High-speed detection of minute motion of channel proteins that sense heat and pain（ Press Release）
- Release Date
- 15 Dec, 2020
- BL40XU (High Flux)
Dec.15, 2020
The University of Tokyo
University of Tsukuba
Japan Synchrotron Radiation Research Institute (JASRI)
Japan Science and Technology Agency (JST)
Japan Agency for Medical Research and Development (AMED)
Key achievements
・The research group carried out, for the first time in the world, the real-time observation of intramolecular motion of protein transient receptor potential (TRP) channels used to sense the heat and pain felt by humans and demonstrated that the motion is very small.
・The TRP channels bind to capsaicin, a constituent of hot pepper, and send signals through their clockwise twisting motion.
・Because the TRP channels are related to the mechanism of pain in humans, the achievement of this study is expected to contribute to the understanding of the mechanism of pain perception and the development of new pain relievers.
A research group led by Professor Yuji Sasaki of Advanced Materials Science, Graduate School of Frontier Sciences, The University of Tokyo, has succeeded in the real-time observation of the intramolecular motion of TRP channels (Note 1), which play a role in sending heat and pain signals, on the microsecond order for the first time in the world. TRP channels, which consist of four subunits (Note 2) each having six transmembrane segments, are related to the responses to sensory stimuli in humans and various animal species. Their detailed structure has been clarified recently; their research has been actively pursued and TRP channels have been attracting considerable attention. When capsaicin, which activates the TRP channels, was added, the TRP channels opened up to allow the flow of ions while the channels twisted clockwise. In contrast, when a drug that inhibits the activity of TRP channels was added, the channels twisted counterclockwise. In addition, in the experiment using mutant TRP channels that do not react with capsaicin, a counterclockwise twisting motion that inhibited the channel motion was observed similarly to the case of adding a drug that inhibits their activity. As explained above, the research group has succeeded in observing, in real time, the dynamic clockwise and counterclockwise motions of TRP channels under various conditions. The diffusion constant (Note 3) of these motions was found to be extremely small (20-30 pm2/ms). The research group used a diffracted X-ray tracking (DXT) method (Note 4) to detect the intramolecular dynamics of a molecule labeled with Au nanocrystals in a video of a single molecule. The experiment was carried out using BL40XU (Note 5) at a large synchrotron radiation facility, SPring-8, located at Nishiharima, Hyogo Prefecture. The DXT method is highly expected to contribute to the development of new technologies for drug discovery utilizing molecular motions, which has not been realized by the conventional determination of molecular structures and the structural analysis of reaction pockets. This achievement published in Journal of Physical Chemistry, an established international journal in physical chemistry, on 9th December 2020. Reference |
Fig. 1 Setup of X-ray experiment of transient receptor potential vanilloid type 1 (TRPV1)
Data were obtained with a temporal resolution of 100 µs/frame using X-rays with a broad energy band. BL40XU, with the highest brilliance among the 64 beamlines available at SPring-8, was used for the measurement by DXT.
Fig. 2 Schematic of DXT
Active TRPV1 was monitored in an aqueous solution. TRPV1 and Au crystals were specifically bonded using a methionine tag. TRPV1 and a substrate were specifically bonded using a histidine tag. The diffraction spots of Au nanocrystals obtained using X-rays were analyzed.
Fig. 3 Motion of diffraction spots and results
The research group succeeded in the world’s first observation of the twisting molecular motion of TRPV1 activated by capsaicin. The diffusion constant of the motion was calculated to be 12.8 pm2/ms (A). The motion of diffraction spots obtained from Au nanocrystals was tracked and classified on the basis of the rate of rotation [fast-moving (B) and slow-moving (C) diffraction spots], and the rotational motion of TRPV1 was confirmed (D).
Glossary
Note1: Transient receptor potential (TRP) channels
TRP channels are formed as homotetramers and each subunit includes six transmembrane segments and cation channels detected in mammals as the homologue of the causative gene trp of a photoresponse variant of the gene in Drosophila. TRP channels act as sensors of various physicochemical stimuli, such as temperature, mechanical stimulation, spices, and acids/bases. Twenty-seven homologues have been reported for humans.
Note2: Subunit
A proteins is generally considered as a unit of molecules; however, it is sometimes considered as a combination of several polypeptide chains. One polypeptide chain is called a subunit in the sense that it is a type of unit. A giant protein molecule generally consists of multiple subunits.
Note3: Diffusion constant
Intramolecular motion is generally discussed in terms of Brownian motion. Brownian motion was discovered by Robert Brown in 1827 and is characterized by the irregular motion of fine particles in liquid. The diffusion constant is frequently used to express the magnitude of Brownian motion. The unit of the diffusion constant in the SI system is m2/s. For example, the diffusion constant of aerosols with a size of 0.1 µm in air is 0.07 µm2/ms. The diffusion constant obtained in the experiment of this research group was 12.8 pm2/ms. The unit “pm” stands for picometer and is 1/1000 of 1 nanometer (nm). The intramolecular motion of TRP channels is extremely small.
Note4: Diffracted X-ray tracking (DXT)
Proteins are labeled with nanocrystals with a diameter of 20–30 nm, and the motion of the nanocrystals that is associated with the intramolecular motion of the proteins is tracked at a high speed in a time-division manner as the Laue diffraction pattern using X-rays. This method was developed and published by Professor Yuji Sasaki in 2000 and has been successfully used in the observation of the intramolecular motion of various proteins (reported in journals such as Physical Review Letter, Cell, and Sci. Rep.).
Note5: SPring-8 (BL40XU)
SPring-8 is a large synchrotron radiation facility managed by RIKEN and is located in Harima Science Garden City, Hyogo Prefecture. It delivers the most powerful synchrotron radiation in the world. Japan Synchrotron Radiation Research Institute (JASRI) is in charge of supporting users. The name ‘SPring-8’ is derived from ‘Super Photon ring-8 GeV’. Synchrotron radiation, which is electromagnetic radiation consisting of narrow, powerful beams, is produced when electron beams, accelerated to nearly the speed of light, are forced to travel along a curved path by electromagnets. Various research studies, including those on nanotechnology, biotechnology, and industrial applications, are conducted using synchrotron radiation at SPring-8. The research group used BL40XU, which boasts a high brilliance among beamlines available at SPring-8.
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