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

P2 X7受体（P2 X7 R）是配体门控的非选择性阳离子通道，其被炎症和组织损伤期间释放的高浓度细胞外核苷酸激活。这些受体在巨噬细胞中表现出最高的表达，在巨噬细胞中它们对于先天免疫是不可或缺的。P2 X7受体的激活不仅导致瞬时阳离子电流，而且导致形成非选择性孔，可渗透高达900 Da的分子，最终导致细胞裂解。刺激P2 X7 R后的下游事件包括膜起泡、膜金属蛋白酶和半胱天冬酶的活化，导致膜磷脂与磷脂酰丝氨酸暴露的混乱、微泡脱落和凋亡性细胞死亡。P2 X7受体刺激后的细胞死亡包括坏死以及凋亡的迹象，包括初始细胞收缩和随后的细胞肿胀。负责Ca 2+活化磷脂扰乱的蛋白质现已被鉴定为anoctamin 6（TMEM 16 F; Ano 6）。它已被证明产生Ca 2+激活的Cl-和非选择性阳离子电流。我们认为Ano 6在P2 X7受体的刺激过程中被激活。我们假设Ano 6在P2 X7受体刺激后的下游事件中具有核心作用，如细胞收缩、孔形成、脂质紊乱和凋亡。这些过程对于巨噬细胞的迁移、吞噬作用和细菌杀伤是必不可少的。因此，Ano 6可能是P2 X7级联、巨噬细胞功能和防御的中心组分。我们将研究离子电流激活，体积调节，细胞迁移，细胞凋亡，吞噬活性和细菌杀死从野生型和Ano 6基因敲除小鼠分离的腹腔巨噬细胞。在小鼠巨噬细胞中获得的发现将在THP-1人类巨噬细胞中得到证实，我们将进一步评估离子电流和信号转导事件，例如Ca 2+增加和磷脂扰乱。在重组细胞系统，如过表达HEK 293细胞和卵母细胞从非洲爪蟾，我们将研究详细的分子方面的Ano 6激活，如Ca 2+和P2 X7 R C-末端孔形成的作用，并将尝试确定新的目标蛋白质必不可少的这种生物级联反应，使用最近开发的方法。我们希望了解Ano 6对P2 X7受体生理学的核心作用，以及其对先天免疫的根本贡献。

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.