Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during microglial activation

ATP-gated purinergic P2X7 receptors are crucial ion channels involved in inflammation. They sense abnormal ATP release during stress or injury and are considered promising clinical targets for therapeutic intervention. However, despite their predominant expression in immune cells such as microglia, there is limited information on P2X7 membrane expression and regulation during inflammation at the single-molecule level, necessitating new labeling approaches to visualize P2X7 in native cells. Here, we present X7-uP, an unbiased, affinity-guided P2X7 chemical labeling reagent that selectively and covalently biotinylates endogenous P2X7 in BV2 cells, a murine microglial cell line, allowing subsequent labeling with streptavidin-Alexa 647 tailored for super-resolution imaging. We uncovered a nanoscale microglial P2X7 redistribution mechanism where evenly spaced individual receptors in quiescent cells undergo upregulation and clustering in response to the pro-inflammatory agent lipopolysaccharide and ATP, leading to synergistic interleukin-1{beta} release. Our method thus offers a new approach to revealing endogenous P2X7 expression at the single-molecule level. Impact statementAn affinity-guided chemical strategy enabling highly specific biotinylation of P2X7 receptors reveals, by super-resolution microscopy, how the nanoscale organization of endogenous P2X7 in BV2 microglial cells dynamically changes upon activation.