Mechanism of Inositol Trisphosphate Action

描述(由申请人提供)：细胞质内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做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，介导细胞内钙离子的激素动员。该分子与一个特定的受体(IP3R)相互作用，该受体已被纯化，并被证明是一个配体门控的钙通道。这项建议的中心主题是研究IP3受体的结构、功能和调节。需要检验的一个主要假设是，C-末端和N-末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。该提案的具体目的是调查：1)C-末端结构域在通道门控中的作用。从C-末端尾部缺失60aa或从TM结构域4和5之间的胞浆暴露环缺失10aa会导致通道功能丧失。将进行点突变以定位这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被用来检验C末端、TM4.5环和N末端结构域的区域密切相关的假设。2]N端抑制域在通道门控中的作用。N-末端的Aa1-224缺失导致IP3结合的显著刺激，但通道功能丧失。突变将被用来确定该区域的关键氨基酸。将使用内源性色氨酸荧光和FRET方法研究IP3诱导的配基结合区域的构象变化。3)确定在通道功能中起关键作用的跨膜区5和6之间的残基。将进行定点突变，以识别属于毛孔前庭或毛孔本身的残基。突变体的电导特性和离子选择性将利用膜片钳细胞核的电生理学方法进行研究。这些研究旨在提供对导电孔的分子结构的见解。4)鉴定IP3R中的高活性硫醇基团。使用大的马来酰亚胺聚乙二醇衍生物的半胱氨酸取代和凝胶位移分析将被用来识别IP3R中表面可访问的硫醇。可及性的变化将被用作探针来监测天然膜中IP3R的构象变化。这一建议的长期目标是了解IP3R通道如何在分子水平上发挥作用，并利用这一知识了解细胞在钙信号中产生复杂的时空模式的机制，这些模式是细胞分裂、细胞增殖、细胞凋亡、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。

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.