Three-dimensional reconstruction using transmission electron microscopy reveals a swollen, bell-shaped structure of transient receptor potential melastatin type 2 cation channel
Three-dimensional reconstruction using transmission electron microscopy reveals a swollen, bell-shaped structure of transient receptor potential melastatin type 2 cation channel
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DOI：
10.1074/jbc.m705694200
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发表时间：
2007-12-21
影响因子：
4.8
通讯作者：
Sato, Chikara
中科院分区：
文献类型：
--
作者：
Maruyama, Yuusuke;Ogura, Toshihiko;Sato, Chikara
瞬时受体电位melastatin 2型（TRPM 2）是一种氧化还原敏感的，钙渗透性的阳离子通道，由各种信号激活，如腺苷二磷酸核糖（ADPR）作用于ADPR焦磷酸酶（ADPRase）结构域和环状ADPR。在这里，我们纯化了FLAG标记的四聚体TRPM2通道，使用负染电子显微镜对其进行分析，并以2.8 nm分辨率重建了三维结构。这种多模态传感器分子具有18 nm宽和25 nm高的钟状形状。整体结构类似于另一个多模态传感器通道，TRP规范类型3（TRPC 3）。在这两种结构中，小的细胞外结构域是一个密集的半圆形，而大的细胞质结构域具有稀疏的双层结构，具有多个内部空腔。然而，在TRPM 2的胞质结构域下观察到独特的方形棱柱突起。在ADPRase结构域的C末端融合的FLAG表位被抗体分配到靠近突起的位置。这表明激动剂结合ADPRase结构域和跨膜区中的离子门分别位于分子中。
Transient receptor potential melastatin type 2 (TRPM2) is a redox-sensitive, calcium-permeable cation channel activated by various signals, such as adenosine diphosphate ribose ( ADPR) acting on the ADPR pyrophosphatase (ADPRase) domain, and cyclic ADPR. Here, we purified the FLAG-tagged tetrameric TRPM2 channel, analyzed it using negatively stained electron microscopy, and reconstructed the three-dimensional structure at 2.8-nm resolution. This multimodal sensor molecule has a bell-like shape of 18 nm in width and 25 nm in height. The overall structure is similar to another multimodal sensor channel, TRP canonical type 3 (TRPC3). In both structures, the small extracellular domain is a dense half-dome, whereas the large cytoplasmic domain has a sparse, double-layered structure with multiple internal cavities. However, a unique square prism protuberance was observed under the cytoplasmic domain of TRPM2. The FLAG epitope, fused at the C terminus of the ADPRase domain, was assigned by the antibody to a position close to the protuberance. This indicates that the agonist-binding ADPRase domain and the ion gate in the transmembrane region are separately located in the molecule.