Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor

Loerinczi, Eva, Bhargava, Yogesh, Marino, Stephen F., Taly, Antoine, Kaczmarek-Hajek, Karina, Barrantes-Freer, Alonso, Dutertre, Sebastien, Grutter, Thomas, Rettinger, Juergen and Nicke, Annette (2012) Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor. Proceedings of the National Academy of Sciences of the United States of America, 109 28: 11396-11401. doi:10.1073/pnas.1118759109


Author Loerinczi, Eva
Bhargava, Yogesh
Marino, Stephen F.
Taly, Antoine
Kaczmarek-Hajek, Karina
Barrantes-Freer, Alonso
Dutertre, Sebastien
Grutter, Thomas
Rettinger, Juergen
Nicke, Annette
Title Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor
Journal name Proceedings of the National Academy of Sciences of the United States of America   Check publisher's open access policy
ISSN 0027-8424
1091-6490
Publication date 2012-07-01
Sub-type Article (original research)
DOI 10.1073/pnas.1118759109
Open Access Status Not Open Access
Volume 109
Issue 28
Start page 11396
End page 11401
Total pages 6
Place of publication Washington, DC United States
Publisher National Academy of Science
Collection year 2013
Language eng
Formatted abstract
P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120-I125) were specifically labeled by tetramethyl-rhodamine-maleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoyl-benzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP-induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain.
Keyword Atp-Binding Sites
Ion-Channel
P2X(1) Receptor
Agonist Binding
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2013 Collection
Institute for Molecular Bioscience - Publications
 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 38 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 40 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Fri, 16 Nov 2012, 01:45:32 EST by System User on behalf of Institute for Molecular Bioscience