NOVEL INTRACELLULAR SENSOR FOR CYCLIC AMP

用于循环放大器的新型细胞内传感器

• 批准号: 2775302
• 负责人: THOMAS C RICH
• 金额: $ 3.67万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2775302
• 关键词: adenylate cyclase; analytical method; bioengineering /biomedical engineering; biological signal transduction; biomedical equipment development; biosensor device; calcium channel; calcium flux; cell line; cell membrane; cyclic AMP; electrophysiology; enzyme activity; intracellular; kidney cell; optics; phosphodiesterases; second messengers; voltage /patch clamp

This proposal describes the development and use of a novel sensor for cAMP to examine the interplay between the Ca2+ and cAMP signaling pathways. Dynamic Ca2+ signaling pathways are intimately related to cAMP signaling pathways, with numerous avenues for co-regulation. However, the current method for measuring cAMP in vivo only allows identification of slow changes in cAMP throughout the cytoplasm. A novel cAMP sensor, a modified CNG channel, has largely been developed. Optical and electrophysiological techniques will be used to address the following questions in the further development and use of this novel sensor. 1. Can Ca2+ flux through the olfactory CNG channel pore generate local [Ca2+] high enough to inhibit channel activity or is Ca2+ regulation of the channel dependent upon other Ca2+ sources? Can the disruption of the Ca 2+- calmodulin binding site remove Ca2+ effects on channel activity? 2. Can the olfactory CNG channel be used as a real- time measure of adenylyl cyclase (AC) activity in the human embryonic kidney cell line. HEK293, and the C6-2B glioma cell line (which expresses predominantly a Ca2+ inhibitable cyclase)? If so, the sensor will be used to investigate the interplay between AC and phosphodiesterase (PDE) activities in regulating cAMP levels. 3. Are Ca2+ and cAMP signaling interrelated in pituitary GH3 cell line? What are the critical molecular components contributing to their interactions in these excitable cells? Does [cAMP] oscillate in conjunction with [Ca2+]?

该提案描述了一种新型传感器的开发和使用 cAMP 用于检查 Ca2+ 和 cAMP 信号传导之间的相互作用 途径。 动态 Ca2+ 信号通路与以下因素密切相关： cAMP 信号通路，具有多种共同调节途径。 然而，目前体内测量 cAMP 的方法仅允许 识别整个细胞质中 cAMP 的缓慢变化。 一个 新型 cAMP 传感器（一种改进的 CNG 通道）已得到广泛开发。 光学和电生理学技术将用于解决 在进一步开发和使用这部小说时存在以下问题 传感器。 1、通过嗅觉CNG通道孔的Ca2+通量能否产生 局部 [Ca2+] 高到足以抑制通道活性或者是 Ca2+ 通道的调节依赖于其他 Ca2+ 来源吗？ 可以吗 Ca 2+- 钙调蛋白结合位点的破坏消除了 Ca2+ 对 渠道活动？ 2、嗅觉CNG通道可以作为真实的通道吗？ 人类胚胎腺苷酸环化酶（AC）活性的时间测量 肾细胞系。 HEK293 和 C6-2B 神经胶质瘤细胞系（ 主要表达 Ca2+ 可抑制环化酶）？如果是这样，则传感器 将用于研究 AC 和 磷酸二酯酶 (PDE) 活性调节 cAMP 水平。 3. 是 Ca2+ 和 cAMP 信号在垂体 GH3 细胞系中相互关联吗？ 什么 是促成其相互作用的关键分子成分 在这些兴奋细胞中？ [cAMP] 是否与 [钙2+]？