Dysfunction of the cystic fibrosis transmembrane con-ductance regulator(CFTR) chloride channel causes cys-tic fibrosis, while inappropriate activity of this channeloccurs in secretory diarrhea and polycystic kidney di...Dysfunction of the cystic fibrosis transmembrane con-ductance regulator(CFTR) chloride channel causes cys-tic fibrosis, while inappropriate activity of this channeloccurs in secretory diarrhea and polycystic kidney dis-ease. Drugs that interact directly with CFTR are there-fore of interest in the treatment of a number of diseasestates. This review focuses on one class of small mol-ecules that interacts directly with CFTR, namely inhibi-tors that act by directly blocking chloride movementthrough the open channel pore. In theory such com-pounds could be of use in the treatment of diarrheaand polycystic kidney disease, however in practice allknown substances acting by this mechanism to inhibitCFTR function lack either the potency or specificity forin vivo use. Nevertheless, this theoretical pharmaco-logical usefulness set the scene for the developmentof more potent, specific CFTR inhibitors. Biophysically,open channel blockers have proven most useful as ex-perimental probes of the structure and function of theCFTR chloride channel pore. Most importantly, the useof these blockers has been fundamental in developing afunctional model of the pore that includes a wide innervestibule that uses positively charged amino acid sidechains to attract both permeant and blocking anionsfrom the cell cytoplasm. CFTR channels are also subjectto this kind of blocking action by endogenous anionspresent in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physio-logical control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease states, of CFTR chan-nel structure and function, and of how CFTR activity is controlled by its local environment.展开更多
目的:探讨1,4,5-三磷酸肌醇1型受体(inositol 1,4,5-trisphosphate receptor type 1,IP3R1)调控钙/钙调蛋白依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)和电压依赖性阴离子通道1(voltage-dependent anion ...目的:探讨1,4,5-三磷酸肌醇1型受体(inositol 1,4,5-trisphosphate receptor type 1,IP3R1)调控钙/钙调蛋白依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)和电压依赖性阴离子通道1(voltage-dependent anion channel 1,VDAC1)在海洛因(heroin,HE)致心肌细胞节律异常中的作用。方法:联合蛋白组学和GEO(Gene Expression Omnibus)数据库分析心律失常芯片数据,寻找关键调控因子。构建IP3R1基因敲减慢病毒并感染原代乳大鼠心肌细胞(neonatal rat cardiomyocytes,NRCMs),实验分为对照(control)组、HE组和HE+shIP3R1组。结晶紫染色观察心肌细胞形态;ELISA法检测乳酸脱氢酶(lactate dehydrogenase,LDH)和天冬氨酸转氨酶(aspartate aminotransferase,AST)水平;透射电镜观察线粒体形态学变化;Fluo-4/AM探针法检测细胞内Ca^(2+)浓度;DCFH-DA荧光探针检测细胞内活性氧(reactive oxygen species,ROS)含量;JC-1染色法检测线粒体膜电位(mitochondrial membrane potential,MMP)水平;ATP检测试剂盒检测细胞内ATP水平;免疫共沉淀(co-immunopre-cipitation,Co-IP)分析IP3R1与CaMKⅡδ和VDAC1蛋白之间的相互作用;Western blot检测IP3R1、CaMKⅡδ、p-CaM-KⅡδ(T287)和VDAC1的蛋白水平。结果:结合蛋白质组学和基因表达谱数据集GSE89410分析,筛选得到80个差异共表达分子,基于基因本体论(Gene Ontology,GO)功能注释和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析结果,最终筛选出关键因子IP3R1,且通过STRING数据库获得IP3R1结合蛋白:CaMKⅡδ和VDAC1。Co-IP结果验证IP3R1与CaMKⅡδ和VDAC1存在相互作用,且HE干预后NRCMs中IP3R1与CaMKⅡδ和VDAC1之间的相互作用增强。体外细胞实验显示,与control组相比,HE组NRCMs数量急剧减少,细胞膜变窄,伪足减少,细胞核结构模糊;LDH和AST水平均显著上升(P<0.05);线粒体超微结构损伤严重,证实HE对NRCMs具有毒性作用并导致线粒体损伤。与control组相比,HE组心肌细胞内Ca^(2+)浓度、ROS水平、MMP以及IP3R1、p-CaMKⅡδ(T287)和VDAC1蛋白水平均显著升高(P<0.05),而HE+shIP3R1组这些指标均显著减低(P<0.05);ATP水平则相反。这证实沉默IP3R1表达可减轻HE干预后NRCMs的钙超载及线粒体损伤。结论:IP3R1通过调控CaMKⅡ和VDAC1引起心肌细胞钙超载和ROS生成增多,参与HE诱导的心肌细胞节律异常。展开更多
文摘Dysfunction of the cystic fibrosis transmembrane con-ductance regulator(CFTR) chloride channel causes cys-tic fibrosis, while inappropriate activity of this channeloccurs in secretory diarrhea and polycystic kidney dis-ease. Drugs that interact directly with CFTR are there-fore of interest in the treatment of a number of diseasestates. This review focuses on one class of small mol-ecules that interacts directly with CFTR, namely inhibi-tors that act by directly blocking chloride movementthrough the open channel pore. In theory such com-pounds could be of use in the treatment of diarrheaand polycystic kidney disease, however in practice allknown substances acting by this mechanism to inhibitCFTR function lack either the potency or specificity forin vivo use. Nevertheless, this theoretical pharmaco-logical usefulness set the scene for the developmentof more potent, specific CFTR inhibitors. Biophysically,open channel blockers have proven most useful as ex-perimental probes of the structure and function of theCFTR chloride channel pore. Most importantly, the useof these blockers has been fundamental in developing afunctional model of the pore that includes a wide innervestibule that uses positively charged amino acid sidechains to attract both permeant and blocking anionsfrom the cell cytoplasm. CFTR channels are also subjectto this kind of blocking action by endogenous anionspresent in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physio-logical control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease states, of CFTR chan-nel structure and function, and of how CFTR activity is controlled by its local environment.
