Receptor-regulated Calcium Entry in Exocrine Secretion

描述（由申请人提供）：细胞外Ca2+的增强进入是多种激素和神经递质作用于与磷脂酶C （PLC）偶联的受体产生的细胞Ca2+信号的主要组成部分。我们的研究重点是了解这种Ca2+进入的性质，及其在整体细胞内信号传导机制中的作用。对这一领域的兴趣在很大程度上是由所谓的存储操作Ca2+通道（例如CRAC通道）主导的，其门控完全依赖于细胞内Ca2+存储的耗尽。然而，最近，其他与储存无关的通路已被证明发挥了关键作用——特别是在较低的、与生理更相关的刺激水平下。其中，我们在8年前首次描述的花生四烯酸调节的Ca2+选择性（ARC）通道仍然是最彻底的表征。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年中，由于STIM和Orai蛋白的发现，这种情况已经从根本上改变了。因此，研究表明，STIM1位于内质网膜上，感知细胞内Ca2+储存的消耗，并激活由Orai1亚基的四聚体组成的CRAC通道。令人惊讶的是，我们最近发现STIM和Orai蛋白也以平行但完全不同的方式影响ARC通道活性。ARC通道的活性受到STIM1的调控，但起作用的是驻留在质膜中的这种蛋白池，而ARC通道孔由Orai1和Orai3亚基的异质复合物组成。这些密切的分子关系表明，商店操作的CRAC通道和商店独立的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，这两个通道进化到在不同的刺激条件下运作，并在激动剂激活的Ca2+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丰富的新工具和方法，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），以及它们的活性如何调节低浓度激动剂刺激细胞中产生的振荡Ca2+信号的机制（Aim 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). PUBLIC HEALTH RELEVANCE: The receptor-activated entry of calcium ions into cells represents a key component in the generation of intracellular calcium signals that are known to regulate a host of different cellular functions - including cell division and proliferation, secretion, motility, and cell death. Misregulation, or errors in these signals underlie a variety of different disease states including cancer, immunodeficiency, pancreatitis, and various muscular and neuronal diseases. Defining the molecular bases of the relevant entry pathways and their regulation, will undoubtedly help elucidate the critical mechanisms involved in their activity, and identify potential targets for their clinical manipulation.