Background:The decreased slowly activating delayed rectifier K+current(IKs)is the molecular basis of arrhythmia caused by myocardial hypertrophy.The aim of this study was to investigate the mechanism of IKs down-regul...Background:The decreased slowly activating delayed rectifier K+current(IKs)is the molecular basis of arrhythmia caused by myocardial hypertrophy.The aim of this study was to investigate the mechanism of IKs down-regulation related to the channel number,as well as the regulation of channel number under pathological conditions.Methods:The HEK 293 cell co-transfected with KCNQ1/KCNE1 genes was cultured conventionally.After the cells incubated with angiotensin II(AngII)(24 h),AngII(72 h),bisindolylmaleimide I(Bis),brefeldin A and dynasore,the effect and relevant mechanism of long term incubation of AngII on the IKs tail current and KCNQ1 channel total protein were investigated by electrophysiology method and western blotting.In the experiment,the Bis,brefeldin A and dynasore could inhibit the protein kinase C(PKC)activity,the forward transport of KCNQ1 channel and the endocytosis of KCNQ1 channel,respectively.At last,the Rab GTPases 11(Rab11)dominant-negative mutant dsRed-Rab11/S25N was infected into the cells to investigate the effect and relevant mechanism of long term AngII incubation on the IKs tail current and KCNQ1 channel total protein.Results:Our results showed that the decreased IKs tail current and the KCNQ1 channel total protein caused by long term AngII incubation were attenuated by Bis treatment,which inhibited PKC activity.In addition,the inhibited IKs tail current and KCNQ1 channel total protein were also alleviated by brefeldin A and dynasore treatment.At last,the expression of Rab11 dominant-negative mutant dsRed-Rab11/S25N could weak the inhibition of IKs tail current and the KCNQ1 channel total protein caused by long term AngII incubation.Conclusion:The long term incubation of AngII inhibited the IKs tail current and KCNQ1 channel total protein was achieved by PKC activation and the disorder of the channel trafficking by Rab11.展开更多
The KCNQ potassium channels play a crucial role in modulating neural excitability,and their dysfunction is closely associated with epileptic disorders.While variants in KCNQ2 have been extensively studied,KCNQ3-relate...The KCNQ potassium channels play a crucial role in modulating neural excitability,and their dysfunction is closely associated with epileptic disorders.While variants in KCNQ2 have been extensively studied,KCNQ3-related disorders have rarely been reported.With advances in next-generation sequencing technologies,an increasing number of cases of KCNQ3-related disorders have been identified.However,the correlation between genotype and phenotype remains poorly understood.In this study,we established a variant library consisting of 24 missense mutations in KCNQ3 and introduced these mutations into three different template types:KCNQ3,KCNQ3-A315T(Q3^(*)),and KCNQ3-KCNQ2 tandem(Q3-Q2).We then analyzed the effects of these mutations on the KCNQ3 channel function using patch-clamp recording.The most informative parameter across all three backgrounds was the current density of the mutant channels.The current density patterns in the Q3^(*)and Q3-Q2 backgrounds were similar,with most mutations resulting in an almost complete loss of function(LOF),they were concentrated in the pore-forming domain of KCNQ3.In contrast,mutations in the voltage-sensing domain or C-terminus did not show significant differences from the wild-type channel.Interestingly,these LOF mutations were typically associated with self-limited familial neonatal epilepsy,while neurodevelopmental disorders(NDD)were more closely associated with mutations that did not significantly differ from the wild-type.V1/2,another important parameter of the electrophysiological properties,could not be accurately determined in the majority of KCNQ3 mutations due to its nearly complete LOF in the Q3^(*)and Q3-Q2 backgrounds.Intriguingly,the V1/2 of functional mutations were primarily leftward shifted,indicating a gain-of-function(GOF)effect,which was typically associated with NDD.In addition to previously reported mutations,we identified G553R as a novel GOF mutation.In the co-transfection background,parameters such as V1/2 could be determined,but the dysfunctional effects of these mutations were mitigated by the co-expression of wild-type KCNQ3 and KCNQ2 subunits,resulting in no significant differences between most mutations and the wild-type channel.Furthermore,we applied KCNQ modulators to reverse the electrophysiological abnormalities caused by KCNQ3 variants.The LOF mutations were reversed by the application of Pynegabine(HN37),a KCNQ opener,while the GOF mutation responded well to Amitriptyline(AMI),a KCNQ inhibitor.These findings provide essential insights into the pathogenic mechanisms underlying KCNQ3-related disorders and may inform clinical decision-making.展开更多
文摘长非编码RNA KCNQ1OT1对多种癌症的发生发展中起着重要的促进作用。然而,目前还没有研究在泛癌中对KCNQ1OT1进行系统的分析。本研究通过对KCNQ1OT1在泛癌组织中的表达水平以及对肿瘤患者的预后情况分析,阐明KCNQ1OT1在肿瘤诊断和预后中的价值,通过分析KCNQ1OT1在胃癌中的调控机制,为胃癌的诊疗提供新的分子靶点。使用Sangerbox 3.0、临床生信之家以及UALCAN数据库,发现KCNQ1OT1在7种肿瘤组织中表达增高(均P<0.05)。在Sangerbox 3.0数据库中发现KCNQ1OT1与多种肿瘤的预后不良相关。使用R软件分析胃癌患者中KCNQ1OT1高表达组和低表达组的差异基因(P<0.05,log2FoldChange>1),并使用基因本体(gene ontology,GO)和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)功能富集分析发现,KCNQ1OT1参与了胃癌谷氨酰胺的代谢过程。细胞计数和Western印迹检测发现,敲低KCNQ1OT1后胃癌细胞的活性、SLC1A5表达水平以及其介导的谷氨酰胺转运过程均显著下降(P<0.01)。生物信息学、RNA免疫沉淀和双荧光素酶分析验证了KCNQ1OT1竞争性结合miR-138-5p,并促进SLC1A5的表达。最后,染色质免疫沉淀测序数据检测KCNQ1OT1的基因位点具有高H3K27ac信号,并通过染色质免疫沉淀定量PCR验证了P300介导的增强子活性调控了胃癌中KCNQ1OT1的高表达。KCNQ1OT1在多种肿瘤中可以作为一种独立的诊断标志物和预后预测因子。靶向KCNQ1OT1/miR-138-5p/SLC1A5信号轴调控的谷氨酰胺代谢,为胃癌的治疗提供了新的策略和分子靶点。
基金supported by the National Natural Science Foundation of China(NSFC)Youth Project(No.82204397 and No.22203063).
文摘Background:The decreased slowly activating delayed rectifier K+current(IKs)is the molecular basis of arrhythmia caused by myocardial hypertrophy.The aim of this study was to investigate the mechanism of IKs down-regulation related to the channel number,as well as the regulation of channel number under pathological conditions.Methods:The HEK 293 cell co-transfected with KCNQ1/KCNE1 genes was cultured conventionally.After the cells incubated with angiotensin II(AngII)(24 h),AngII(72 h),bisindolylmaleimide I(Bis),brefeldin A and dynasore,the effect and relevant mechanism of long term incubation of AngII on the IKs tail current and KCNQ1 channel total protein were investigated by electrophysiology method and western blotting.In the experiment,the Bis,brefeldin A and dynasore could inhibit the protein kinase C(PKC)activity,the forward transport of KCNQ1 channel and the endocytosis of KCNQ1 channel,respectively.At last,the Rab GTPases 11(Rab11)dominant-negative mutant dsRed-Rab11/S25N was infected into the cells to investigate the effect and relevant mechanism of long term AngII incubation on the IKs tail current and KCNQ1 channel total protein.Results:Our results showed that the decreased IKs tail current and the KCNQ1 channel total protein caused by long term AngII incubation were attenuated by Bis treatment,which inhibited PKC activity.In addition,the inhibited IKs tail current and KCNQ1 channel total protein were also alleviated by brefeldin A and dynasore treatment.At last,the expression of Rab11 dominant-negative mutant dsRed-Rab11/S25N could weak the inhibition of IKs tail current and the KCNQ1 channel total protein caused by long term AngII incubation.Conclusion:The long term incubation of AngII inhibited the IKs tail current and KCNQ1 channel total protein was achieved by PKC activation and the disorder of the channel trafficking by Rab11.
文摘The KCNQ potassium channels play a crucial role in modulating neural excitability,and their dysfunction is closely associated with epileptic disorders.While variants in KCNQ2 have been extensively studied,KCNQ3-related disorders have rarely been reported.With advances in next-generation sequencing technologies,an increasing number of cases of KCNQ3-related disorders have been identified.However,the correlation between genotype and phenotype remains poorly understood.In this study,we established a variant library consisting of 24 missense mutations in KCNQ3 and introduced these mutations into three different template types:KCNQ3,KCNQ3-A315T(Q3^(*)),and KCNQ3-KCNQ2 tandem(Q3-Q2).We then analyzed the effects of these mutations on the KCNQ3 channel function using patch-clamp recording.The most informative parameter across all three backgrounds was the current density of the mutant channels.The current density patterns in the Q3^(*)and Q3-Q2 backgrounds were similar,with most mutations resulting in an almost complete loss of function(LOF),they were concentrated in the pore-forming domain of KCNQ3.In contrast,mutations in the voltage-sensing domain or C-terminus did not show significant differences from the wild-type channel.Interestingly,these LOF mutations were typically associated with self-limited familial neonatal epilepsy,while neurodevelopmental disorders(NDD)were more closely associated with mutations that did not significantly differ from the wild-type.V1/2,another important parameter of the electrophysiological properties,could not be accurately determined in the majority of KCNQ3 mutations due to its nearly complete LOF in the Q3^(*)and Q3-Q2 backgrounds.Intriguingly,the V1/2 of functional mutations were primarily leftward shifted,indicating a gain-of-function(GOF)effect,which was typically associated with NDD.In addition to previously reported mutations,we identified G553R as a novel GOF mutation.In the co-transfection background,parameters such as V1/2 could be determined,but the dysfunctional effects of these mutations were mitigated by the co-expression of wild-type KCNQ3 and KCNQ2 subunits,resulting in no significant differences between most mutations and the wild-type channel.Furthermore,we applied KCNQ modulators to reverse the electrophysiological abnormalities caused by KCNQ3 variants.The LOF mutations were reversed by the application of Pynegabine(HN37),a KCNQ opener,while the GOF mutation responded well to Amitriptyline(AMI),a KCNQ inhibitor.These findings provide essential insights into the pathogenic mechanisms underlying KCNQ3-related disorders and may inform clinical decision-making.
