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Mechanism of Inositol Trisphosphate Action

三磷酸肌醇的作用机制

基本信息

批准号：
8034977
负责人：
SURESH K JOSEPH
金额：
$ 10.68万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8034977
关键词：
Agonist Amino Acids Apoptosis Architecture Binding Biological Assay C-terminal COS-7 Cell Calcium Cell Nucleus Cell Proliferation Cell division Cells Chimeric Proteins Complex Cysteine Cytosol Development Electrophoretic Mobility Shift Assay Fertilization Fluorescence Fluorescence Resonance Energy Transfer Goals Growth Factor Hormonal Hormones ITPR1 gene Inositol Ion Channel Ions Knowledge Learning Length Ligand Binding Ligand Binding Domain Ligands Maleimides Mediating Membrane Memory Methods Molecular Monitor Muscle Contraction Mutagenesis Mutation N-terminal Neurotransmitters Pattern Physiological Processes Play Polyethylene Glycols Property Protein Isoforms Recombinants Regulation Role Signal Transduction Site Site-Directed Mutagenesis Smooth Muscle Structure Sulfhydryl Compounds Surface System Tail Testing Transmembrane Domain Tryptophan Vestibule crosslink insight mutant patch clamp receptor

项目摘要

DESCRIPTION (provided by applicant): An elevation of the free calcium concentration in the cytoplasmic compartment is an integral component of the mechanism by which cells respond to hormones, growth-factors and certain neurotransmitters. D-myo-lnositol 1,4,5-trisphosphate (IP3) is an intracellular messenger mediating the hormonal mobilization of Ca2+ from intracellular stores. This molecule interacts with a specific receptor (IP3R) that has been purified and shown to be a ligand-gated Ca2+ channel. The central theme of this proposal is to study the structure, function and regulation of IP3 receptors. A major hypothesis to be tested is that interactions between the C- and N-terminal domains are fundamental to the mechanism by which ligand-binding leads to channel opening. The specific aims of the proposal are to investigate: 1] The role of the C- terminal domain in channel gating. Deletion of 60aa from the C-terminal tail or 10aa from the cytosol- exposed loop between TM domains 4 & 5 cause loss of channel function. Point mutants will be made to locate the critical amino acids involved in both regions. GST-fusion proteins and targeted cysteine cross linking studies will be used to test the hypothesis that regions of the C-terminus, TM4.5 loop and N-terminal domains are in close association. 2] The role of the N-terminal suppressor domain in channel gating. Deletion of aa1-224 of the N-terminus results in a marked stimulation of IP3 binding but loss of channel function. Mutagenesis will be used to identify critical amino-acids in this region. Conformational changes induced by IP3 in the ligand-binding domain will be studied using intrinsic tryptophan fluorescence and FRET methods. 3] Identify residues between transmembrane domains 5&6 which play a key role in channel function. Site-directed mutations will be made to identify residues that are part of the vestibule of the pore or the pore itself. Conductance properties and ion selectivity of the mutants will be investigated using electrophysiological approaches utilizing patch-clamped nuclei. These studies are intended to provide insights into the molecular architecture of the conduction pore. 4] Identify highly reactive thiol groups in the IP3R. Cysteine substitution and gel-shift assays using large maleimide polyethylene glycol derivatives will be used to identify surface accessible thiols in the IP3R. Changes in accessibility will be used as a probe to monitor conformational changes in the IP3R in native membranes. The long-term goal of this proposal is to understand how IP3R channels function at a molecular level and to use this knowledge to understand the mechanism by which cells generate the complex spatial and temporal patterns in Ca2+ signaling that underlie a multitude of physiological processes as diverse as cell division, cell proliferation, apoptosis, fertilization, development, secretion, smooth muscle contraction, memory and learning.

描述（由申请人提供）：细胞质区室中游离钙浓度的升高是细胞对激素、生长因子和某些神经递质作出反应的机制的一个组成部分。 D-肌醇 1,4,5-三磷酸 (IP3) 是一种细胞内信使，介导细胞内储存的 Ca2+ 的激素动员。该分子与特定受体 (IP3R) 相互作用，该受体已被纯化并显示为配体门控 Ca2+ 通道。该提案的中心主题是研究IP3受体的结构、功能和调控。需要测试的一个主要假设是，C 端和 N 端结构域之间的相互作用是配体结合导致通道开放的机制的基础。该提案的具体目标是研究： 1] C 端域在通道选通中的作用。从 C 末端尾部删除 60 个氨基酸或从 TM 结构域 4 和 5 之间的胞质暴露环中删除 10 个氨基酸会导致通道功能丧失。将进行点突变来定位两个区域中涉及的关键氨基酸。 GST 融合蛋白和靶向半胱氨酸交联研究将用于检验 C 末端区域、TM4.5 环和 N 末端结构域密切相关的假设。 2] N 端抑制域在通道门控中的作用。 N 末端 aa1-224 的缺失会导致 IP3 结合的显着刺激，但通道功能丧失。诱变将用于鉴定该区域的关键氨基酸。将使用内在色氨酸荧光和 FRET 方法研究 IP3 在配体结合域中诱导的构象变化。 3] 鉴定跨膜结构域 5 和 6 之间的残基，这些残基在通道功能中起关键作用。将进行定点突变以鉴定属于孔前庭或孔本身的一部分的残基。将使用利用膜片钳核的电生理学方法来研究突变体的电导特性和离子选择性。这些研究旨在深入了解传导孔的分子结构。 4] 识别 IP3R 中的高反应性硫醇基团。使用大马来酰亚胺聚乙二醇衍生物的半胱氨酸取代和凝胶迁移测定将用于鉴定 IP3R 中的表面可接近的硫醇。可及性的变化将用作监测天然膜中 IP3R 构象变化的探针。该提案的长期目标是了解 IP3R 通道如何在分子水平上发挥作用，并利用这些知识来了解细胞在 Ca2+ 信号传导中产生复杂的空间和时间模式的机制，这些模式是细胞分裂、细胞增殖、凋亡、受精、发育、分泌、平滑肌收缩、记忆和学习等多种生理过程的基础。

项目成果

期刊论文数量（17）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis.

DOI：
10.1083/jcb.200505022
发表时间：
2005-09-26
期刊：
The Journal of cell biology
影响因子：
0
作者：
Madesh M;Hawkins BJ;Milovanova T;Bhanumathy CD;Joseph SK;Ramachandrarao SP;Sharma K;Kurosaki T;Fisher AB
通讯作者：
Fisher AB

Characteristics of GTP-mediated microsomal Ca2+ release.

GTP 介导的微粒体 Ca2 释放的特征。