Molecular mechanisms for functional and spatial regulation of the G protein-gated potassium channel

G蛋白门控钾通道功能和空间调节的分子机制

基本信息

批准号：
15209008
负责人：
KURACHI Yoshihisa
金额：
$ 30.87万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15209008/
关键词：
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

项目摘要

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.

G蛋白门控钾（K_G）通道受G蛋白的接收器依赖性激活的控制。该G蛋白-K_G通道系统由RGS（G蛋白信号传导的调节剂）蛋白质负面控制，通常受到磷脂酰-3,4,5-三磷酸盐（PIP_3）的抑制，但可以通过CA^<2+>/calmodulin（CAM）对各种生物刺激（E.G.）激活（抑制）（抑制）。本研究的目的是描述K_G通道功能调控的基础的分子机制，并获得了以下结果。 1。RGS4蛋白被表达，纯化，并研究了其与磷脂和CAM的相互作用。已经证明（1）PIP_3选择性地与RGS4的RGS域结合，（2）PIP_3与RGS4的结合通过Ca^<2+>/CAM竞争性地移动，并且（3）（3）RGS域中的积极电荷保留似乎与PIP_3和CAM之间的有竞争力的绑定有关。 2.使用FRET（荧光共振能量转移）技术进一步分析了RGS4蛋白和CAM的分子动力学。 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缺乏脂质筏 - 平局所需的棕榈酰化位点。本研究为PIP_3和CAM对RGS蛋白的相互调控提供了分子基础，这有助于更好地理解G蛋白K_G通道系统功能调节的分子机制。