Interaction of Purinergic P2X4 and P2X7 Receptor Subunits

Abstract

P2X4 and P2X7 are members of the P2X receptor family, comprising seven isoforms (P2X1–P2X7) that form homo- and heterotrimeric non-specific cation channels gated by extracellular ATP. P2X4 and P2X7 are widely coexpressed, particularly in secretory epithelial cells and immune and inflammatory cells, and regulate inflammation and nociception. Although functional heteromerization has been established for P2X2 and P2X3 subunits expressed in sensory neurons, there are contradictory reports regarding a functional interaction between P2X4 and P2X7 subunits. To resolve this issue, we coexpressed P2X4 and P2X7 receptor subunits labeled with green (EGFP) and red (TagRFP) fluorescent proteins in Xenopus laevis oocytes and investigated a putative physical interaction between the fusion proteins by Förster resonance energy transfer (FRET). Coexpression of P2X4 and P2X7 subunits with EGFP and TagRFP located in the extracellular receptor domains led to significant FRET signals. Significant FRET signals were also measured between C-terminally fluorophore-labeled full-length P2X41-384 and C-terminally truncated fluorescent P2X71-408 subunits. We furthermore used the two-electrode voltage clamp technique to investigate whether human P2X4 and P2X7 receptors (hP2X4, hP2X7) functionally interact at the level of ATP-induced whole-cell currents. Concentration–response curves and effects of ivermectin (P2X4-potentiating drug) or BzATP (P2X7-specific agonist) were consistent with a model in which coexpressed hP2X4 and hP2X7 do not interact. Similarly, the effect of adding specific inhibitors of P2X4 (PSB-15417) or P2X7 (oATP, A438079) could be explained by a model in which only homomers exist, and that these are blocked by the respective antagonist. In conclusion, we show that P2X4 and P2X7 subunits can form heterotrimeric P2X4/P2X7 receptors. However, unlike observations for P2X2 and P2X3, coexpression of P2X4 and P2X7 subunits does not result in a novel electrophysiologically discriminable P2X receptor phenotype.

Macrophage P2X4 receptors augment bacterial killing and protect against sepsis

“…Our current results demonstrating in vivo augmentation of bacterial killing by P2X4Rs illustrates, however, that both P2X4Rs and P2X7Rs augment bacterial killing in vivo. There is abundant evidence for physical interactions between P2X4Rs and P2X7Rs in the literature (73,74). The question of how these receptors interact in sepsis will be the subject of future studies.…”

The P2X7 Receptor: Central Hub of Brain Diseases

“…In addition, the P2X7 receptor usually assembles as homotrimer (Sluyter and Stokes, 2011). However, it can also form heteromeric interactions with P2X4 receptor subunits as evidenced in 2007 by Guo et al (2007) and later confirmed by Schneider et al (2017).…”

P2 Receptors as Therapeutic Targets in the Salivary Gland: From Physiology to Dysfunction

“…Physical interactions between P2X4Rs and P2X7Rs have been demonstrated, although the nature of this interaction remains controversial (Kopp et al, 2019). Some studies suggest that P2X4R and P2X7R subunits form heteromeric channels (Guo et al, 2007;Schneider et al, 2017), while others conclude that P2X4 and P2X7 receptors interact in their respective homotrimeric form (Nicke, 2008;Boumechache et al, 2009;Antonio et al, 2011). Furthermore, P2X4R expression has been localized to lysosomal membranes, whereas P2X7Rs primarily reside at the plasma membrane (Guo et al, 2007;Huang et al, 2014).…”