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

Abstract

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.

Cross-Talk between P2X and NMDA Receptors

“…While it would be interesting to see the extent of P2X–NMDAR inhibition at lower concentrations, such as EC 10-20 ., such studies are out of the scope of this current work. However, it is important to note that non-additive interactions between P2X2 and 5-HT 3A have been, reported at both low and saturating agonist concentrations [ 22 , 23 , 24 ].…”

“…P2X cross-talk can rely on physical interactions between the intracellular domains of the two different receptors and may also regulate the subcellular targeting of receptors in neurons. Cross-talk between several P2X subtypes has been shown to modulate the activity of GABA-A receptors [ 17 , 18 , 19 , 20 ], nicotinic ACh receptors [ 21 ] and 5-Hydroxytryptamine Receptor 3A (5-HT 3A ) [ 22 , 23 , 24 ]. Alternatively, P2X can also have slow but long-lasting modulatory effects on the function or surface trafficking of receptors; activation of post-synaptic P2X by ATP released from glia has been shown to trigger changes in the surface trafficking of α-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid receptors (AMPAR), which leads to long lasting changes in synaptic efficacy at glutamatergic synapses.…”

Regulation of membrane NMDA receptors by dynamics and protein interactions

“…In the hippocampus, the activation of postsynaptic P2X2 or P2X4 triggers surface AMPAR internalization, leading to P2X-mediated long-term synaptic depression ( Pougnet et al, 2014 , 2016 ). P2X receptors further modulate the activity of GABAa receptors ( Boué-Grabot et al, 2004 ; Toulmé et al, 2007 ; Jo et al, 2011 ), nACh receptors ( Khakh et al, 2000 ), and 5-hydroxytryptamine receptor 3A ( Boué-Grabot et al, 2003 ; Emerit et al, 2016 ; Soto et al, 2020 ). Coexpression of P2X receptors and NMDARs in oocytes showed that they interact via the C-terminal domain of NMDAR, and their activation inhibits NMDAR-dependent currents ( Rodriguez et al, 2020 ).…”