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J. Biol. Chem., Vol. 283, Issue 37, 25725-25734, September 12, 2008

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The P2X₇ Carboxyl Tail Is a Regulatory Module of P2X₇ Receptor Channel Activity^*

Daniel Becker¹, Ronja Woltersdorf¹, Wolfgang Boldt, Stephan Schmitz, Ursula Braam, Günther Schmalzing²³, and Fritz Markwardt²⁴

From the Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle, Magdeburger Straße 6, D-06097 Halle/Saale and the Department of Molecular Pharmacology, Rheinisch-Westfaelische Technische Hochschule Aachen (RWTH), Aachen University, Wendlingweg 2, D-52074 Aachen, Germany

P2X₇ receptors are ATP-gated cation channels composed of three identical subunits, each having intracellular amino and carboxyl termini and two transmembrane segments connected by a large ectodomain. Within the P2X family, P2X₇ subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X₇ subunit as two complementary fragments, a carboxyl tail-truncated receptor channel core (residues 1-436 or 1-505) and a tail extension (residues 434-595) in Xenopus laevis oocytes. P2X₇ channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only 5% of the full-length P2X₇ receptor current. Co-assembly of channel core subunits with full-length P2X₇ subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X₇ receptor activity. Co-expression of the tail extension as a discrete protein increased ATP-gated current amplitudes of P2X₇ channel cores 10-20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X₇ receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X₇ channel core. We conclude that a non-covalent association of crucial functional importance exists between the carboxyl tail of the channel core and the tail extension. Using a slightly shorter P2X₇ subunit core and subfragments of the tail extension, this association could be narrowed down to include residues 409-436 and 434-494 of the split receptor. Together, these results identify the tail extension as a regulatory gating module, potentially making P2X₇ channel gating sensitive to intracellular regulation.

Received for publication, May 20, 2008 , and in revised form, June 18, 2008.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants Ma1581/12-1 (to F. M.), Schm536/6-3 and SFB 542 (to G. S.), and Medical Faculty of the Martin-Luther-University Roux Program Grants 5/09, 10/01, and 13/07 (to F. M.). 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 Both authors contributed equally to this article.

² Both authors contributed equally to this work as senior authors.

³ To whom correspondence may be addressed. Tel.: 49-241-8089130; Fax: 49-241-80-82433; E-mail: gschmalzing{at}ukaachen.de.

⁴ To whom correspondence may be addressed. Tel.: 49-345-5571390; Fax: 49-345-557-4019; E-mail: Fritz.Markwardt{at}medizin.uni-halle.de.

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