Petrotchenko Evgeniy V, Yamaguchi Naohiro, Pasek Daniel A, Borchers Christoph H, Meissner Gerhard
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA.
Res Rep Biol. 2011 Jan;2011(2):13-21. doi: 10.2147/RRB.S15776.
The tetrameric skeletal muscle ryanodine receptor ion channel complex (RyR1) contains a large number of free cysteines that are potential targets for redox-active molecules. Here, we report the mass spectrometric analysis of free thiols in RyR1 using the lipophilic, thiol-specific probe monobromobimane (MBB). In the presence of reduced glutathione, MBB labeled 14 cysteines per RyR1 subunit in tryptic peptides in five of five experiments. Forty-six additional MBB-labeled cysteines per RyR1 subunit were detected with lower frequency in tryptic peptides, bringing the total number of MBB-labeled cysteines to 60 per RyR1 subunit. A combination of fluorescence detection and mass spectrometry of RyR1, labeled in the presence of reduced and oxidized glutathione, identified two redox-sensitive cysteines (C1040 and C1303). Regulation of RyR activity by reduced and oxidized glutathione was investigated in skeletal muscle mutant RyR1s in which 18 cysteines were substituted with serine or alanine, using a [(3)H]ryanodine ligand binding assay. Three single-site RyR1 mutants (C1781S, C2436S, and C2606S) and two multisite mutants with five and seven substituted cysteines exhibited a reduced redox response compared with wild-type RyR1. The results suggest that multiple cysteines determine the redox state and activity of RyR1.
四聚体骨骼肌兰尼碱受体离子通道复合物（RyR1）含有大量游离半胱氨酸，这些半胱氨酸是氧化还原活性分子的潜在作用靶点。在此，我们报告了使用亲脂性、硫醇特异性探针单溴代双马来酰亚胺（MBB）对RyR1中的游离硫醇进行质谱分析的结果。在存在还原型谷胱甘肽的情况下，在五个实验中的每一个实验里，MBB在胰蛋白酶肽段中标记了每个RyR1亚基14个半胱氨酸。在胰蛋白酶肽段中还以较低频率检测到每个RyR1亚基另外46个MBB标记的半胱氨酸，使得每个RyR1亚基上MBB标记的半胱氨酸总数达到60个。对在还原型和氧化型谷胱甘肽存在下标记的RyR1进行荧光检测和质谱分析相结合，鉴定出两个氧化还原敏感的半胱氨酸（C1040和C1303）。使用[³H]兰尼碱配体结合试验，在18个半胱氨酸被丝氨酸或丙氨酸取代的骨骼肌突变型RyR1中，研究了还原型和氧化型谷胱甘肽对RyR活性的调节。与野生型RyR1相比，三个单位点RyR1突变体（C1781S、C2436S和C2606S）以及两个分别有五个和七个取代半胱氨酸的多位点突变体表现出降低的氧化还原反应。结果表明，多个半胱氨酸决定了RyR1的氧化还原状态和活性。
