 expression;however,when cells treated together with proteasome inhibitor?MG132?and CHX,western blot results showed no significant changes in Kv7.1.In addition,after interfering with ubiquitin?Ub?,Kv7.1expression increased,while in the MG132 treatment group,Kv7.1 expression did not change significantly after interfering with Ub.These findings indicated that Kv7.1was modified by ubiquitin.4.FAT10 inhibited the ubiquitination of Kv7.1Co-IP results showed that Ub-Kv7.1 conplexes expression decreased after overexpression of FAT10,and conversely,Ub-Kv7.1 conplexes expression increased after interference with FAT10.Conclusion:1.FAT10 is involved in regulation Kv7.1 protein expression2.Kv7.1 is degraded in cardiomyocytes by the ubiquitin–proteasome system?UPS?3.FAT10 stabilizes Kv7.1 expression by antagonizing its ubiquitination-mediated degradation in cardiomyocytes.