Nav1.5钠离子通道P环E375残基侧链负电荷决定通道的电导与稳态失活  

Negative Charge of Residue E375 in the P-Loop of Sodium Channel Nav1.5 Determines Channel Conductance and Steady-State Inactivation

作　　者：胡振盛[1] 郭佳[1] 陈文[1] 庄重[1] 赵钟钟[1,2] 张朝[1,2] 

机构地区：[1]江苏省分子医药生物技术重点实验室南京师范大学生命科学学院,南京210046 [2]江苏省医药超分子材料及应用重点实验室,南京210046

出　　处：《生物物理学报》2011年第12期1045-1053,共9页Acta Biophysica Sinica

基　　金：南京师范大学优秀人才启动基金项目(2007104GJBO117);江苏高校优势学科建设工程项目(164320H106);国家自然科学基金项目(30570662;30871228)

摘　　要：钠离子通道结构域中的P环在通道的门控和离子选择性中起着重要作用。本文采用基因定点突变、电生理、定量PCR和Western bloting等实验技术,将钠离子通道Nav1.5结构域I中P环375位高度保守的负电性谷氨酸残基(E375),分别突变为电中性的丙氨酸(A)、正电性的赖氨酸(K)和负电性的天冬氨酸(D),记录不同钠通道突变体在HEK293细胞中表达后的全细胞电流,并对其动力学性质进行了分析。结果显示,E375A和E375K突变体的全细胞钠电流显著减小,稳态激活曲线和稳态失活曲线发生改变;而E375D突变体的电流和稳态失活曲线则恢复至接近野生型的状态。上述结果表明,E375残基侧链所带负电荷对钠离子通道的电导和稳态失活过程起决定作用。P-loops in the domains of sodium channel play an essential role in channel gating and ion selectivity. Using combined techniques of site-directed mutagenesis and electrophysiology, a negatively charged residue Glu （E375）, conservatively located in P-loop of domain I of sodium channel Navl.5, was mutated to a neutrally charged residue Ala （A）, a positively charged residue Lys （K） and another negatively charged residue Asp （D）, individually. Whole-cell currents were recorded and analyzed from HEK293 cells transfected with wild-type or mutant channels. Results demonstrated that E375A and E375K mutations caused dramatic current reduction, a leftward shift in steady-state activation and a rightward shift in steady-state inactivation curves as compared to wild-type channel, while E375D mutation restored the amplitude of current and steady-state inactivation kinetics. These demonstrate that the negative charge of residue E375 determines the conductance and steady-state inactivation of sodium channel Navl.5.

关 键 词：钠离子通道 基因定点突变 门控 稳态失活 

分 类 号：Q735[生物学—分子生物学]