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

• Applicant: Professor Dr. Karl Kunzelmann
• Subject Area: Anatomy and Physiology ; Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2014 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 256953317

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.

• DFG Programme: Research Grants
• International Connection: Portugal
• Participating Person: Professorin Margarida D. Amaral