The human (h) P2X3 receptor and its mutants deficient in one out of four N-glycosylation sites were expressed in HEK293 cells. Concentration-response curves were generated by whole-cell recordings of α,β-methylene ATP (α,β-meATP)-induced currents. A gradual change of external pH from the alkaline 8.0 to the acidic 5.0 successively decreased the maximum current amplitude (Emax) without affecting the EC50 value. The replacement of Asn-139 and -170 by Asp (N139D, N170D) abolished the pH sensitivity of the wild-type (WT) hP2X3 receptor. In the case of N194D, the Emax was again the highest at the alkaline pH value with no change from 7.4 to 6.5, whereas in the case of N290D, there was an inverse pH sensitivity, with an increase of Emax in the acidic range. However, this effect appeared to be due to enhanced protonation by the insertion of Asp into the receptor, because replacement of Asn by the neutral Thr resulted in a comparable potency of α,β-meATP at any of the pH values investigated. In accordance with the reported finding that His-206 is involved in the modulation of WT P2X3 receptors by protons, we showed that the normal change of Emax by an acidic, but not alkaline pH was abolished after substitution of this His by Ala. However, the double mutant H206A + N290D did not react to acidification or alkalinization with any change in Emax. In conclusion, only fully N-glycosylated P2X3 receptors recognize external pH with a modified sensitivity towards α,β-meATP.
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience