MECHANISM OF INOSITOL TRISPHOSPHATE ACTION

描述：（逐字来自申请人的摘要） 细胞质室中的钙浓度是一个组成部分， 细胞对激素、生长因子和 某些神经递质D-肌肌醇1，4，5-三磷酸盐（IP 3）是一种 细胞内信使介导的激素动员的Ca 2+从 细胞内储存。该分子与特定受体（IP 3R）相互作用 已经被纯化并显示为配体门控的Ca 2+通道。中央 本提案的主题是研究的结构，功能和调节 IP 3受体。该提案的具体目的是调查：1） Ca 2+激活的分子机制。Ca 2+是IP 3R的主要调节因子 并对通道功能产生双相作用。2种已知的酸性氨基酸 IP 3R的钙结合区域将被突变。变异的受体 瞬时转染COS-7细胞及其功能特性 将使用测量Ca 2+对IP 3介导的 ~（45）Ca ~（2+）通量、（3 H）-IP 3结合和与钙调蛋白（CaM）琼脂糖凝胶结合。 这些实验将检验由Ca 2+激活IP 3Rs的假设。 这是Ca 2+与蛋白质中高亲和力位点直接结合的结果。2)的 Ca 2+抑制的分子机制。过度表达野生型的影响 和突变的CaM以及已知CaM突变的功能后果 结合位点，以及推定的IQ结构域钙调蛋白结合位点将被 考察实验将检验IP 3R具有多个 CaM结合位点，并且CaM结合负责Ca 2+抑制， IP3R。3)确定跨膜结构域5和6之间的残基， 渠道功能的关键作用。将进行定点突变，以确定 作为孔前庭或孔本身的一部分的残基。离子 突变体的选择性将使用电生理学方法进行研究 利用膜片钳核或重建成平面脂质的方法 双层。这些研究旨在提供对分子生物学的深入了解。 传导孔的结构。4)C-末端与 受体的N-末端结构域。重组融合蛋白和体外 翻译的跨膜结构域将用于映射相互作用位点， C端和N端结构域。这些区域随后发生突变， 破坏相互作用将被用来测试假设， 相互作用是配体结合导致 渠道开放本提案的长期目标是了解IP 3R如何 通道在分子水平上起作用，并利用这些知识来了解 细胞产生复杂的时空模式的机制 在众多生理反应的基础上的Ca 2+信号中。

DESCRIPTION: (Verbatim from the Applicant's Abstract) An elevation of 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-inositol 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. The specific aims of the proposal are to investigate: 1) the molecular mechanism of Ca2+ activation. Ca2+ is the principal regulator of IP3R and exerts a biphasic effect on channel function. Acidic amino acids in 2 known calcium-binding regions of the IP3R will be mutated. The mutated receptors will be transiently transfected into COS-7 cells and their functional properties will be assessed using assays that measure the effects of Ca2+ on IP3-mediated 45Ca2+ fluxes, (3H)-IP3 binding and binding to calmodulin (CaM) sepharose. These experiments will test the hypothesis that activation of IP3Rs by Ca2+ is the result of direct Ca2+ binding to high affinity sites in the protein. 2) The molecular mechanism of Ca2+ inhibition. The effect of over-expressing wild type and mutated CaM and the functional consequence of mutation of the known CaM binding site, as well as putative IQ domain calmodulin binding sites will be examined. The experiments will test the hypothesis that the IP3R has multiple CaM binding sites and that CaM binding is responsible for Ca2+ inhibition of the IP3R. 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. Ion selectivity of the mutant will be investigated using electrophysiological approaches utilizing patch-clamped nuclei or reconstitution into planar lipid bilayers. These studies are intended to provide insights into the molecular architecture of the conduction pore. 4) Interactions between C-terminal and N-terminal domains of the receptor. Recombinant fusion proteins and in vitro translated transmembrane domains will be used to map the interaction sites on the C-terminal and N-terminal domains. Subsequent mutation of these regions to disrupt the interaction will be used to test the hypothesis that the interaction is fundamental to the mechanism by which ligand binding leads to channel opening. 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 responses.