超氧中介巯基氧化剂对骨骼肌型钙释放通道的调控作用

Modulation Activity of Superoxide Medium Thiol Oxidant to Skeletal Muscle Type Ca^(2+) Release Channel

目的通过研究可氧化巯基的萘醌(NQ)和硒(Se)化合物对骨骼肌型钙释放通道(RyR1)的调控作用,探讨以O2.-巯基为基础的氧化胁迫对细胞钙调控机制/钙信号通路的影响。方法在NQ和Se的激活及抑制浓度下,考察骨骼肌肌质网(SR)囊泡系的O2.-的产量及其对[3H]-ryanodine结合、钙释放动力学、RyR1的氧化还原电势Eryr、单通道活动和通道蛋白的自由巯基群规模等的影响。结果RyR1的自由巯基逐渐被NQ或Se氧化,表现为先激活后抑制通道;导致Eryr分别向更加还原和氧化方向移动;氧化巯基同时伴有浓度依赖性的O2.-产生,后者参与氧化RyR1自由巯基。结论O2.-在NQ和Se类巯基氧化剂调控RyR1的机制中起到重要作用,其主要作用目标之一是RyR1中那些对氧化环境敏感的自由巯基职能群。

Objective To study the modulation activities of thiol oxidants such as naphthoquinon （NO） and selenium compound （Se） on skeletal muscle type ryanodine receptor （RyR1）/Ca^2＋ release channel to reveal the effects of superoxide thiols on RyRi in the redox mechanism. Methods The isolated rabbit skeletal muscle SR vesicles were used in this research. The generated amount of O2^.- and its [^3H]-ryanodine binding, Ca^2＋ flux from SR vesicles, the redox potential Eryr of RyR1, single channel activity in bi-layer membrane, channel protein and change of free thiol groups on RyRt were studied under the conditions of active and inhibitive concentrations of NQ or Se. Results The thiol oxidants （ NQ, Se） could oxidize free thiols on RyR1 gradually, firstly activated then inhibited and cause Eryr to more negative or positive direction. These oxidants simultaneously generated O2^.- which activated the RyR1 in a concentration dependent manner and exerted biphasic effects on the channel activity. However, the addition of superoxide dismutase （SOD） greatly prevented all of these changes. Conclusion O2^.- is involved in the effects of NQ and Se on their modulations to RyR1 activity by oxidizing different group of functional Tree thiols on the channel. It is proposed that ROS may play a key role as intracellular signal molecules that participate in modulating intracellular Ca^2＋ mobility and Ca^2＋ homeostasis.