Anoctamin 6 mediates Effects downstream of P2X7-Receptors, essential for Macrophage function and Innate Immunity

P2X7受体(P2X7R)是一种配体门控的非选择性阳离子通道，可被高浓度的细胞外核苷酸激活，在炎症和组织损伤时释放。这些受体在巨噬细胞中的表达最高，在巨噬细胞中它们是天然免疫所必需的。P2X7受体的激活不仅导致瞬时阳离子电流，而且形成一个非选择性的孔道，可被高达900Da的分子渗透，最终导致细胞溶解。P2X7R刺激后的下游事件包括膜起泡、膜金属蛋白酶和半胱氨酸酶的激活，导致膜磷脂与磷脂酰丝氨酸的接触，微泡脱落和细胞凋亡。受P2X7受体刺激后的细胞死亡包括坏死和凋亡的迹象，包括最初的细胞收缩和随后的细胞肿胀。负责钙激活的磷脂扰乱的蛋白质现已被鉴定为Anoctamin 6(TMEM16F；Ano6)。已证明它能产生钙激活的氯离子和非选择性阳离子电流。我们认为，在刺激P2X7受体的过程中，Ano6被激活。我们推测，Ano6在刺激P2X7受体后的下游事件中发挥核心作用，如细胞收缩、孔洞形成、脂质扰乱和细胞凋亡。这些过程对于巨噬细胞的迁移、吞噬和杀菌是必不可少的。因此，Ano6可能是P2X7级联反应、巨噬细胞功能和防御的中心成分。我们将检测从野生型和Ano6基因敲除小鼠分离的巨噬细胞的离子电流激活、体积调节、细胞迁移、细胞凋亡、吞噬活性和细菌杀灭。在小鼠巨噬细胞中获得的发现将在THP-1人巨噬细胞中得到证实，在那里我们将进一步评估离子电流和信号转导事件，如钙升高和磷脂争抢。在重组细胞系统中，例如非洲爪哇的HEK293细胞和卵母细胞，我们将研究Ano6激活的详细分子方面，如钙和P2X7R C末端在孔道形成中的作用，并将尝试利用最近发展的方法识别这一生物级联反应所必需的新的靶蛋白。我们希望了解Ano6在P2X7受体生理学中的核心作用，以及它在天然免疫中的基础作用。

P2X7 receptors (P2X7R) are ligand gated non-selective cation channels that are activated by high concentrations of extracellular nucleotides, released during inflammation and tissue injury. These receptors show highest expression in macrophages, where they are indispensable for innate immunity. Activation of P2X7 receptors not only leads to an instantaneous cationic current, but also to formation of a non-selective pore, permeable to molecules up to 900 Da, which eventually leads to cell lysis. Downstream events after stimulation of P2X7R comprise membrane blebbing, activation of membrane metalloproteases and caspases, leading to scrambling of membrane phospholipids with exposure of phosphatidylserine, shedding of microvesicles and apoptotic cell death. Cell death upon stimulation of P2X7 receptors includes signs of necrosis as well as apoptosis, including initial cellular shrinkage and subsequent cell swelling. The protein in charge of Ca2+ activated phospholipid scrambling has now been identified as anoctamin 6 (TMEM16F; Ano6). It has been demonstrated to produce Ca2+ activated Cl- and nonselective cation currents. We propose that Ano6 is activated during stimulation of P2X7 receptors. We hypothesize that Ano6 has a central role in downstream events following stimulation of P2X7 receptors, such as cell shrinkage, pore formation, lipid scrambling and apoptosis. These processes are essential for migration, phagocytosis and bacterial killing by macrophages. Thus Ano6 may be a central component of the P2X7 cascade, macrophage function and defense. We will examine ion current activation, volume regulation, cell migration, apoptosis, phagocytic activity and bacterial killing by peritoneal macrophages isolated from wild type and Ano6 knockout mice. The findings obtained in mouse macrophages will be confirmed in THP-1 human macrophages, where we will further assess ion currents and signal transduction events such as Ca2+ increase and phospholipid scrambling. In recombinant cell systems such as overexpressing HEK293 cells and oocytes from Xenopus laevis we will study detailed molecular aspects of Ano6 activation such as the role of Ca2+ and P2X7R C-terminus for pore formation, and will try to identify novel target proteins essential for this biological cascade, using a recently developed method. We expect to understand the central role of Ano6 for P2X7 receptor physiology, and its fundamental contribution to innate immunity.