The mechanism of secondary damage spread after brain trauma remains unsolved. In this work, we redirected the attention to astrocytic communication pathways. Using an in vitro trauma model that consists of a scratch injury applied to an astrocyte monolayer, we found a significant and transient induction of connexin43 (Cx43) hemichannel activity in regions distal from the injury, which was maximal ∼1 h after scratch. Two connexin hemichannel blockers, La(3+) and the peptide Gap26, abolished the increased activity, which was also absent in Cx43 KO astrocytes. In addition, the scratch-induced increase of hemichannel activity was prevented by inhibition of P2 purinergic receptors. Changes in hemichannel activity took place with a particular spatial distribution, with cells located at ∼17 mm away from the scratch presenting the highest activity (dye uptake). In contrast, the functional state of gap junction channels (dye coupling) was not significantly affected. Cx43 hemichannel activity was also enhanced by the acute extracellular application of 60 mM K(+) . The increase in hemichannel activity was associated with an increment in apoptotic cells at 24 h after scratch that was totally prevented by Gap26 peptide. These findings suggest that Cx43 hemichannels could be a new approach to prevent or reduce the secondary cell damage of brain trauma.

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Significance Pannexin1 is a glycoprotein that has been shown to form functional plasma membrane channels and mediate many cellular signaling pathways. However, the formation and function of pannexin1-based intercellular cell–cell channels in mammalian cells and vertebrate tissue is a question of substantial debate. This work provides robust electrophysiological evidence to demonstrate that endogenously expressed human pannexin1 forms cell–cell channels and lays the groundwork for studying a potential new type of electrical synapses between many mammalian cell types that endogenously express pannexin1.

Interacting receptors at the neuronal plasma membrane represent an additional regulatory mode for intracellular transduction pathways. P2X4 receptor triggers fast neurotransmission responses via a transient increase in intracellular Ca 2+ levels. It has been proposed that the P2X4 receptor interacts with the 5-HT 3 A receptor in hippocampal neurons, but their binding stoichiometry and the role of P2X4 receptor activation by ATP on this crosstalking system remains unknown. Via pull-down assays, total internal reflection fluorescence (TIRF) microscopy measurements of the receptors colocalization and expression at the plasma membrane, and atomic force microscopy (AFM) imaging, we have demonstrated that P2X4/5-HT 3 A receptor complexes can interact with each other in a 1:1 stoichiometric manner that is preserved after ATP binding. Also, macromolecular docking followed by 100 ns molecular dynamics (MD) simulations suggested that the interaction energy of the P2X4 receptor with 5-HT 3 A receptor is similar at the holo and the apo state of the P2X4 receptor, and the interacting 5-HT 3 A receptor decreased the ATP binding energy of P2X4 receptor. Finally, the P2X4 receptor-dependent Ca 2+ mobilization is inhibited by the 5-HT 3 A interacting receptor. Altogether, these findings provide novel molecular insights into the allosteric regulation of P2X4/5-HT 3 A receptor complex in lipid bilayers of living cells via stoichiometric association, rather than accumulation or unspecific clustering of complexes.