【中文摘要】 当人体处于极端环境和病理生理状态下,体内的氧化还原平衡被打破,氧化水平升高,可以形成胞内局部的氧化胁迫环境。研究表明一些心肌和骨骼肌疾病都与肌质网膜的离子通道的氧化损伤有关。而肌质网肌质网(sarcoplasmicreticulum,SR)中的钙释放通道(ryanodine receptor,RyR)富含巯基,对氧化还原环境异常敏感,是氧化胁迫的作用目标。前期工作表明,在氧化胁迫条件下RyR被严重抑制,蛋白内的自由巯基数量剧减。钙释放通道RyR的功能状态与其它膜蛋白和RyR之间的相互作用密切相关,因此,我们选择蛋白间的相互作用作为主要研究内容,初步探讨氧化胁迫对RyR的影响如何和造成这种影响的机制。本研究以氧化胁迫浓度下的Na_2SeO_3和1,4NQ模拟氧化胁迫环境,以兔骨骼肌肌质网(SR)囊泡和钙释放通道(RyR1)为研究对象,利用【3H】-ryanodine结合实验,SDS-PAGE和Western Blotting,光子相干光谱法(PCS)和DPH荧光偏振法,考察氧化胁迫条件下氧化型通道调控剂对RyR1通道活性,SR蛋白分布,RyR1颗粒粒径分布和SR膜流动性的影响。实验表明,...更多氧化胁迫浓度的Na_2SeO_3和1,4NQ的预处理导致了RyR1通道活性和SR膜的流动性降低;并导致了SR膜蛋白交联,形成大分子交联复合物电泳条带并被DTT逆转;RyR、钙泵、myosin和钙结合蛋白等蛋白参与了复合物的形成;进而发现,Na_2SeO_3和1,4NQ处理使得RyR蛋白之间形成了大粒径的聚合物,导致RyR的平均粒径增大,而DTT也可以部分逆转这种粒径的增大。上述结果显示,氧化胁迫浓度的氧化剂对RyR1通道的抑制作用,可能是由于氧化了负责关闭通道的职能巯基群,并由此产生了蛋白间错误交联而形成蛋白异常堆积,因此可能改变了钙释放通道的功能结构;SR膜流动性的下降,也可能诱导了蛋白和脂质分子、以及脂质分子间的交联,由此也可能导致蛋白变构的灵活性下降。另外所导致的RyR之间相互连接的增强也可能影响RyR之间的正常的协同性。 

【英文摘要】 When human was in extreme environment under pathophysiological conditions,the redox of the body was elevated.Then regional oxidative stress followed intracellularly. Researches showed that some myocardial and skeletal muscle diseases were related to the oxidative damage of the ion channels on sarcoplasmic reticulum.The calcium release channel(ryanodine receptor,RyR) in sarcoplasmic reticulum was rich in sulfhydryls.As the target of oxidative stress,the calcium release channel was very sensitive to redox environment.Previous researches showed that RyRs were severely inhabited under oxidative stress conditions and the number of sulfhydryls greatly decreased.The function of RyRs was closely related to the interaction of RyR and other membrane proteins.Thus,we chose the interaction between those proteins as our main study to explore the influences of the oxidative stress to RyRs as well as its mechanism.In this study,high concentration of 1,4 NQ and Na_2SeO_3 was used to simulate oxidative... stress,and the vesicle of sarcoplasmic reticulum(SR) and the calcium release channel(RyRl) of rabbit skeletal muscle were chosen as research objects. Many methods were used including[3H]-ryanodine binding assay,SDS-PAGE and western blotting,photon correlation spectroscopy(PCS) and DPH fluorescence polarization to explore the influences of oxidation modulators on the channel activity and the particle size of RyRl,the distribution of SR protein,and fluidity of SR membrane under oxidative stress.The activity of RyRl channel and the fluidity of SR membrane were lowered by high concentration of 1.4-NQ and Na_2SeO_3.Some macromolecular cross-linked complexes consisting of RyR,Ca2+ and myosin protein emerged on the SR membrane. More researches showed that DTT can decompose the cross-linked complexes. Na_2SeO_3 and 1,4 NQ led to cross-linked polymers of the RyRls,and increased the average particle size,while DTT can partially decompose the polymers.Above results showed that the inhibition of RyRl channel caused by the high concentration of oxidant modulators was probably due to the functional sulfhydryls which are responsible for the closure of the channels were oxidized,and the ensued incorrect cross-linking between proteins changed the structure of the calcium release channel.Furthermore,the enhancing interaction between RyRs probably affected the cooperativity of RyRs. 

【中文关键词】 氧化调控剂; 钙释放通道; 巯基; 大分子交联复合物; 氧化胁迫
【英文关键词】 oxidation modulator; calcium release channel; sulfhydryl; macromolecular cross-linked complex; oxidative stress