Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA092

Poster Communications

P2X4 re-sensitisation depends on recycling via acidic organelles

G. Fois1, K. J. Föhr2, C. Kling1, M. Fauler1, O. H. Wittekind1, P. Dietl1, M. Frick1

1. Institute of general physiology, University of Ulm, Ulm, Germany. 2. Department of Anesthesiology, University of Ulm, Ulm, Germany.

P2X receptors constitute a family of seven different cation-permeable, ligand-gated ion channels present on the plasma membrane of most cells. These receptors are activated upon binding of ATP; this activation is followed by a desensitization phase and a deactivation phase upon removal of ATP. To return in its resting state the receptor needs a re-sensitization phase. Thus this cycle of de-sensitization - re-sensitization is crucial in the receptor signal transduction. Furthermore, since some receptors of the P2X family are mainly expressed on intracellular organelles, it has been suggested that cellular trafficking might also regulate P2X receptors signaling. P2X4 is predominately localized on the limiting membrane of late endosomes, lysosomes and lysosome-related organelles, which are all characterized by the acidic luminal pH. Since acidic pH inhibits activation of these receptors, the physiological role of their expression on acidic compartments has long remained elusive. In this study, we investigate the trafficking of P2X4 receptor to acidic compartments and how trafficking affects re-sensitization of the receptors. To characterize the effect of acidic pH on channel re-sensitization, we overexpressed (wt)P2X4-EGFP in HeLa cells and we performed intracellular calcium measurements ([Ca2+]i). We compared the responses to two subsequent stimulation of ATP (5 min interval) exposing the cells to bath solution with decreasing pH and we observed a pH dependent re-sensitization of P2X4. We subsequently increased the time interval between the two stimulations and we observed that after 30 min the recovery was almost complete. We than blocked organelles acidification, clathrin-mediated endocytosis and exocytosis. All of these perturbations significantly reduced the Ca2+-peak following second ATP (< 19% for all conditions), indicating that inhibition of recycling and/or acidification inhibits P2X4 re-sensitization. We generated P2X4 mutants were all the histidine residues on the extracellular loops were substituted by alanine, arginine or aspartate. All 3 mutants have significant effects for full recovery of P2X4 suggesting that protonation and de-protonation of critical histidine residues is crucial. In our study, we provide evidence that targeting of P2X4 receptors to acidic organelles is essential for receptor re-sensitization. Our findings indicate that the internalization/recycling via acidic organelles facilitate protonation/de-protonation of critical histidine residues within the extracellular loop that is required for receptor recovery, in particular H286.

Where applicable, experiments conform with Society ethical requirements