Mechanism of Inositol Trisphosphate Action

描述（由申请人提供）：细胞质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的反应机制的一个组成部分。d -肌糖醇1,4,5-三磷酸（IP3）是细胞内信使，介导细胞内储存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。本课题的中心主题是研究IP3受体的结构、功能和调控。要验证的一个主要假设是，C端和n端结构域之间的相互作用是配体结合导致通道打开的机制的基础。该提案的具体目的是研究：1)C端域在通道门控中的作用。从c端尾部缺失60aa或从TM结构域4和5之间的细胞质暴露环缺失10aa会导致通道功能的丧失。将制造点突变体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验证c端、TM4.5环和n端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端a1-224的缺失导致IP3结合的显著刺激，但通道功能丧失。诱变将用于鉴定该区域的关键氨基酸。IP3在配体结合区域引起的构象变化将使用固有色氨酸荧光和FRET方法进行研究。3]鉴定在通道功能中起关键作用的跨膜结构域5和6之间的残基。位点定向突变将用于识别作为孔前庭的一部分或孔本身的残基。突变体的电导特性和离子选择性将使用电生理学方法利用片夹核进行研究。这些研究旨在深入了解传导孔的分子结构。[4]在IP3R中鉴定高活性巯基。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移位测定将用于鉴定IP3R中表面可接近的硫醇。可及性的变化将被用作监测天然膜中IP3R构象变化的探针。本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，并利用这些知识来理解细胞在Ca2+信号中产生复杂的时空模式的机制，这些模式是多种生理过程的基础，如细胞分裂、细胞增殖、细胞凋亡、受精、发育、分泌、平滑肌收缩、记忆和学习。

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.