GBF1调控Nav1.5转运的分子机制及其纠正Nav1.5转运障碍突变体功能的机制研究

心脏钠通道Nav1.5转运障碍是导致心律失常的重要机制。但Nav1.5从内质网输出后转运途径不清，尚缺乏纠正Nav1.5转运障碍突变体功能的广谱措施。鸟苷酸交换因子GBF1调控COPI囊泡形成，促进蛋白向高尔基体转运。我们发现上调GBF1增加野生型及不同Nav1.5转运障碍突变体在细胞膜的分布及电流，因此假设GBF1通过COPI囊泡形成促进Nav1.5向高尔基体及在高尔基体内部转运及折叠，是纠正Nav1.5转运障碍突变体功能的广谱关键分子。本研究拟以iPSC-CMs和Nav1.5-HEK细胞为对象，通过分子生物学及膜片钳等方法阐明GBF1调控Nav1.5转运及折叠的分子机制，探索Nav1.5结构与其转运途径的关系；构建Nav1.5基因突变小鼠及多种Nav1.5转运障碍突变体，验证GBF1纠正Nav1.5转运障碍突变体功能的广谱作用及分子机制。预期结果将为心脏钠通道病的防治提供新的干预靶点。

Defects in cell surface trafficking of cardiac voltage-gated sodium channel type 5 (Nav1.5) have been demonstrated to be an important molecular mechanism underlying the pathogenesis of varies congenital and acquired arrhythmias. However, the mechanism of how Nav1.5 traffic from endoplasmic reticulum (ER) to Golgi apparatus (GA) and within Golgi is not clear. And also, no effective treatments are available to restore the function of Nav1.5 defective-trafficking mutant. Guanine-nucleotide exchange factor GBF1 contributes to the formation of COPI vesicles and thus promotes protein traffic from ER to GA. We found that GBF1 could increase the expression and sodium current of wild-type and defective-trafficking Nav1.5 mutants. Therefore, we hypothesize that GBF1 promotes the transport of wild-type Nav1.5 from ER to GA and rescues the function of defective-trafficking Nav1.5 mutants. Nav1.5-HEK cells and human induced pluripotent stem cell derived cardiomyocytes were used as experimental models. Using molecular biology, patch clamp and multi-electrode recording techniques, to elucidate the underlying molecular mechanism of GBF1 promoting Nav1.5 transport and folding, and then, to further explore the how the molecular structure of Nav1.5 regulate its intracellular transport. Next, the SCN5a gene mutation mouse model and different defective-trafficking Nav1.5 mutants were used to test the effect of GBF1 on these Nav1.5 mutant and the underlying molecular mechanism. Upon conducting the proposal successfully, we expected to explore the underlying mechanisms of Nav1.5 trafficking and optimize therapy target for cardiac sodium channel disease.