南开大学生命科学学院,南开大学物理学院,南开大学生命科学学院,南开大学生命科学学院,南开大学生命科学学院,南开大学物理学院,天津师范大学生命科学学院,天津市动植物抗性重点实验室
国家自然科学基金资助项目(31071714,31371974),天津市动植物抗性重点实验室开放基金资助项目
College of Life Sciences,Nankai University,School of Physics,Nankai University,College of Life Sciences,Nankai University,College of Life Sciences,Nankai University,College of Life Sciences,Nankai University,School of Physics,Nankai University,Tianjin Key Laboratory of Animal and Plant Resistance,College of Life Sciences,Tianjin Normal University
This work was supported by grants from The National Natural Science Foundation of China (31371974, 31071714) and The Open Fund of Tianjin Key Laboratory of Animal and Plant Resistance
T型钙通道(Cav3)广泛分布于各类细胞,其显著的电生理学特点是低电位激活和快速的电压依赖性失活.失活在通道的生理功能调节中起十分重要的作用,但具体参与通道失活的分子基础目前并不完全清楚.为明确Cav3.1通道中调控电压依赖性失活的结构域,用Cav1.2通道(无电压依赖性失活)结构域Ⅰ和Ⅱ中的S1~S4、S5~S6区及Ⅰ和Ⅱ间的联系区替换Cav3.1中的相应区域,构建嵌合通道,并在卵母细胞中表达,用电压钳技术分析通道的电生理学特性.结果表明,替换Ⅰ中的S1~S4或S5~S6区可使Cav3.1的失活特性显著改变,但这种改变主要是由激活-失活偶联所致.Ⅱ的替换使通道的失活曲线参数发生显著改变,表明结构域Ⅱ,包括S1~S4和S5~S6均参与Cav3.1失活过程的调控.Ⅰ、Ⅱ间的联系区及Ⅰ中的S5~S6主要调控Cav3.1的失活速率,Ⅰ和Ⅱ中的S1~S4对通道失活速率无影响.综上所述,结构域Ⅱ是调控Cav3.1电压依赖性失活的关键因素,结构域Ⅰ不参与该通道失活过程的调控.Ⅰ、Ⅱ间的联系区及Ⅰ中的S5~S6主要调控Cav3.1通道的失活速率,Ⅰ、Ⅱ中的S1~S4对通道失活速率无影响.
The notable features for inactivation of Cav3 channels are fast inactivating rate and strong voltage-dependence. We have investigated the molecular basis for determining the voltage-dependence of inactivation for Cav3.1, focusing on domain Ⅰ and Ⅱ. We made chimeras between Cav3.1 and Cav1.2. Chimeras were expressed in oocytes and currents were recorded by voltage clamp. For domain Ⅰ, replacement of S1~S4 or S5~S6 shifted the steady state inactivation curve significantly. These changes were mainly or partially caused by activation-inactivation coupling, rather than molecular modification. Replacement of domain Ⅱ shifted the inactivation curve significantly and these changes refer to molecular modification, indicating that domain Ⅱ contributed to the voltage-dependence of inactivation for Cav3.1. Furthermore, both voltage sensor region S1~S4 and pore region S5~S6 in domain Ⅱ were also involved, but Ⅰ-Ⅱ linker has no contribution. In addition, we found that the Ⅰ-Ⅱ linker and S5~S6 in domain Ⅰ contributed strongly to inactivation rate for Cav3.1, while S1~S4 in domain Ⅰ and Ⅱ was not involved. Taken collectively, our results suggest that domain Ⅱ plays a key role in determining the voltage-dependence of inactivation for Cav3.1, which was different from the molecular determinants for inactivating rate and for voltage-dependence of activation.
贺秉军,胡芬,商学良,韩丽鑫,吴广彦,李俊英,孙金生.调控T型钙通道Cav3.1电压依赖性失活的分子结构域[J].生物化学与生物物理进展,2014,41(9):877-886
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