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J. Biol. Chem., Vol. 279, Issue 51, 53109-53115, December 17, 2004

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Identification of Negative Residues in the P2X₃ ATP Receptor Ectodomain as Structural Determinants for Desensitization and the Ca²+-sensing Modulatory Sites^*

Elsa Fabbretti, Elena Sokolova, Lara Masten, Marianna D'Arco, Alessandra Fabbro, Andrea Nistri, and Rashid Giniatullin

From the Neurobiology Sector, International School for Advanced Studies (SISSA), Via Beirut 4, 34014 Trieste, Italy

On nociceptive neurons, one important mechanism to generate pain signals is the activation of P2X₃ receptors, which are membrane proteins gated by extracellular ATP. In the presence of the agonist, P2X₃ receptors rapidly desensitize and then recover slowly. One unique property of P2X₃ receptors is the recovery acceleration by extracellular Ca²⁺ that can play the role of the gain-setter of receptor function only when P2X₃ receptors are desensitized. To study negatively charged sites potentially responsible for this action of Ca²⁺, we mutated 15 non-conserved aspartate or glutamate residues in the P2X₃ receptor ectodomain with alanine and expressed such mutated receptors in human embryonic kidney cells studied with patch clamping. Unlike most mutants, D266A (P2X₃ receptor numbering) desensitized very slowly, indicating that this residue is important for generating desensitization. Recovery appeared structurally distinct from desensitization because E111A and D266A had a much faster recovery and D220A and D289A had a much slower one despite their standard desensitization. Furthermore, E161A, E187A, or E270A mutants showed lessened sensitivity to the action of extracellular Ca²⁺, suggesting that these determinants were important for the effect of this cation on desensitization recovery. This study is the first report identifying several negative residues in the P2X₃ receptor ectodomain differentially contributing to the general process of receptor desensitization. At least one residue was important to enable the development of rapid desensitization, whereas others controlled recovery from it or the facilitating action of Ca²⁺. Thus, these findings outline diverse potential molecular targets to modulate P2X₃ receptor function in relation to its functional state.

Received for publication, August 25, 2004 , and in revised form, October 5, 2004.

^* This work is supported by Grant GGP04037 from Telethon Italy. 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.

Both authors contributed equally to this work.

To whom correspondence should be addressed: SISSA, Via Beirut 4, 34014 Trieste, Italy. Tel.: 39-040-3787-228; Fax: 39-040-3787-528; E-mail: nistri{at}sissa.it.

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