| Project/Area Number |
15209008
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (A)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
General physiology
|
|---|
| Research Institution | Osaka University |
|---|
Principal Investigator |
KURACHI Yoshihisa Osaka University, Graduate School of Medicine, Professor, 医学系研究科, 教授 (30142011)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
INANOBE Atsushi Osaka University, Graduate School of Medicine, Associate Professor, 医学系研究科, 助教授 (00270851)
HIBINO Hiroshi Osaka University, Graduate School of Medicine, Assistant, 医学系研究科, 助手 (70314317)
ISHII Masaru Osaka Minami Medical Center, Department of Clinical Research, Clinical Fellow, 免疫異常疾患研究所, 研究員 (10324758)
山田 充彦 信州大学, 医学部, 教授 (10263237)
|
|---|
| Project Period (FY) |
2003 – 2005
|
| Project Status |
Completed (Fiscal Year 2005)
|
| Budget Amount *help |
¥48,230,000 (Direct Cost: ¥37,100,000、Indirect Cost: ¥11,130,000)
Fiscal Year 2005: ¥10,400,000 (Direct Cost: ¥8,000,000、Indirect Cost: ¥2,400,000)
Fiscal Year 2004: ¥12,350,000 (Direct Cost: ¥9,500,000、Indirect Cost: ¥2,850,000)
Fiscal Year 2003: ¥25,480,000 (Direct Cost: ¥19,600,000、Indirect Cost: ¥5,880,000)
|
|---|
| Keywords | G protein-gated potassium channel / RGS protein / PDZ protein / Calmodulin / Lipid raft / FRET / G蛋白質 / カリウムチャネル / FRET / G蛋白質制御カリウムチャネル / カルモディリン / カルモデュリン |
|---|
| Research Abstract |
G protein-gated potassium (K_G) channels are under the control of receptor-dependent activation of G protein. This G protein-K_G channel system is negatively controlled by RGS (regulators of G protein signaling) proteins, which are normally inhibited by phosphatidyl-3,4,5-triphosphate (PIP_3) but can be activated (disinhibited) by Ca^<2+>/calmodulin (CaM) upon various biological stimulations (e.g., depolarization). The objective of the present study is to delineate molecular mechanisms underlying the functional regulation of K_G channels, and the following results were obtained. 1. RGS4 protein was expressed, purified and its interactions with phospholipids and CaM were investigated. It was demonstrated that (1)PIP_3 selectively bound to the RGS domain of RGS4, (2)the binding of PIP_3 to RGS4 was competitively displaced by Ca^<2+>/CaM and (3)the positively charged residues within the RGS domain seemed to be involved in the competitive binding between PIP_3 and CaM. 2.Molecular dynamics of RGS4 protein and CaM were further analyzed using FRET (Fluorescent resonance energy transfer) technique. It was demonstrated that (1)an elevation of intracellular Ca^<2+> concentration by ionomycin specifically increased the assembly of RGS protein and CaM in the living cells, but such increases were not observed (2)in the cells pre-treated with methyl-β-cyclodextrin, which depletes membrane cholesterol and disrupts lipid rafts or (3)in the cells expressing RGS4 mutant which lacks palmitoylation site necessary for the lipid raft-localization. The present study provide molecular basis for the reciprocal regulation of RGS protein by PIP_3 and CaM, which contribute to better understanding the molecular mechanisms for functional regulation of G protein- K_G channel system.
|
