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J. Biol. Chem., Vol. 283, Issue 29, 20126-20136, July 18, 2008

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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^*

Jonathan A. Roberts, Helen R. Digby, Madina Kara, Sam El Ajouz, Michael J. Sutcliffe¹, and Richard J. Evans²

From the Department of Cell Physiology and Pharmacology, Henry Wellcome Building, and the Department of Biochemistry, University of Leicester, University Road, Leicester LE1 9HN, United Kingdom

The agonist binding site of ATP-gated P2X receptors is distinct from other ATP-binding proteins. Mutagenesis on P2X₁ 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 P2X₂ and P2X₄ receptors have similar effects and if these mutant receptors can be regulated by charged methanethiosulfonate (MTS) compounds. All the mutants (except the P2X₂ 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.

Received for publication, January 11, 2008 , and in revised form, May 16, 2008.

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^* This work was supported by the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: School of Chemical Engineering and Analytical Science, University of Manchester.

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² To whom correspondence should be addressed. Tel.: 44-116-229-7059; Fax: 44-116-252-5045; E-mail: rje6{at}le.ac.uk.

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