摘要
目的采用基因沉默技术抑制HL-1细胞的桥粒蛋白(DSP)基因表达以明确DSP与Nav1.5的结构和功能关系。方法用基因沉默技术抑制DSP基因的表达,然后采用Western blotting检测HL-1细胞DSP和Nav1.5蛋白的表达,用双免疫荧光方法检测DSP与Nav1.5蛋白的表达与定位情况,并用全细胞膜片钳技术检测细胞钠通道的电生理特征。结果与空白组和对照组相比,siRNA-DSP组的DSP和Nav1.5蛋白表达量降低。免疫荧光检测发现空白组和对照组DSP与Nav1.5蛋白存在共定位情况,而siRNA-DSP组DSP和Cx43蛋白共定位则遭到破坏,并且膜片钳检测发现siRNA-DSP组峰值电流从(156.3±6.2)pA/pF减少至(41.8±3.1)pA/pF(P<0.05),电压依赖的失活曲线V0.5从-42 mV左移至-61 mV(P<0.05)和从失活恢复时间延长。结论 DSP表达抑制不仅使DSP与Nav1.5的共定位特征遭到破坏,而且还改变了Nav1.5电生理特征。
Objective To investigate the association of desmoplakin with the distribution and function of Nav 1.5 by RNA silencing technology in HL-1 cells. Methods HL-1 cells with desmoplakin expression suppression by RNA silencing were examined for desmoplakin and Nav 1.5 protein expressions by Western blotting, and the distribution and co- location of desmoplakin and Nav 1.5 protein were detected by immunoflurescence staining. Patch-clamp recording was applied to analyze the changes in whole- cell sodium current after desmoplakin silencing. Results Compared with the untreated group and negative control group, the cells with desmoplakin silencing showed obviously reduced expressions of desmoplakin and Nav 1.5 proteins. Co- localization of desmoplakin and Nav 1.5 was detected at cell- cell contact in untreated and control conditions, and desmoplakin expression silencing induced a drastic redistribution of Nav 1.5 with decreased peak current density （156.3±6.2 vs 41.8±3.1, n=6, P0.05）, a shift in voltage dependence of steady-state inactivation （-42 mV vs -61 mV, n=5, P 0.05）, and prolonged time of recovery from inactivation. Conclusion Desmoplakin silencing caused redistribution of Nav 1.5 protein and also changes in its electrophysiological properties in HL-1 cells.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2013年第7期983-989,共7页
Journal of Southern Medical University
基金
广东省自然科学基金(10151008002000011)
