Na channel reconstructionTRPM2

TRPチャネル中でも、特に温度や酸化ストレスのセンサーであるTRPM2は注目されている。このチャネルを精製・負染色電顕撮影・単粒子画像解析を行った。その結果、TRPC3と似た隙間だらけの膨れあがったベル型2重入れ子構造を持つことを解明した。解明された三次元構造では、張り出した構造が3次元的な結合スペースをつくり出し、様々な制御タンパク質を同時に結合し、TRPM2が多様な刺激のセンサーであることを可能にしている。さらには酸化ストレスを感知する酵素部位NUDT9-Hドメインは、ベルから細胞内に突き出ている突起と考察された。本チャンネルは、特に糖尿病の発症機構と深く関連することが知られており、更なる高分解能での構造解明を目指したい。


Abstract from: THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 51, pp. 36961–36970, December 21, 2007

 

Three-dimensional Reconstruction Using Transmission Electron Microscopy Reveals a Swollen, Bell-shaped Structure of Transient Receptor Potential Melastatin Type 2 Cation Channel

Yuusuke Maruyama‡§, Toshihiko Ogura‡¶, Kazuhiro Mio‡, Shigeki Kiyonaka, Kenta Kato, Yasuo Mori, and Chikara Sato‡§1

From the ‡Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Umezono 1-1-4, Tsukuba, Ibaraki 305-8568, the §Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai,Tsukuba, Ibaraki 305-8577, ¶PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, and the Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan

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.

 

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タンパク質構造決定技術、膜タンパク質精製技術、情報技術、電子顕微鏡技術、神経細胞の培養技術等.