Abstract
Purinergic P2X7 receptors (P2X7R) are fundamental to innate immune response. In macrophages, transient stimulation of P2X7R activates several transport mechanisms and induces the scrambling of phospholipids with subsequent membrane blebbing and apoptosis. These processes support phagocytosis and subsequent killing of phagocytosed bacteria. Here we demonstrate that the stimulation of P2X7 receptors activates anoctamin 6 (ANO6, TMEM16F), a protein that functions as Ca(2+) dependent phospholipid scramblase and Ca(2+)-activated Cl(-) channel. Inhibition or knockdown of ANO6 attenuates ATP-induced cell shrinkage, cell migration and phospholipid scrambling. In mouse macrophages, Ano6 produces large ion currents by stimulation of P2X7 receptors and contributes to ATP-induced membrane blebbing and apoptosis, which is largely reduced in macrophages from Ano6-/- mice. ANO6 supports bacterial phagocytosis and killing by mouse and human THP-1 macrophages. Our data demonstrate that anoctamin 6 is an essential component of the immune defense by macrophages.
Highlights
Purinergic P2X7 receptors (P2X7R) are fundamental to innate immune response
P2X7 receptors show highest expression in macrophages, where they are important for innate immunity during infectious diseases[26,27], while ANO6 was found to be expressed in spleen, osteoclasts and immune cells[15,17,22,28], suggesting an overlapping expression
We expressed hP2X7R in Xenopus oocytes and observed very large delayed activated currents on continuous stimulation by 1 mM ATP (Fig. 1a,b). These pore currents were not observed in water-injected oocytes, and were not due to activation of endogenous Ca2 þ activated Cl À currents, since whole-cell currents activated by the Ca2 þ ionophore ionomycin were negligible when compared with P2X7R pore currents (Fig. 1c, Supplementary Fig. 1a)
Summary
Purinergic P2X7 receptors (P2X7R) are fundamental to innate immune response. In macrophages, transient stimulation of P2X7R activates several transport mechanisms and induces the scrambling of phospholipids with subsequent membrane blebbing and apoptosis. ANO6 (anoctamin 6, TMEM16F) is a Ca2 þ -dependent phospholipid scramblase[15,16,17] and a putative Ca2 þ -activated Cl À and non-selective cation channel[18,19,20,21] that is activated during apoptotic cell death[22,23,24]. A cascade of cellular events is triggered through the stimulation of P2X7R, with the initial activation of non-selective cation currents and a subsequent pore conductance This is followed by sequential cell shrinkage/swelling, phospholipid scrambling, membrane blebbing and cell death[2,29,30,31]. These questions were tackled by analysing the activation of ANO6 through stimulation of P2X7R in overexpressing Xenopus oocytes and HEK293 cells, and in human and mouse macrophages from wild-type (wt) and Ano6-knockout mice
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