Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Proceedings of the National Academy of Sciences of the United States of America Année : 2012

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

Résumé

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.
Fichier principal
Vignette du fichier
PNAS-Lörinczi-2012.pdf (1.85 Mo) Télécharger le fichier
Origine : Fichiers éditeurs autorisés sur une archive ouverte
Loading...

Dates et versions

hal-02306930 , version 1 (07-10-2019)

Identifiants

Citer

Éva Lörinczi, Yogesh Bhargava, Stephen Marino, Antoine Taly, Karina Kaczmarek-Hájek, et al.. 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, 2012, 109 (28), pp.11396-11401. ⟨10.1073/pnas.1118759109⟩. ⟨hal-02306930⟩
197 Consultations
32 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More