Receptor-regulated Calcium Entry in Exocrine Secretion

描述(申请人提供)：细胞外钙离子进入增强是由多种激素和神经递质作用于与磷脂酶C(PLC)偶联的受体所产生的细胞钙信号的主要成分。我们研究的重点是了解这种钙离子进入的性质，以及它在整个细胞内信号机制中的作用。对这一领域的兴趣主要由所谓的钙离子通道(如CRAC通道)主导，其门控完全依赖于细胞内钙离子存储的耗尽。然而，最近，其他不依赖商店的通路被证明发挥了关键作用--特别是在更低的、更具生理学意义的刺激水平上。在这些通道中，我们在大约8年前首次描述的花生四烯酸调节的钙选择性(ARC)通道仍然是最彻底的特征。研究CRAC通道和ARC通道的一个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，STIM和Orai蛋白的发现从根本上改变了这种情况。因此，已经证明STIM1位于内质网膜上，感受细胞内钙储备的耗竭，并激活由Orai1亚基组成的同源四聚体组成的CRAC通道。令人惊讶的是，我们最近发现STIM和Orai蛋白也以并行但完全不同的方式发挥作用，以影响ARC通道活动。ARC通道活性由STIM1调节，但负责的是位于质膜上的这种蛋白质池，ARC通道孔由Orai1和Orai3亚基的异构体复合体组成。这些紧密的分子关系表明，商店经营的CRAC频道和商店独立的ARC频道代表了一个全新的频道家族的创始成员--“基于Orai的频道”。然而，我们的功能研究表明，这两个通道进化为在不同的刺激条件下工作，并在激动剂激活的钙信号调节中发挥独特的作用。重要的是，这些新的分子洞察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(目标1)，花生四烯酸激活通道的分子机制(目标2)，它们被PKA依赖的磷酸化调节的分子基础(目标3)，以及它们的活性调节在低浓度激动剂刺激的细胞中产生的振荡钙信号的机制(目标4)。 与公共健康相关：受体激活的钙离子进入细胞是产生细胞内钙信号的关键组成部分，已知该信号调节一系列不同的细胞功能-包括细胞分裂和增殖、分泌、运动和细胞死亡。这些信号的错误调节或错误导致了各种不同的疾病状态，包括癌症、免疫缺陷、胰腺炎和各种肌肉和神经疾病。确定相关进入途径的分子基础及其调控，无疑将有助于阐明其活性所涉及的关键机制，并确定其临床操作的潜在靶点。

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.