Topology of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (RyR1)
Topology of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (RyR1)
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DOI：
10.1073/pnas.012688999
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发表时间：
2002-12-24
影响因子：
11.1
通讯作者：
MacLennan, DH
中科院分区：
文献类型：
--
作者：
Du, GG;Sandhu, B;MacLennan, DH
为了确定骨骼肌兰尼碱受体（RyR 1）的拓扑结构，增强型GFP（EGFP）与RyR 1的C末端框内融合，取代一系列C末端缺失，这些缺失开始于预测的跨膜螺旋M1-M10的开始或结束附近。构建体在HEK-293（人胚肾细胞系293）或小鼠胚胎成纤维细胞（MEF）细胞中表达，并且使用完整和皂苷透化的细胞的共聚焦显微镜来确定截短的融合蛋白的亚细胞位置。在M3之后截短的融合蛋白表现出均匀的细胞质荧光，其在透化后消失，表明所提出的M '、M”、M1、M2和M3序列不是膜结合的。在M4-M5环末端截短并含有M4的融合蛋白是膜结合的。所有较长的截短融合蛋白也与细胞内膜相关。通过蛋白酶消化和提取分离的微粒体进行的定位表明，位于M5、M7的N-末端一半（M7 a）或M8之后的EGFP位于管腔中，并且位于M4、M6、M7的C-末端一半（M7 b）或M10之后的EGFP位于细胞质中。这些结果表明，RyR 1含有八个跨膜螺旋，组织为四个发夹环。第一个发夹可能由M4a-M4 b组成。然而，它可以由M3-M4组成，即使M3单独不与膜结合，M3-M4也可能形成发夹环。其他三个发夹环由M5-M6、M7 a-M7 b和M8-M10形成。M9不是跨膜螺旋，但它可能在M8和M10之间形成选择性过滤器。
To define the topology of the skeletal muscle ryanodine receptor (RyR1), enhanced GFP (EGFP) was fused in-frame to the C terminus of RyR1, replacing a series of C-terminal deletions that started near the beginning or the end of predicted transmembrane helices M1-M10. The constructs were expressed in HEK-293 (human embryonic kidney cell line 293) or mouse embryonic fibroblast (MEF) cells, and confocal microscopy of intact and saponin-permeabilized cells was used to determine the subcellular location of the truncated fusion proteins. The fusion protein truncated after M3 exhibited uniform cytoplasmic fluorescence, which was lost after permeabilization, indicating that proposed M', M", M1, M2, and M3 sequences are not membrane-associated. The fusion protein truncated at the end of the M4-M5 loop and containing M4 was membrane-associated. All longer truncated fusion proteins were also associated with intracellular membranes. Mapping by protease digestion and extraction of isolated microsomes demonstrated that EGFP positioned after either M5, the N-terminal half of M7 (M7a), or M8 was located in the lumen, and that EGFP positioned after either M4, M6, the C-terminal half of M7 (M7b) , or M10 was located in the cytoplasm. These results indicate that RyR1 contains eight transmembrane helices, organized as four hairpin loops. The first hairpin is likely to be made up of M4a-M4b. However, it could be made up from M3-M4, which might form a hairpin loop even though M3 alone is not membrane-associated. The other three hairpin loops are formed from M5-M6, M7a-M7b, and M8-M10. M9 is not a transmembrane helix, but it might form a selectivity filter between M8 and M10.