| Project/Area Number |
14570087
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General pharmacology
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| Research Institution | SHOWA UNIVERSITY |
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Principal Investigator |
OYAMADA Hideto SHOWA UNIVERSITY, SCHOOL OF MEDICINE, DEPARTMENT OF PHARMACOLOGY, RESEARCH ASSISTANT, 医学部, 助手 (50266160)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | calcium ion / calcium release channel / ryanodine receptor / green fluorescent protein / dominant-negative / 緑色蛍光蛋白質(GFP) / CICR |
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| Research Abstract |
Intracellular calcium ion (Ca^<2+>) is an important signal as an internal second messenger to regulate so many diverse cellular processes. Now a days, it is essential to know "When, where, and how to, the Ca^<2+> signals are developed in the living cells." We have tried to establish the dominant -negative inhibition of the ryanodine receptor (RyR) as an intracellular Ca^<2+> release channel.
1.We divided the type 1 of RyR (RyR1) cDNA (about 15000bp) into 11 cassettes by utilization of unique restriction endonuclease sites introduced at intervals of 1500 bp.
2.We mutated the 4032Glutamic acid of RyR1 to Alanine (E4032ARyR1) fused with an enhanced green fluorescent protein (EGFP) to be visualized at the single -cell revel under living conditions.
3.Any 4 -chrolo -3 -ethylphenol (4 -CEP), RyR stimulating agent, induced Ca^<2+> increment were not observed in the E4032ARyR1-EGFP transfected cells while significant elevations of the Ca^<2+> by 4-CEP were seen in the wild type of RyR1 expressed cells.
4.Co-expression of E4032ARyR1-EGFP in addition to the wtRyR1 caused the inhibition of the 4-CEP induced Ca^<2+> increase in the EGFP fluorescent cells as E4032ARyR1 expressed cells.
These results suggested that the mutated E4032ARyR1-EGFP could specifically inhibit the Ca^<2+> release channel / RyR1 as the dominant -negative inhibition of RyR1, which could be recognized as the EGFP fluorescent protein at the single -cell level under living conditions.
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