Junctophilin-MORN 结构域与心肌小电导Ca2+激活K+通道功能性耦联的研究

Small conductance Ca2+-activated K+ channels (SK) are recognized as a novel ion channel candidate in atrial fibrillation (AF) and involved in electrical remodeling of AF. Searching for molecular modulations of SK channels have important implication in our understandings of pathphysiology for arrhythmias. Junctophilin (JP), a protein contributed to the formation of the junctional membrane complexes, is essential for stabilizing the close apposition of the plasma membrane and the sarcoplasmic reticulum membrane in excitable cells. We have previously documented the interaction between SK2 channel and JP2 by the binding assay in vitro. Furthermore, the interaction was also proved by coimmunoprecipitation in vivo combing with MALDI–TOF mass spectrometry analysis. However, molecular mechanism and biological roles underlying the coupling of JP2 to the SK2 channel are unknown. .JP contains MORN motifs (membrane occupation and recognition nexus) at its N-terminal cytoplasm region that is thought to interact specifically with the plasma membrane/surface membrane. Till now no report has showed a coupling of SK2 channel with JP2 protein via JP2-MORN motifs in the cardiac muscle yet. In this present study we will define that the JP2-MORN motif is the critical domain of JP2 interaction the SK channels in the myocardium using Yeast two-hybrid assay in vitro combing with Site-Directed Mutagenesis and document the interaction between JP2-MORN motif with the SK2 channel. Next, we'll determine that the cell-surface expression of the SK2 channel is regulated by its interaction with JP2-MORN motif using TIRF microscopic imaging, Cell-Surface Biotinylation and confocal microscopy. Moreover, we will examine mRNA and protein levels of SK2 in the cardiomyocytes treated with adenovirus -mediated siRNA targeted against JP2-MORN motif by qPCR and Western blot analysis. Finally, we'll further test the role of JP2-MORN knockdown on the SK2 channel mediated Ca2+-activated K+ current in the cardiomyocytes transduced with adenovirus -mediated siRNA specific to JP2-MORN motif using a whole-cell patch clamp technique. Our study will present functional interaction of JP2-MORN motif with the SK2 channel in the cardiac myocytes. These studies will reveal molecular mechanisms of junctophilin modulation the SK2 channel in the myocardium. The new insights may provide drug targets for the treatment of arrhythmias.

心肌小电导Ca2+激活K+通道（SK）涉及房颤的电重构机制。研究SK通道的分子调控对揭示心律失常的病理机制有重要的科学意义。我们前期研究表明具有耦联胞浆膜与肌质网膜特性的Junctophilin2(JP2)与胞浆膜SK2通道具有相互作用，但不清楚其分子基础及生物效应。JP-MORN结构域被预测为与细胞胞浆膜结合的部位，JP2与SK2通道的相互作用是否与JP-MORN结构域相关，国内外尚未见报道。本项目拟在前期工作基础上鉴定JP-MORN结构域是否为JP调控心肌SK通道的关键位点:1.研究SK2与JP2相互作用的关键位点；2.研究JP2-MORN结构域与SK2的相互作用；3.研究JP2-MORN结构域在SK2通道靶向膜表达中的作用；4.研究心肌细胞JP2-MORN结构域敲减对心肌SK2通道表达及功能的影响。从理论上阐明JP2调控心肌SK通道的分子机制，为心律失常的防治和药物筛选提供新靶点。

本项目在前期阐明小鼠心肌细胞JP2与SK2有相互作用的基础上，进一步探讨JP2与SK2相互作用的结构域，以及JP2对SK2调节的分子机制。根据GenBank 中小鼠 JP2 和SK2基因序列，将JP2分为三个片段，即JP2-N1 (aa: 1-253，含JP2-N端MORN1结构域)， JP2-N2（aa: 216-350，含MORN2结构域）和 JP2-C（aa: 340-696，含JP2-C端）；将SK2分为SK2-N（aa: 1-145）、SK2-M（aa: 141-390）和 SK2-C（aa: 380-574）三个片段；免疫共沉淀和GST pulldown实验显示包含JP2 MORN1 结构域的JP2-N1与SK2-C之间有相互作用。为了进一步确定JP2 MORN1区对细胞膜上SK2通道表达的影响，我们在JP2 MORN1区选择N101R和Y141H两个位点进行突变，建立SK2、SK2+JP2-N1和SK2+JP2-N1mut HEK293稳转细胞株，经表面-生物素标记、细胞免疫荧光组织化学和全内反射荧光显微镜技术揭示JP2 MORN1 结构域有助于SK2通道蛋白转运到细胞膜表面或减少它的逆向转运；并且JP2 MORN1突变可能会减少JP2和SK2的相互作用；又经siRNA敲减H9c2心肌细胞株JP2的表达也揭示SK2通道膜表达和定位减少，而不影响总蛋白表达。我们围绕JP2可能是通过影响SK2的转运影响膜上的表达和定位。在转染SK2+JP2-N1mut质粒的HEK293细胞，SK2与早期核小体标记物EEA1、Rab5有明显共定位，而与高尔基体标记物GM130共定位较弱；同时，用primaquine选择性抑制离子通道转运的循环途径，结果均提示JP2异常通过影响SK2通道的内吞循环转运途径而使SK2通道膜蛋白表达减少。在这些研究基础上，我们进一步研究病理情况下两种蛋白之间的相互作用，测定出房颤患者右心耳JP2和SK2蛋白表达均减少，用血管紧张素Ⅱ诱导建立的H9c2心肌细胞肥厚模型SK2通道电流增多，敲减肥厚心肌细胞JP2表达进一步增加电流密度，而过表达JP2导致SK2通道电流减少。这些研究成果为揭示心脏疾病的发生机制开辟了新途径，提供新视点，为心脏疾病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。