Publication | eLife

Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation

The Ion Channel Engineering (ICE) team, in collaboration with the Smart Nanoparticles (SNP) team, UMR 5297, and CNRS UPR 3212, is pleased to announce that the article entitled “Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation” has now been published in its final version (version of record) in eLife.

In this work, we developed a new chemical method enabling the specific biotinylation of P2X7 receptors, allowing their labeling with Alexa Fluor 647–conjugated streptavidin. Implemented in BV2 cells, a murine microglial cell line, and combined with super-resolution imaging (dSTORM), this labeling strategy makes it possible to reveal and track the redistribution of P2X7 receptors at the single-molecule level in response to inflammatory signaling. The results demonstrate, for the first time, a clustering of P2X7 receptors following this inflammatory response. These findings open new therapeutic perspectives by identifying the nanoscale reorganization of P2X7 receptors as a potential target for the treatment of inflammation.

 

 

 

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Abstract image
Affinity-guided labeling strategy for P2X7. (A) Schematic representation of the P2X7 labeling strategy using ligand-directed N-cyanomethyl NASA chemistry. A P2X7 ligand (Lg) binds to allosteric sites on the receptor, bringing the N-cyanomethyl NASA warhead into close proximity with endogenous lysine (K) residues. This spatial arrangement enables the covalent transfer of a biotin moiety via amide bond formation, resulting in highly specific biotinylation of P2X7. The biotin tag allows super-resolution imaging of nanoscale P2X7 localization using a Streptavidin-Alexa 647 probe (Strept-A 647). Notably, following biotin transfer, the ligand can dissociate from P2X7, leaving the allosteric sites unoccupied. (B) Crystal structure of panda P2X7 (pdP2X7) shown in ribbon representation, bound to AZ10606120 depicted as spheres (PDB:5U1W; Karasawa and Kawate, 2016). One of the three ATP-binding sites and the approximate location of the membrane are also indicated. Inset, enlarged view of the AZ10606120-binding pocket, rotated 180°. Distances (in Å) between the α-carbon of selected lysines and the hydroxyl group of AZ10606120 are displayed. Note that K300 is not visible in this view. (C) Chemical structure of X7-uP.