CALCIUM CHANNELS IN NONEXCITABLE CELLS

该研究项目的长期目标是了解与激动剂诱导的哺乳动物不可兴奋细胞中Ca2+信号传导相关的分子机制。在各种细胞类型中，膜受体的刺激引起Ca2+从内部储存的释放和伴随的Ca2+从细胞外空间的内流。尽管肌醇-1,4,5-三磷酸（Ins(1,4,5)P3）诱导Ca2+从内部储存释放的机制已经确立，但与Ca2+进入相关的分子机制仍然未知。在许多细胞中，Ca2+内流是继发于内部Ca2+储存的消耗，即所谓的容性Ca2+进入（CCE）模型。尽管已经记录了储存耗尽所产生的膜电流，但由于缺乏对相关通道的分子特性的了解，我们理解导致CCE激活的生化事件的能力受到阻碍。对果蝇光转导的研究为它们的身份提供了线索。光刺激果蝇光感受器细胞会导致膜电导的增加，这需要磷脂酶C (PLC)，并反映瞬时受体电位通道（Trp）和Trp样通道（TrpL）的活性。最初认为Ins(1,4,5) p3诱导的Ca2+释放激活了Trp和TrpL，其机制类似于哺乳动物不可兴奋细胞中的CCE。然而，最近的数据表明，情况并非如此。在目前的应用中，我们考虑了激活Trp家族离子通道的替代模型，该模型需要PLC，但独立于内部Ca+存储。具体来说，我们提出不是Ins(1,4,5)P3本身的释放，而是PIP2的水解负责Trp的激活。利用生物化学、分子生物学和电生理技术的结合，本提案的具体目标将测试有关色氨酸通道的结构、功能和调节的几个假设。具体目的是：1)确定Trp家族离子通道受受体刺激调控的机制。2)确定Trp通道受[Ca2+]i升高调控的机制。3)确定高度保守的三脯氨酸和存在于NH2-端和cooh -端的酸性区域分别对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.