分拣连接蛋白调控心脏电生理稳态的机制研究

The precise spatial arrangements of the ion channels in the cardiac myocyte is the foundation to maintain the electrophysiological homeostasis of the heart . The Phox homolog (PX) protein domain, which specifically binds to membrane inositol phospholipids, is the important structural base for the implementation of the directional transport of proteins within cells. Sorting connexins (SNXs) are the major family members of the molecules that contain the PX domain. We found that SNXs had differential subcellular distribution in human cardiac myocytes. Among them, sorting nexin 17 mainly located in the endosomal system of cardiac myocytes. We also found that up-regulation of SNX-17 in cardiac myocytes leaded to down-regulation of a variety of membrane ion channel proteins and transporters such as type L calcium channel and sodium hydrogen exchanger, which indicated that SNXs have the potential of multidirectional regulating cardiac myocyte membrane ion channel and transporter stability. Based on the results we have already gained, we propose to utilize various molecular biology, electrophysiology techniques and model organism zebra fish to explore the expression and subcellular localization of the SNXs molecules in the heart and then to systematically examine the mechanisms of SNXs regulating the cardiac electrophysiology homeostasis. Our research will provide new theories on cardiac electrophysiology homeostatic regulation, and offer new targets for prevention/treatment of abnormal heart rhythms.

离子通道在心肌细胞中的精准空间分布是维持心脏电生理稳态的前提。特异结合到膜肌醇磷脂的一种蛋白结构域（Phox homolog，PX）是执行蛋白定向运输功能的重要结构基础之一。分拣连接蛋白（sorting nexin，SNX）是包含PX结构域的最主要分子家族。我们发现，SNXs在人类心肌细胞中存在着差异性分布，其中SNX17主要定位在心肌细胞内小体系统。我们还发现，上调心肌细胞内SNX17可以导致多种离子通道和交换体（例如L型钙通道和钠氢交换体等）的表达水平的下降。提示SNXs具有多向性调节心肌细胞离子通道和交换体稳态的潜能。我们拟在前期研究基础之上，采用分子生物学、电生理学、和斑马鱼模式生物学等实验技术，进一步确立特定SNX分子在心肌细胞中的表达与分布特征和对心脏电活动稳态的影响，进而探索其对心脏电活动稳态的调控机制。我们的研究将为心脏电生理稳态调控提供新的视角和潜在靶标。

心肌细胞膜表面离子通道蛋白动力学稳态是心脏规律性发放与扩布电脉冲的关键与前提。分选连接蛋白是目前已知胞内蛋白质靶向运输至亚细胞与细胞膜区间的调控体系之一。我们系统分析了人类心房与心室肌组织中分选连接蛋白分子家族成员的表达模式，发现SNX13在心房与心室均高表达，具有FERM结构的SNX17在心室优势表达。通过分析SNX13与SNX17基因敲除的模式斑马鱼与模式大鼠，我们证明SNX13基因缺陷明显抑制心室的机械功能，对心脏电活动无明显影响；SNX17基因敲除导致大鼠胚胎致死，其杂合子发育正常且心脏结构与功能无明显改变。心电遥测分析发现，SNX17部分缺失大鼠基础心电活动正常，但是在应激状态下室性快速心律失常的发生明显地增加。心脏电压敏感的光标测分析提示，SNX17缺陷不改变基础心室动作电位，但明显影响心室复极化储备。单个心室肌细胞电生理参数分析显示，SNX17缺陷选择性延缓了瞬时外向钾电流的失活。细胞免疫化学与生化检测发现，SNX17缺陷导致Kv1.4通道（主要参与瞬时外向钾电流失活的通道）蛋白的膜聚集，而SNX17蛋白中的FERM结构域起着关键性调控作用。为此，我们的研究提示，分选连接蛋白在心脏电活动稳态中起着重要作用。该发现为临床心律失常的干预策略提供了新的思路与理论基础。