| Project/Area Number |
12357001
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
General physiology
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| Research Institution | Osaka University |
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Principal Investigator |
YANAGIDA Toshio Osaka University, Graduate School of Frontier Biosciences, Professor, 生命機能研究科, 教授 (30089883)
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| Co-Investigator(Kenkyū-buntansha) |
IWANE Atsuko Osaka University, Graduate School of Frontier Biosciences, Assistant Professor, 生命機能研究科, 助手 (30252638)
SAKO Yasushi Osaka University, Graduate School of Frontier Biosciences, Associate Professor, 生命機能研究科, 助教授 (20215700)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥46,000,000 (Direct Cost: ¥40,000,000、Indirect Cost: ¥6,000,000)
Fiscal Year 2002: ¥10,400,000 (Direct Cost: ¥8,000,000、Indirect Cost: ¥2,400,000)
Fiscal Year 2001: ¥15,600,000 (Direct Cost: ¥12,000,000、Indirect Cost: ¥3,600,000)
Fiscal Year 2000: ¥20,000,000 (Direct Cost: ¥20,000,000)
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| Keywords | electrophsiology / artificial membrane / ion channels / total internal reflection / fluorescence micorscopy / single-molecule detection / GPCR / chemical sensing / 人口膜 / 全反射蛍光顕微鏡 / CARI / CRAC / イオンチャネル / 全反射顕微鏡 / 蛋白構造 / パッチクランプ |
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| Research Abstract |
We have developed a novel microscope to measure optical and electrical signals simultaneously from a single protein incorporated into artificial or bio membranes. Using this microscope, we measured lateral movement and ion transport activity of a single ion channel molecule and signal transduction of a G-protein coupled receptor (GPCR) in living cells. During the period of this grant, we obtained the following achievements.
(1) Simultaneous measurement of optical and electrical signal of single ion channels : we have developed single channel recording apparatus that can be equipped with a single-molecule imaging microscope. The artificial membranes were formed horizontally in an aqueous environment or on thin agarose layer. Single molecules in the bilayer were observed under an epi-fluorescence or an objective-type total internal reflection fluorescence microscope. Using this microscope, lateral diffusion movement of single alamethicin, Ca-activated K channel and cardiac ryanodine receptor was observed simultaneously with their single-channel gating fluctuation.
(2) Single-molecule imaging of a GPCR in living cells : A cAMP receptor for chemotaxis of Dictyostelium cells, CAR1 and a cytoplasmic protein, CRAC working downstream of CAR1 were fused with GFP and observed in living cells in single-molecules. Binding of cAMP conjugated with a fluorophore Cy3 and CAR1 was analyzed in single-molecules. After concentration jump of cAMP using flash photolysis of caged cAMP, the affinity between Cy3-cAMP and CAR1 was transiently increased suggesting a positive feedback loop of the binding of cAMP and CAR1.
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