| Co-Investigator(Kenkyū-buntansha) |
MIYAWAKI Atsushi The Institute of Medical Science, The University of Tokyo, Asistant Professor, 医科学研究所, 助手 (80251445)
SATTELLE Dav ケンブリッジ大学, 動物学教室, 主任研究員
SATTELLE David B. Department of Zoology, University of Cambridge, Senior Principal Scientific Offi
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| Budget Amount *help |
¥8,000,000 (Direct Cost: ¥8,000,000)
Fiscal Year 1995: ¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 1994: ¥4,100,000 (Direct Cost: ¥4,100,000)
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| Research Abstract |
The inositol 1,4,5-trisphosphate receptor (IP_3R) exisits as a tetrameric complex to form a functional inositol 1,4,5-trisphosphate-gated Ca^<2+> channel. Molecular cloning studies have shown that there are at least three types of IP_3R subunits, designated type 1, type 2, and type 3. The levels of expression of IP_3R subunits in various cell lines were investigated by Western blot analysis using type-specific antibodies against 15 C-terminal amino acids of each IP_3R subunits. We found that all the three types of IP_3R subunits were expressed in each cell line examined, but their levels of expression varied. To determine whether IP_3R from heterotetramers, we employed immunoprecipitation experiments using Chinese hamster ovary cells (CHO-K1 cells), in which all three types are abundantly expressed. Each type-specific antibody immunoprecipitated not only the respective cognate type but also the other two types. This result suggests that distinct types of IP_3R subunits assemble to form
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heterotetramers in CHO-K1 cells. We also detected heterotetramers in rat liver, in which IP_3R type 1 and type 2 are expressed abundantly. Previous studies have shown some functional differences among IP_3R types, suggesting the possibility that various compositions of submits show distinct channel properties. The diversity of IP_3R channels may be further increased by the co-assembly of different IP_3R subunits to form homo-or heterotetramers.
Kinetics of Ca^<2+> release by adenophostin, a novel gaonist of inositol 1,4,5-trisphosphate (IP_3) receptor, in the purified and reconstituted IP_3 receptor type 1 (IP_3R1) was investigated using the fluorescent Ca^<2+> indicator fluo-3. Submaximal concentrations of adenophostin caused quantal Ca^<2+> release from the purified IP_3R1 as IP_3 did. Adenophostin-induced Ca^<2+> release by the purified IP_3R1 exhibited a high positive cooperativity (nH=3.9(]SY.+-.])0.2, EC_<50>=11 nM), whereas the IP_3-induced Ca^<2+> release exhibited a moderate one (nH=1.8(]SY.+-.])0.1, EC_<50>=1100 nM). Inhibitation of [^3H] IP_3 binding to the purified IP_3R1 by adenophostin exhibited a positive cooperativity (nH=1.9, K_i=10 nM), whereas IP_3 did not (nH=1.1, K_i=41 nM). Less
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