CALCIUM CHANNELS IN NONEXCITABLE CELLS

非兴奋细胞中的钙通道

• 批准号: 6180604
• 负责人: WILLIAM P SCHILLING
• 金额: $ 33.57万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-11 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180604
• 关键词: SDS polyacrylamide gel electrophoresis; Sf9 cell line; calcium channel; calcium flux; calcium ion; calmodulin; electrophysiology; fluorescent dye /probe; hydrolysis; immunoprecipitation; membrane channels; phosphatidylinositols; phospholipase C; phosphorylation; protein binding; protein structure function; receptor binding; single cell analysis; tissue /cell culture; vascular endothelium; western blottings

The long range goal of this research project is to understand the molecular mechanisms associated with agonist-induced Ca2+ signaling in mammalian non-excitable cells. In a variety of cell types, stimulation of membrane receptors causes the release of Ca2+ from internal stores and the concomitant influx of Ca2+ from the extracellular space. Although the mechanisms responsible for inositol-1,4,5-trisphosphate(Ins(1,4,5)P3)-induced Ca2+ release from internal stores are well established, the molecular mechanisms associated with Ca2+ entry remain unknown. In many cells, Ca2+ influx is secondary to the depletion of the internal Ca2+ store, i.e. the so-called capacitative Ca2+ entry (CCE) model. Although the membrane current generated by depletion of the store has been recorded, our ability to understand the biochemical events leading to activation of CCE is hampered by our lack of knowledge concerning the molecular identity of the channels involved. A clue to their identity derives from studies of Drosophila phototransduction. Stimulation of Drosophila photoreceptor cells by light causes an increase in membrane conductance that requires phospholipase C (PLC) and reflects the activity of both the transient receptor potential channel (Trp) and the Trp-like channel (TrpL). It was originally thought that an Ins(1,4,5)P3-induced Ca2+ release activated Trp and TrpL via a mechanism analogous to CCE in mammalian non-excitable cells. More recent data however, suggests that this is not the case. In the present application, we consider an alternative model for the activation of the Trp family of ion channels that requires PLC, but is independent of the internal Ca+ stores. Specifically, we propose that it's not the release of Ins(1,4,5)P3 per se, but rather the hydrolysis of PIP2 that is responsible for activation of Trp. Using a combination of biochemical, molecular biological, and electrophysiological techniques, the specific aims of this proposal will test several hypotheses concerning the structure, function and regulation of the Trp channels. The specific aims are: 1) To determine the mechanism by which the Trp family of ion channels are regulated by receptor stimulation. 2) To determine the mechanism by which Trp channels are regulated by a rise in [Ca2+]i. 3) To determine the role of the highly conserved triple proline and acidic regions that exist NH2- and COOH-terminal to the PIP2 binding domain, respectively.

该研究项目的远距离目标是了解与激动剂诱导的Ca2+信号传导相关的分子机制。在多种细胞类型中，刺激膜受体会导致内部存储中Ca2+的释放，并从细胞外空间中散发Ca2+​​的流入。尽管负责从内部商店中诱导的Ca2+释放的负责肌醇1,4,5-三磷酸（INS（1,4,5）P3）的机制尚不清楚。在许多细胞中，Ca2+流入量是内部Ca2+储存的耗竭的继发的，即所谓的电容CA2+进入（CCE）模型。尽管已经记录了通过耗尽商店产生的膜电流，但由于我们对所涉及的通道的分子身份缺乏知识，我们了解导致CCE激活的生化事件的能力受到阻碍。其身份的线索来自果蝇光转导的研究。通过光刺激果蝇感光细胞会导致需要磷脂酶C（PLC）的膜电导增加，并反映了瞬时受体电位通道（TRP）和TRP样通道（TRPL）的活性。最初认为，INS（1,4,5）P3诱导的Ca2+释放活化的TRP和TRPL通过类似于哺乳动物的非脱离细胞CCE的机制。但是，最新数据表明事实并非如此。在本应用中，我们考虑了需要PLC的离子通道的激活的替代模型，但与内部CA+存储无关。具体而言，我们建议它本身不是INS（1,4,5）P3的释放，而是导致TRP激活的PIP2的水解。使用生化，分子生物学和电生理技术的结合，该提案的具体目的将检验有关TRP通道的结构，功能和调节的几种假设。具体目的是：1）确定由受体刺激调节离子通道的TRP家族的机制。 2）确定由[Ca2+] i的增加来调节TRP通道的机制。 3）分别确定与PIP2结合结构域分别存在NH2-和COOH末端的高度保守的三脯氨酸和酸性区域的作用。