Receptor-regulated Calcium Entry in Exocrine Secretion

描述（由申请人提供）：细胞外Ca 2+的增强进入是由多种激素和神经递质作用于磷脂酶C（PLC）偶联受体产生的细胞Ca 2+信号的主要成分。我们研究的重点是了解这种Ca 2+进入的性质，以及它在整个细胞内信号传导机制中的作用。对该领域的兴趣主要由所谓的钙库操纵的Ca 2+通道（例如CRAC通道）主导，其门控完全依赖于细胞内Ca 2+库的消耗，并且在细胞内Ca 2+库的消耗之后。然而，最近，已经显示出其他不依赖于储存的途径起关键作用-特别是在较低的、更生理相关的刺激水平下。其中，花生四烯酸调节的钙离子选择性（ARC）通道，我们第一次描述了一些8年前，仍然是最彻底的特点。研究CRAC通道和ARC通道的主要障碍是缺乏关于这些通道的分子性质的任何信息。在过去的2-3年里，这种情况已经从根本上改变了发现的STIM和奥赖蛋白。因此，已经表明，位于内质网膜中的STIM 1感测细胞内Ca 2+储存的耗尽，并激活CRAC通道，其孔由Orai 1亚基的同源四聚体组成。令人惊讶的是，我们最近发现STIM和奥赖蛋白也以平行但完全不同的方式影响ARC通道活性。ARC通道活性由STIM 1调节，但负责的是驻留在质膜中的这种蛋白质的库，并且ARC通道孔由Orai 1和Orai 3亚基的异聚复合物组成。这些紧密的分子关系表明，存储操作的CRAC通道和存储独立的ARC通道代表了一个全新的通道家族的创始成员-“基于Orai的通道”。然而，我们的功能研究表明，这两个通道的发展，在不同的条件下的刺激和服务的激动剂激活的Ca 2+信号的调节中发挥独特的作用。重要的是，这些新的分子见解创造了大量新的工具和方法，我们现在建议使用这些工具和方法来确定ARC通道孔的详细分子组织（Aim 1），花生四烯酸激活通道的分子机制（Aim 2），PKA依赖性磷酸化调节通道的分子基础（Aim 3），以及它们的活性调节低浓度激动剂刺激的细胞中产生的振荡Ca 2+信号的机制（目的4）。

DESCRIPTION (provided by applicant): The enhanced entry of extracellular Ca2+ is a major component of cellular Ca2+ signals generated by a variety of hormones and neurotransmitters acting on receptors coupled to phospholipase C (PLC). The focus of our research is to understand the nature of this Ca2+ entry, and its roles in overall intracellular signaling mechanisms. Interest in this field has been largely dominated by so-called store- operated Ca2+ channels (e.g. the CRAC channels) whose gating is entirely dependent on, and subsequent to, the depletion of intracellular Ca2+ stores. More recently however, other store- independent pathways have been shown to play a key role - particularly at lower, more physiologically relevant, levels of stimulation. Of these, the arachidonic acid-regulated Ca2+-selective (ARC) channels, that we first described some 8 years ago, remain the most thoroughly characterized. A major impediment to study of both the CRAC channels and the ARC channels has been the lack of any information regarding the molecular nature of these channels. In the past 2-3 years, this situation has been fundamentally transformed by the discovery of the STIM and Orai proteins. Thus, it has been shown that STIM1 located in the membrane of the endoplasmic reticulum, senses the depletion of intracellular Ca2+ stores, and activates the CRAC channels whose pore is comprised of a homotetramer of Orai1 subunits. Surprisingly, we have recently shown that STIM and Orai proteins also function in parallel, yet entirely distinct, ways to affect ARC channel activity. ARC channel activity is regulated by STIM1, but it is the pool of this protein that is resident in the plasma membrane that is responsible, and the ARC channel pore is comprised of a heteromeric complex of both Orai1 and Orai3 subunits. These close molecular relationships indicate that the store-operated CRAC channels and the store-independent ARC channels represent the founding members of an entirely new family of channels - the "Orai-based channels". However, our functional studies have demonstrated that these two channels evolved to operate under distinct conditions of stimulation and to serve unique roles in the regulation of agonist-activated Ca2+ signals. Importantly, these new molecular insights have created a wealth of novel tools and approaches that we now propose to use to determine the detailed molecular organization of the ARC channel pore (Aim 1), the molecular mechanisms underlying activation of the channels by arachidonic acid (Aim 2), the molecular basis for their regulation by PKA-dependent phosphorylation (Aim 3), and the mechanisms by which their activity acts to modulate the oscillatory Ca2+ signals generated in cells stimulated with low agonist concentrations (Aim 4).