Role of aromatic and charged ectodomain residues in the P2X₄ 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 P2X₄ receptor, substitution of the K67, F185, K190, F230, R278, D280, R295, and K313 ectodomain residues with alanine generated...

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Published in:Journal of neurochemistry 2007-08, Vol.102 (4), p.1139-1150
Main Authors: Zemkova, Hana, Yan, Zonghe, Liang, Zhaodong, Jelinkova, Irena, Tomic, Melanija, Stojilkovic, Stanko S
Format: Article
Language:English
Subjects:
Adenosine triphosphatase
ATP
Binding sites
Biochemistry
Biological and medical sciences
Cell receptors
Cell structures and functions
Central nervous system
Central neurotransmission. Neuromudulation. Pathways and receptors
deactivation
Fundamental and applied biological sciences. Psychology
ivermectin
ligand binding domain
Molecular and cellular biology
Molecular biology
Monoamines receptors (catecholamine, serotonine, histamine, acetylcholine)
Mutation
Neurosciences
P2X
purinergic receptors
Vertebrates: nervous system and sense organs
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Summary: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 P2X₄ 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 P2X₄R. In the presence of ivermectin, all low or non-responsive mutants responded to ATP in a dose-dependent manner, with the EC₅₀ values for ATP of about 1, 2, 4, 20, 60, 125, 270, 420, 1000 and 2300 μmol/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 P2X₄R for agonist binding and/or channel gating.
ISSN:0022-3042
1471-4159
DOI:10.1111/j.1471-4159.2007.04616.x