Abstract
In this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed ATP binding site of the human P2X3 receptor on the agonistic effect of nucleotide analogs. The wild-type and mutant receptors were expressed in HEK293 cells, and the nucleotide effects were measured by means of the whole-cell patch-clamp method. Modifications in the receptor binding site changed the concentration-response relationship, the current kinetics, and the recovery from desensitization during fast, pulsed, local agonist applications. On the basis of this fact, we were able to distinguish binding from other effects, such as gating/desensitization, by using a hidden Markov model that describes the complete channel behavior with a matrix of rate constants. The binding energies of the nucleotide analogs were calculated and compared with the binding energies of ATP at both the wild-type receptor and its alanine mutants. Changes in the binding energies caused by alterations in the receptor and/or agonist structures were concluded to be attributable to the preferential binding of certain structural constituents of ATP to certain amino acid moieties of the receptor. The results were also checked for consistency with a P2X3 homology model that we developed from the known zebrafish P2X4 crystal structure in the closed state. The functional data correlated well with the predictions of the agonist dockings to the structural model.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the Deutsche Forschungsgemeinschaft (Research Unit FOR748) [Grants RI 2092/1-2, IL 20/18-2].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- ASIC
- acid-sensing ion channel
- AA
- amino acid
- NBS
- nucleotide-binding segment
- R.M.S.D.
- root mean square deviation
- wt
- wild-type
- zf
- zebrafish
- me-ATP
- methylene-ATP
- ATP-γ-S
- adenosine 5′-O-(γ-thio)triphosphate
- 2-MeS-ATP
- 2-methylthio-ATP
- TNP-ATP
- trinitrophenyl-ATP
- Bz-ATP
- dibenzoyl-ATP.
- Received January 16, 2012.
- Accepted April 11, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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