Function of P2X4 Receptors Is Directly Modulated by a 1:1 Stoichiometric Interaction With 5-HT3A Receptors

• Main Authors: Paola Soto, Pablo S. Gaete, Christian Fuentes, Benjamin Lozano, Pamela A. Naulin, Xavier F. Figueroa, Nelson Patricio Barrera
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2020-05-01
• Series: Frontiers in Cellular Neuroscience
• Subjects: receptor-receptor interaction; atomic force microscopy; stoichiometry; P2X4 receptor; 5-HT3A receptor; intracellular Ca2+
• Online Access: https://www.frontiersin.org/article/10.3389/fncel.2020.00106/full

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 Ca2+ levels. It has been proposed that the P2X4 receptor interacts with the 5-HT3A 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-HT3A 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-HT3A receptor is similar at the holo and the apo state of the P2X4 receptor, and the interacting 5-HT3A receptor decreased the ATP binding energy of P2X4 receptor. Finally, the P2X4 receptor-dependent Ca2+ mobilization is inhibited by the 5-HT3A interacting receptor. Altogether, these findings provide novel molecular insights into the allosteric regulation of P2X4/5-HT3A receptor complex in lipid bilayers of living cells via stoichiometric association, rather than accumulation or unspecific clustering of complexes.