CALCIUM CHANNELS IN NONEXCITABLE CELLS

本研究项目的长期目标是了解与激动剂诱导的哺乳动物非兴奋细胞中的Ca 2+信号转导相关的分子机制。在多种细胞类型中，膜受体的刺激导致Ca 2+从内部储存中释放，并伴随Ca 2+从细胞外空间流入。虽然负责肌醇-1，4，5-三磷酸（Ins（1，4，5）P3）-诱导的Ca 2+从内部存储的释放的机制已经建立，但与Ca 2+进入相关的分子机制仍然未知。在许多细胞中，Ca 2+内流是继发于内部Ca 2+储存的耗尽，即所谓的容量性Ca 2+进入（CCE）模型。虽然已记录的存储耗尽所产生的膜电流，我们的能力，以了解导致激活的CCE的生化事件是阻碍了我们缺乏知识的分子身份有关的渠道。它们的身份的线索来自果蝇光转导的研究。光刺激果蝇感光细胞引起膜电导增加，这需要磷脂酶C（PLC），并反映瞬时受体电位通道（Trp）和Trp-like通道（TrpL）的活性。最初认为Ins（1，4，5）P3诱导的Ca 2+释放通过类似于哺乳动物非兴奋细胞中CCE的机制激活Trp和TrpL。然而，最近的数据表明，情况并非如此。在本申请中，我们考虑了一种替代模型，用于激活Trp家族的离子通道，其需要PLC，但独立于内部Ca+储存。具体地说，我们认为这不是Ins（1，4，5）P3本身的释放，而是PIP 2的水解导致Trp的激活。使用生物化学，分子生物学和电生理学技术的组合，本提案的具体目标将测试有关Trp通道的结构，功能和调节的几个假设。具体目的是：1）确定受体刺激对Trp离子通道家族的调节机制。2)确定Trp通道通过[Ca 2 +]i升高调节的机制。3)确定分别存在于PIP 2结合结构域的NH 2-和COOH-末端的高度保守的三重脯氨酸和酸性区域的作用。

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.