Junctophilin在小电导Ca2+-激活K+通道与RyR受体功能耦联中的作用

Dysfunction in small conductance Ca2+-activated K+ channels (SK) is close associated with atrial arrhythmias. Searching for molecular modulations of SK channels have important implication in our understandings of inducibility for arrhythmias. Our previous study demenstrated a functional modulation of ryanodine receptor type 2 (RyR2) on the SK2 channel in mouse cardiac myocytes. However, molecular mechanisms underlying the coupling of the SK2 channel to RyR2 are not known. Our newly data show that both SK2 protein and RyR2 form immunocomplexes with junctophilin2（JPH2）that is component of the cardiac junctional complexes ，in native mouse atria by co-immunoprecipitation assays.Till now no report has showed functional coupling of SK2 channel with RyR2 Ca2+ release channels via JPHs in the cardiac muscle yet. In this present study we'll directly documente interactions between JPH2 and these two proteins of SK2 and RyR2. Next,we'll determine whether JPH2 could aid in the proper membrane localization of SK2 channels and contribute RyR2-sensitive Ca2+ release to calcium homeostasis. Moreover, we will examine mRNA and protein levels of SK2 and RyR2 in culture mouse neonatal cardiomyocytes treated with lentivirus-mediated siRNA specific to JPH2. Finally, we will investigate knockdown of JPH2 on functional crosstalk between SK2 channel and RyR2 in the cardiac myocytes. Our study will present those roles of junctophilin2 on coupling of SK2 channel with RyR2 in the cardiac myocytes. These studies will reveal molecular mechanisms of the coupling of SK2 channel with RyR2-sensitive Ca2+ release channel via junctophilin. The new insights may provide drug targets for the treatment of atrial arrhythmias.

小电导Ca2+激活K+通道（SK）功能异常与心律失常的发生密切相关。研究SK通道的分子调控对揭示心律失常的发生机制有重要的科学意义。我们前期工作表明RyR2对心肌SK2通道具有调控作用，但不清楚其机理。预实验提示耦联胞浆膜与肌质网膜的Junctophilin2（JPH2）蛋白与心肌RyR2和SK2可形成免疫共沉淀。RyR2对心肌SK2调控是否与JPHs相关，国内外尚未见报道。本项目拟在此基础上深入研究JPH在心肌SK通道与RyRs功能耦联中的作用:1.研究心肌JPH2与SK2、RyR2蛋白交联及相互作用；2.研究JPH2与SK2通道膜表面定位、与RyR2Ca2+释放的生理性相关；3.研究心肌细胞JPH2敲减与心肌细胞SK2和RyR2表达;4.研究JPH2敲减对SK2与RyR2功能耦联的影响。从理论上阐明JPH是心肌SK通道与RyRs功能耦联的关键分子，为心律失常的防治和药物筛选提供新靶点。

本项目研究了心肌JP2与SK2、JP2与RyR2相互作用；JP2与SK2通道表面定位、JP2与RyR2 Ca2+释放的生理性相关；研究了JP2敲减对心肌细胞SK2和RyR2表达及功能的影响。经蛋白质谱分析表明心肌JP2是与SK2、RyR2等的相关蛋白；经体内和体外实验首次证明了小鼠内源性心肌组织JP2与SK2通道、JP2与RyR2具有相互作用；初步阐明JP2-N-末端MORN motifs与SK2相互作用的结构域、JP2-C-末端跨膜区与RyR2特异性相互作用的结构域；共聚焦显微镜显示小鼠心肌细胞JP2与SK2、JP2与RyR2存在共定位；表面-生物素标记结合共聚焦显微镜证明JP2可明显增加SK2通道表面膜表达和膜定位；通过构建靶向JP2siRNA缐病毒载体, 经Real-time PCR 和Western blot证明JP2 沉默可明显抑制心肌细胞JP2表达；功能性实验证明JP2敲减明显抑制心肌细胞SK2通道电流，也明显抑制RyR2对SK2通道的调控作用。Ca2+ image表明JP2敲减明显降低细胞内Ca2+瞬变和细胞内Ca2+含量。简言之，我们的研究围绕与Ca2+信号相关的离子通道及膜耦联复合体蛋白之间的相互作用及功能调控，揭示了JP2是心肌SK2通道和RyR2的重要相关蛋白，JP2与SK2通道的功能耦联有助于SK2通道在细胞膜的定位，JP2与RyR2的功能耦联可调节细胞内Ca2+信号，从而涉及心肌兴奋-收缩耦联机制，参与心肌SK通道的功能调控。经本项目研究从理论上提出JP2是心肌SK2通道调控的关键分子，在心肌SK2通道与RyR Ca2+ 释放通道功能耦联中具有重要作用。研究成果为心律失常的发生机制提供了新的视点，为心律失常防治策略和药物靶点的筛选提供重要的学术观点和实验依据，具有明显的学术价值和临床意义。