Mio Kazuhiro, Ohkubo Tatsunari, Sasaki Daisuke, Arai Tatsuya, Sugiura Mayui, Fujimura Shoko, Nozawa Shunsuke, Sekiguchi Hiroshi, Kuramochi Masahiro, Sasaki Yuji C
AIST-UTokyo Advanced Operando-Measurement Technology Open Innovation Laboratory (OPERANDO-OIL), National Institute of Advanced Industrial Science and Technology (AIST), 6-2-3 Kashiwanoha, Chiba 277-0882, Japan.
Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
Membranes (Basel). 2023 Jul 30;13(8):708. doi: 10.3390/membranes13080708.
The transient receptor potential vanilloid type 1 (TRPV1) is a multimodal receptor which responds to various stimuli, including capsaicin, protons, and heat. Recent advances in cryo-electron microscopy have revealed the structures of TRPV1. However, due to the large size of TRPV1 and its structural complexity, the detailed process of channel gating has not been well documented. In this study, we applied the diffracted X-ray tracking (DXT) technique to analyze the intracellular domain dynamics of the TRPV1 protein. DXT enables the capture of intramolecular motion through the analysis of trajectories of Laue spots generated from attached gold nanocrystals. Diffraction data were recorded at two different frame rates: 100 μs/frame and 12.5 ms/frame. The data from the 100 μs/frame recording were further divided into two groups based on the moving speed, using the lifetime filtering technique, and they were analyzed separately. Capsaicin increased the slope angle of the MSD curve of the C-terminus in 100 μs/frame recording, which accompanied a shifting of the rotational bias toward the counterclockwise direction, as viewed from the cytoplasmic side. This capsaicin-induced fluctuation was not observed in the 12.5 ms/frame recording, indicating that it is a high-frequency fluctuation. An intrinsiccounterclockwise twisting motion was observed in various speed components at the N-terminus, regardless of the capsaicin administration. Additionally, the competitive inhibitor AMG9810 induced a clockwise twisting motion, which is the opposite direction to capsaicin. These findings contribute to our understanding of the activation mechanisms of the TRPV1 channel.
瞬时受体电位香草酸亚型1（TRPV1）是一种多模态受体，可对多种刺激作出反应，包括辣椒素、质子和热。冷冻电子显微镜技术的最新进展揭示了TRPV1的结构。然而，由于TRPV1的尺寸较大及其结构复杂性，通道门控的详细过程尚未得到充分记录。在本研究中，我们应用衍射X射线追踪（DXT）技术来分析TRPV1蛋白的细胞内结构域动力学。DXT能够通过分析附着的金纳米晶体产生的劳厄斑点轨迹来捕获分子内运动。衍射数据以两种不同的帧率记录：100微秒/帧和12.5毫秒/帧。使用寿命过滤技术，将100微秒/帧记录的数据根据移动速度进一步分为两组，并分别进行分析。在100微秒/帧的记录中，辣椒素增加了C末端MSD曲线的斜率角，从细胞质侧看，这伴随着旋转偏向逆时针方向的转变。在12.5毫秒/帧的记录中未观察到这种辣椒素诱导的波动，表明它是一种高频波动。无论是否施用辣椒素，在N末端的各种速度成分中均观察到固有逆时针扭曲运动。此外，竞争性抑制剂AMG9810诱导了顺时针扭曲运动，这与辣椒素的方向相反。这些发现有助于我们理解TRPV1通道的激活机制。
