Caveolin-1 forms a complex with P2X7 receptor and tunes P2X7-mediated ATP signaling in mouse bone marrow-derived macrophages

The ionotropic purinergic P2X7 receptor responds to extracellular ATP and can trigger proinflammatory immune signaling in macrophages. Caveolin-1 (Cav-1) is known to modulate functions of macrophages and innate immunity. However, it is unknown how Cav-1 modulates P2X7 receptor activity in macrophages. We herein examined P2X7 receptor activity and macrophage functions using bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Cav-1 knockout (KO) mice. ATP (1 mM) application caused biphasic increase in cytosolic [Ca²⁺] and sustained decrease in cytosolic [K⁺]. A specific P2X7 receptor blocker, A-740003, inhibited the maintained cytosolic [Ca²⁺] increase and cytosolic [K⁺] decrease. Total internal reflection fluorescent imaging and proximity ligation assays revealed a novel molecular complex formation between P2X7 receptors and Cav-1 in WT BMDMs that were stimulated with lipopolysaccharides. This molecular coupling was increased by ATP application. Specifically, the ATP-induced Ca²⁺ influx and K⁺ efflux through P2X7 receptors were increased in Cav-1 KO BMDMs, even though the total and surface protein levels of P2X7 receptors in WT and Cav-1 KO BMDMs were unchanged. Cell-impermeable dye (TO-PRO3) uptake analysis revealed that macropore formation of P2X7 receptors was enhanced in Cav-1 KO BMDMs. Cav-1 KO BMDMs increased ATP-induced IL-1β secretion, reactive oxygen species production, Gasdermin D (GSDMD) cleavage, and lactate dehydrogenase release indicating pyroptosis. A-740003 completely prevented ATP-induced pyroptosis. In combination, these datasets show that Cav-1 has a negative effect on P2X7 receptor activity in BMDMs and that Cav-1 in macrophages may contribute to finely tuned immune responses by preventing excessive IL-1β secretion and pyroptosis.NEW & NOTEWORTHY In bone marrow-derived macrophages, Cav-1 suppresses the macropore formation of P2X7 receptors through their direct or indirect interactions, resulting in reduced membrane permeability of cations (Ca²⁺ and K⁺) and large cell-impermeable dye (TO-PRO3) induced by ATP. Cav-1 also inhibits ATP-induced IL-1β secretion, ROS production, GSDMD cleavage, and pyroptosis. Cav-1 contributes to the maintenance of proper immune responses by finely tuning IL-1β secretion and cell death in macrophages.