Molecular and functional properties of the TRPM2 catioin channel

描述(申请人提供)：瞬时受体潜力(Trp)离子通道家族最近出现，因为它含有关键的通道蛋白，参与感觉感知、镁稳态或细胞凋亡等过程。我们发现TRPM2是Trp家族的一员，它是一种钙离子通透性的阳离子通道，由新的第二信使ADP-核糖(ADPR)通过通道的内源性焦磷酸酶结构域调控。TRPM2表达于多种细胞类型，包括胰岛β细胞和中性粒细胞。最近，围绕ADPR的代谢网络中的分子对TRPM2功能特性的作用已经浮出水面。在这里，新的钙释放化合物cADPR和其他腺嘌呤二核苷酸是TRPM2生理过程中的关键因素，因此涉及细胞内钙稳态的过程。基于ADPR和活性氧物种的初步激活，TRPM2可能在细胞应激中发挥作用，并仍然是参与细胞凋亡过程的首选候选分子。为了进一步了解TRPM2在细胞生理学中的作用，我们建议在特定目标1中进行实验，重点研究TRPM2在腺嘌呤-核苷酸启动的事件中的功能。我们利用TRPM2过表达的细胞系统和TRPM2基因敲除的小鼠模型，推测TRPM2可能是钙内流和钙释放的双重通道。我们将探索激动剂-受体之间的相互作用，涉及生成腺嘌呤二核苷酸的主要候选者，即CD38。我们将使用一种基于链脲佐菌素的1型糖尿病小鼠模型来研究TRPM2在细胞死亡中的作用。这些实验将在表达系统和从野生型、CD38、PARP和TRPM2基因敲除小鼠模型中分离的小鼠中性粒细胞和β细胞中进行，使用膜片钳、成像和凋亡研究相结合的方法。在特定的目标2中，我们将研究TRPM2激动剂结合的分子决定因素，并解决钙调素是否是TRPM2钙依赖易化的媒介的问题。了解TRPM2和腺嘌呤二核苷酸在钙信号转导中的作用将加深我们对表达这一离子通道的组织中钙调节的认识，并可能为涉及TRPM2功能的过程提供新的治疗干预靶点，包括糖尿病引起的胰岛β细胞死亡。

DESCRIPTION (provided by applicant): The transient receptor potential (TRP) ion channel family recently emerged as harboring crucial channel proteins involved in processes such as sensory perception, magnesium homeostasis or apoptosis. We identified TRPM2, a member of the TRP family, as a calcium-permeable cation channel gated by the novel second messenger ADP-ribose (ADPR) through the channel's intrinsic pyrophosphatase domain. TRPM2 is expressed in a variety of cell types, including pancreatic beta cells and neutrophils. Recently, the role of the molecules in the metabolic network surrounding ADPR on the functional properties of TRPM2 have emerged. Here, the novel calcium-release compound cADPR and other adenine dinucleotides materialize as crucial factors in TRPM2 physiology and hence processes involving cellular calcium homeostasis. Based on its primary activation by ADPR and reactive oxygen species, TRPM2 may have a function in cellular stress and remains a prime candidate for being involved in the apoptotic process. To further our understanding of TRPM2 in cell physiology, we propose experiments in Specific Aim 1 that will focus on the function of TRPM2 in events initiated by adenine-nucleotides. We hypothesize that TRPM2 may function as dual calcium influx and calcium release channel by taking advantage of a TRPM2 overexpressing cell system and a TRPM2 knock-out mouse model. We will explore agonist-receptor interactions engaging the main candidate in generating adenine dinucleotides, namely CD38. We will investigate the involvement of TRPM2 in cell death using a streptozotocin-based mouse model for diabetes type-1. These experiments will be performed in an expression system and mouse neutrophils and beta cells isolated from wild-type, CD38, PARP and TRPM2 knock-out mice models using a combination of patch-clamp, imaging and apoptotic studies. In Specific Aim 2 we will investigate the molecular determinants of TRPM2 agonist binding and address the question whether calmodulin is the mediator of the calcium-dependent facilitation of TRPM2. Understanding TRPM2 and adenine-dinucleotide function in calcium signaling will refine our knowledge of calcium regulation in tissues expressing this ion channel and may provide new targets for therapeutic interference in processes involving TRPM2 function, including diabetes-causing cell death of pancreatic beta cells.