Role of aromatic and charged ectodomain residues in the P2X(4) receptor functions.
The localization of ATP binding site(s) at P2X receptors and the molecular rearrangements associated with opening and closing of channels are still not well understood. At P2X(4) receptor, substitution of the K67, F185, K190, F230, R278, D280, R295, and K313 ectodomain residues with alanine generated low or non-responsive mutants, whereas the F294A mutant was functional. The loss of receptor function was also observed in K67R, R295K, and K313R mutants, but not in F185W, K190R, F230W, R278K, and D280E mutants. To examine whether the loss of function reflects decreased sensitivity of mutants for ATP, we treated cells with ivermectin, an antiparasitic agent that enhances responsiveness of P2X(4)R. In the presence of ivermectin, all low or non-responsive mutants responded to ATP in a dose-dependent manner, with the EC(50) values for ATP of about 1, 2, 4, 20, 60, 125, 270, 420, 1000 and 2300 micromol/L at D280A, R278A, F185A, K190A, R295K, K313R, R295A, K313A, K67A and K67R mutants, respectively. These results indicate that lysines 67 and 313 and arginine 295 play a critical role in forming the proper three-dimensional structure of P2X(4)R for agonist binding and/or channel gating.
Section on Cellular Signaling, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
Hana
H
Zemkova
Zonghe
Z
Yan
Zhaodong
Z
Liang
Irena
I
Jelinkova
Melanija
M
Tomic
Stanko S
SS
Stojilkovic
D000255:Adenosine Triphosphate / Q000494:pharmacology; D024322:Amino Acids, Aromatic / Q000502:physiology*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002461:Cell Line, Transformed; D004305:Dose-Response Relationship, Drug; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism; D006801:Humans; D007031:Hypothalamus / Q000166:cytology; D007559:Ivermectin / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000528:radiation effects; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009474:Neurons / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding / Q000187:drug effects; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000502:physiology*; D058848:Receptors, Purinergic P2X4; D013329:Structure-Activity Relationship; D014162:Transfection
D024322:Amino Acids, Aromatic; C548226:P2RX4 protein, human; C548227:P2rx4 protein, mouse; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D007987:Gonadotropin-Releasing Hormone; D007559:Ivermectin; D000255:Adenosine Triphosphate