Dutka T L, Mollica J P, Lamboley C R, Weerakkody V C, Greening D W, Posterino G S, Murphy R M, Lamb G D
School of Life Sciences, La Trobe University, Melbourne, Victoria, Australia.
Institute of Sport, Exercise and Active Living (ISEAL), Victoria University, Melbourne, Victoria, Australia; and.
Am J Physiol Cell Physiol. 2017 Mar 1;312(3):C316-C327. doi: 10.1152/ajpcell.00334.2016. Epub 2016 Dec 14.
Nitric oxide is generated in skeletal muscle with activity and decreases Ca sensitivity of the contractile apparatus, putatively by nitrosylation of an unidentified protein. We investigated the mechanistic basis of this effect and its relationship to the oxidation-induced increase in Ca sensitivity in mammalian fast-twitch (FT) fibers mediated by glutathionylation of Cys134 on fast troponin I (TnI). Force-[Ca] characteristics of the contractile apparatus in mechanically skinned fibers were assessed by direct activation with heavily Ca-buffered solutions. Treatment with nitrosylating agents, nitrosoglutathione (GSNO) or nitroso--acetyl-penicillamine (SNAP), decreased pCa ( = -log [Ca] at half-maximal activation) by ~-0.07 pCa units in rat and human FT fibers without affecting maximum force, but had no effect on rat and human slow-twitch fibers or toad or chicken FT fibers, which all lack Cys134. The Ca sensitivity decrease was ) fully reversed with dithiothreitol or reduced glutathione, ) at least partially reversed with ascorbate, indicative of involvement of S-nitrosylation, and ) irreversibly blocked by low concentration of the alkylating agent, -ethylmaleimide (NEM). The biotin-switch assay showed that both GSNO and SNAP treatments caused nitrosylation of TnIglutathionylation pretreatment blocked the effects of nitrosylation on Ca sensitivity, and vice-versa. nitrosylation pretreatment prevented NEM from irreversibly blocking glutathionylation of TnI and its effects on Ca sensitivity, and likewise glutathionylation pretreatment prevented NEM block of nitrosylation. Following substitution of TnI into rat slow-twitch fibers, nitrosylation treatment caused decreased Ca sensitivity. These findings demonstrate that nitrosylation and glutathionylation exert opposing effects on Ca sensitivity in mammalian FT muscle fibers, mediated by competitive actions on Cys134 of TnI.
一氧化氮在骨骼肌活动时产生，并降低收缩装置对钙的敏感性，推测是通过对一种未鉴定蛋白质的亚硝基化作用实现的。我们研究了这种效应的机制基础及其与哺乳动物快肌（FT）纤维中氧化诱导的钙敏感性增加之间的关系，后者是由快速肌钙蛋白I（TnI）上Cys134的谷胱甘肽化介导的。通过用高钙缓冲溶液直接激活来评估机械剥离纤维中收缩装置的力-[钙]特性。用亚硝基化剂亚硝基谷胱甘肽（GSNO）或亚硝基-α-乙酰青霉胺（SNAP）处理，可使大鼠和人FT纤维中的pCa（半最大激活时的-log[钙]）降低约0.07个pCa单位，而不影响最大力，但对大鼠和人慢肌纤维或蟾蜍和鸡的FT纤维没有影响，这些纤维都缺乏Cys134。钙敏感性降低：）用二硫苏糖醇或还原型谷胱甘肽可完全逆转，）用抗坏血酸至少部分逆转，表明涉及S-亚硝基化，并且）被低浓度的烷基化剂N-乙基马来酰亚胺（NEM）不可逆地阻断。生物素转换试验表明，GSNO和SNAP处理均导致TnI的亚硝基化，谷胱甘肽化预处理可阻断亚硝基化对钙敏感性的影响，反之亦然。亚硝基化预处理可防止NEM不可逆地阻断TnI的谷胱甘肽化及其对钙敏感性的影响，同样，谷胱甘肽化预处理可防止NEM对亚硝基化的阻断。将TnI替换到大鼠慢肌纤维中后，亚硝基化处理导致钙敏感性降低。这些发现表明，亚硝基化和谷胱甘肽化对哺乳动物FT肌纤维中的钙敏感性发挥相反的作用，是通过对TnI的Cys134的竞争性作用介导的。
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