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

P2X7受体（P2X7R）是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，在炎症和组织损伤期间释放。这些受体在巨噬细胞中表达最高，它们是先天免疫不可或缺的。P2X7受体的激活不仅会导致瞬时阳离子电流，还会形成一个非选择性孔，可通过高达900 Da的分子，最终导致细胞裂解。P2X7R刺激后的下游事件包括膜泡、膜金属蛋白酶和半胱天蛋白酶的激活，导致膜磷脂随着磷脂酰丝氨酸的暴露而混乱、微泡脱落和细胞凋亡。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Ca2+激活的磷脂混乱的蛋白质现已被确定为anotamin 6 （TMEM16F; Ano6）。它已被证明产生Ca2+激活的Cl-和非选择性阳离子电流。我们认为Ano6在刺激P2X7受体时被激活。我们假设Ano6在P2X7受体刺激后的下游事件中起核心作用，如细胞收缩、孔形成、脂质紊乱和细胞凋亡。这些过程是巨噬细胞迁移、吞噬和杀死细菌所必需的。因此，Ano6可能是P2X7级联、巨噬细胞功能和防御的核心组成部分。我们将研究从野生型和Ano6敲除小鼠中分离的腹腔巨噬细胞的离子电流激活、体积调节、细胞迁移、凋亡、吞噬活性和细菌杀伤。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，我们将进一步评估离子电流和信号转导事件，如Ca2+增加和磷脂混乱。在重组细胞系统中，如过表达HEK293细胞和非洲爪蟾卵母细胞，我们将研究Ano6激活的详细分子方面，如Ca2+和P2X7R c端在孔形成中的作用，并将尝试使用最新开发的方法识别这种生物级联所必需的新靶蛋白。我们希望了解Ano6在P2X7受体生理学中的核心作用，以及它对先天免疫的基本贡献。

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.