INTRACELLULAR MESSENGER FUNCTIONS OF INOSITOL PHOSPHATES

磷酸肌醇的细胞内信使功能

• 批准号: 3466975
• 负责人: IAN PARKER
• 金额: $ 8.42万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466975
• 关键词: G protein; Xenopus; calcium channel; cell membrane; chloride channels; density gradient ultracentrifugation; egg /ovum; electrophysiology; fluorescence microscopy; inositol phosphates; intracellular membranes; ion transport; laboratory rat; membrane channels; membrane model; membrane permeability; membrane proteins; messenger RNA; microelectrodes; microinjections; voltage /patch clamp

Inositol phosphates are used as intracellular messengers within a signalling pathway which serves to control many diverse cellular functions, such as neurotransmitter and hormone responses, secretion, muscle contraction and phototransduction. Disorders of this signalling pathway have been implicated in diseases including tumorigenesis and manic depressive illness, and the relevance of this system to clinical studies will almost certainly grow as we come to understand it more. Great progress has recently been made in the biochemical elucidation of the pathways leading to the formation of several different inositol phosphate compounds. However, the physiological study of the actions of these compounds is less well advanced, and although it is thought that they act primarily by raising intracellular free calcium levels, little is known about the specific actions of many of them, or about the mechanisms by which they regulate calcium fluxes across intracellular and cell surface membranes. The main object of this proposal is to study the messenger functions of inositol phosphates, by using electrophysiological and optical techniques to measure membrane currents and intracellular calcium. Xenopus oocytes will be used as a convenient model cell system, as these cells have a well characterized phosphoinositide signalling system, and their large size facilitates many procedures including voltage-clamp recording and micro-injection. The initial aim is to characterize the abilities of different inositol phosphates to liberate intracellular calcium, to activate influx of calcium across the plasma membrane, and to activate membrane conductances independent of calcium. Subsequently, the properties of inositol phosphateactivated calcium channels will be examined in detail by voltage- and patch-clamp recording of currents across the plasma membrane, and by reconstituting channels from internal membranes into lipid bilayers. This will give information about the kinetics and conductances of single channels, their ionic specificity, modulation by inositol phosphates and other second messengers, and blocking by calcium antagonists and other pharmacological agents. Similar studies will also be made of the calcium-activated chloride channels which mediate the final electrical response to phosphoinositide activation in the oocyte, and of any calcium-independent currents which are found to be modulated by inositol phosphates. Recordings of intracellular calcium will be made to determine whether the oscillatory membrane current response to inositol phosphates arises from an oscillatory liberation of calcium, and the feedback mechanism responsible for this process will be studied. The oocyte will also be used as a translation system for exogenous mRNA, to see whether calcium channels, or other components of the phosphoinositide signalling system, can be functionally expressed by mRNA from brain or salivary gland.

肌醇磷酸被用作细胞内信使， 信号通路，用于控制许多不同的细胞 功能，如神经递质和激素反应， 分泌、肌肉收缩和光转导。 障碍 这种信号通路与疾病有关 包括肿瘤发生和躁狂抑郁症， 该系统与临床研究的相关性几乎肯定会 随着我们对它的了解越来越多。 了很大的进展 最近在生物化学阐明中， 导致几种不同肌醇形成的途径 磷酸盐化合物 然而，对这一现象的生理学研究 这些化合物的作用不太先进，虽然它 人们认为它们主要通过提高细胞内自由基来发挥作用， 钙水平，很少有人知道的具体行动，许多 或者它们调节钙的机制 穿过细胞内和细胞表面膜的通量。 主要 这项建议的目的是研究信使的职能， 肌醇磷酸，通过使用电生理学和光学 测量膜电流和细胞内电流的技术 钙 非洲爪蟾卵母细胞将被用作一个方便的模型细胞 系统，因为这些细胞具有充分表征的磷酸肌醇 信号系统，其大尺寸便于许多程序 包括电压钳记录和微注射。 最初的目的是表征不同肌醇的能力， 磷酸盐释放细胞内钙，激活流入 钙离子穿过质膜，并激活膜 电导率与钙无关。 随后 磷酸肌醇激活的钙通道的特性将是 通过电压和膜片钳记录详细检查， 电流穿过质膜，并通过重组 从内膜进入脂质双层的通道。 这将 提供有关单个分子的动力学和电导的信息 通道，其离子特异性，肌醇磷酸调节 和其他第二信使，并被钙拮抗剂阻断 和其它药理学试剂。 类似的研究也将在 由钙激活的氯离子通道组成， 最终的电响应磷酸肌醇激活在 卵母细胞以及发现的任何钙非依赖性电流 被磷酸肌醇调节。 录音 细胞内钙将被确定， 对肌醇磷酸的振荡膜电流反应 产生于钙的振荡释放， 负责这一过程的机制将被研究。 的 卵母细胞也将用作外源性的翻译系统。 mRNA，看看是否钙通道，或其他成分， 磷酸肌醇信号系统可以在功能上 由来自脑或唾液腺的mRNA表达。