Regulation of the pH sensitivity of human P2X receptors by N-linked glycosylation.
The human (h) P2X(3) 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 alpha,beta-methylene ATP (alpha,beta-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 (E(max)) without affecting the EC(50) value. The replacement of Asn-139 and -170 by Asp (N139D, N170D) abolished the pH sensitivity of the wild-type (WT) hP2X(3) receptor. In the case of N194D, the E(max) 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 E(max) 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 alpha,beta-meATP at any of the pH values investigated. In accordance with the reported finding that His-206 is involved in the modulation of WT P2X(3) receptors by protons, we showed that the normal change of E(max) 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 E(max). In conclusion, only fully N-glycosylated P2X(3) receptors recognize external pH with a modified sensitivity towards alpha,beta-meATP.
Wirkner
Kerstin
K
Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Haertelstrasse, Leipzig, Germany.
Stanchev
Doychin
D
Milius
Doreen
D
Hartmann
Lars
L
Kato
Erzsebet
E
Zadori
Zoltan S
ZS
Mager
Peter P
PP
Rubini
Patrizia
P
Nörenberg
Wolfgang
W
Illes
Peter
P
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D019943:Amino Acid Substitution; D000704:Analysis of Variance; D000970:Antineoplastic Agents / Q000494:pharmacology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000187:drug effects; D016296:Mutagenesis; D018408:Patch-Clamp Techniques; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D058469:Receptors, Purinergic P2X
D000970:Antineoplastic Agents; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate