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J Biol Chem 2008 Jul 18;28329:20126-36. doi: 10.1074/jbc.M800294200.
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Cysteine substitution mutagenesis and the effects of methanethiosulfonate reagents at P2X2 and P2X4 receptors support a core common mode of ATP action at P2X receptors.

Roberts JA , Digby HR , Kara M , El Ajouz S , Sutcliffe MJ , Evans RJ .

The agonist binding site of ATP-gated P2X receptors is distinct from other ATP-binding proteins. Mutagenesis on P2X(1) receptors of conserved residues in mammalian P2X receptors has established the paradigm that three lysine residues, as well as FT and NFR motifs, play an important role in mediating ATP action. In this study we have determined whether cysteine substitution mutations of equivalent residues in P2X(2) and P2X(4) receptors have similar effects and if these mutant receptors can be regulated by charged methanethiosulfonate (MTS) compounds. All the mutants (except the P2X(2) K69C and K71C that were expressed, but non-functional) showed a significant decrease in ATP potency, with >300-fold decreases for mutants of the conserved asparagine, arginine, and lysine residues close to the end of the extracellular loop. MTS reagents had no effect at the phenylalanine of the FT motif, in contrast, cysteine mutation of the threonine was sensitive to MTS reagents and suggested a role of this residue in ATP action. The lysine-substituted receptors were sensitive to the charge of the MTS reagent consistent with the importance of positive charge at this position for coordination of the negatively charged phosphate of ATP. At the NFR motif the asparagine and arginine residues were sensitive to MTS reagents, whereas the phenylalanine was either unaffected or showed only a small decrease. These results support a common site of ATP action at P2X receptors and suggest that non-conserved residues also play a regulatory role in agonist action.

PubMed ID: 18487206
PMC ID: PMC2459275
Article link: J Biol Chem
Grant support: [+]

Species referenced: Xenopus laevis
Genes referenced: p2rx1 p2rx2 p2rx4

Article Images: [+] show captions
References [+] :
Akabas, Acetylcholine receptor channel structure probed in cysteine-substitution mutants. 1992, Pubmed, Xenbase