CALCIUM CHANNELS IN NONEXCITABLE CELLS

这项研究的长期目标是了解激动剂在哺乳动物不可兴奋细胞中诱导钙信号的相关分子机制。在各种类型的细胞中，膜受体的刺激导致细胞内钙离子的释放和伴随的细胞外钙离子的内流。尽管inositol-1，4，5-trisphosphate(Ins(1，4，5)P3)-induced钙离子从内库释放的机制已被证实，但与钙离子进入相关的分子机制仍不清楚。在许多细胞中，钙离子内流是次要的，内部钙离子储备库的耗尽，即所谓的容量性钙离子内流(CCE)模型。尽管存储耗尽产生的膜电流已经被记录下来，但由于缺乏有关通道的分子身份的知识，我们理解导致CCE激活的生化事件的能力受到阻碍。关于它们的身份的线索来自对果蝇光转导的研究。光刺激果蝇光感受器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)和类Trp通道(Trpl)的活性。最初认为Ins(1，4，5)P3诱导的钙释放通过类似于CCE的机制激活哺乳动物不可兴奋细胞中的Trp和Trpl。然而，最近的数据表明，情况并非如此。在本申请中，我们考虑了另一种激活Trp家族离子通道的模型，该模型需要PLC，但独立于内部钙存储。具体地说，我们认为不是INS(1，4，5)P3本身的释放，而是PIP2的水解导致了Trp的激活。结合生物化学、分子生物学和电生理学技术，这项提案的具体目的将检验有关Trp通道的结构、功能和调节的几个假说。其具体目的是：1)确定受体刺激调节Trp家族离子通道的机制。2)确定[Ca~(2+)]升高对Trp通道的调节机制。3)确定分别存在NH2-和COOH-末端的高度保守的三聚丙氨酸和酸性区域对PIP2结合域的作用。

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.