Simultaneous measurement for intracellular temperature and activity of thermosensitive TRP channel

2020 Fiscal Year Final Research Report

• Project/Area Number: 18K15024
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 48020:Physiology-related
• Research Institution: Center for Novel Science Initatives, National Institutes of Natural Sciences
• Principal Investigator: Ujisawa Tomoyo 大学共同利用機関法人自然科学研究機構(新分野創成センター、アストロバイオロジーセンター、生命創成探究, 生命創成探究センター, 特別研究員 (10816515)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Keywords: TRP channel / 細胞内温度

Outline of Final Research Achievements

It is hard to determine the accurate temperature thresholds for activation of thermosensitive TRP channels since people measure the temperature in the bath solution closed to the patch-clamped cell expressing thermosensitive TRP channels. Therefore, we attempted to develop the system for the simultaneous measurement of the intracellular temperature and membrane currents using a fluorescent temperature probe. We introduced the fluorescent temperature probe into the HEK293 cells and analyzed the changes in fluorescent ratio upon local stimulation with infrared laser. Then, we combined the patch-clamp system on the confocal microscope with the infrared laser. We clearly observed rat TRPV1-mediated membrane currents with this system upon laser stimulation. Thus, we could establish the system for the simultaneous measurement of intracellular temperature and thermosensitive TRP channel currents.

Free Research Field

生理学

Academic Significance and Societal Importance of the Research Achievements

温度感受性TRPチャネルの活性化温度閾値は膜電流とパッチクランプされた細胞近傍の外溶液温度の同時計測から決定されている。しかし、細胞外からの熱刺激による細胞内温度、さらにはTRPチャネルが発現する形質膜付近の温度変化とチャネル応答を同時記録した研究はない。さらには、IRレーザーを用いて局所温度変化を与えた際の分子の活性変化を報告したものはなく、今後cell-attached法による単一チャネル電流解析から、チャネルgatingへの影響も解析可能であり、細胞膜温度変化からチャネル開口に至る過程の解明に寄与できると期待される。