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J. Biol. Chem., Vol. 281, Issue 51, 39561-39572, December 22, 2006

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P2X₅ Subunit Assembly Requires Scaffolding by the Second Transmembrane Domain and a Conserved Aspartate^*

From the Department of Molecular Pharmacology, Rheinisch-Westfaelische Technische Hochschule (RWTH) Aachen, Wendlingweg 2, D-52074 Aachen, Germany

Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X₁-P2X₇), P2X₅ occurs in humans primarily as a non-functional variant lacking the C-terminal end of the ectodomain and the outer half of the second transmembrane domain. We show that this truncated variant, which results from the splice-skipping of exon 10, is prone to subunit aggregation because the residual transmembrane domain 2 is too short to insert into the membrane. Alleviation of the negative hydrophobic mismatch by the addition of a stretch of moderately hydrophobic residues enabled formation of a second membrane-spanning domain and strictly parallel homotrimerization. Systematic mutagenesis identified only one transmembrane domain 2 residue, Asp³⁵⁵, which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X₅ homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp³⁵⁵. While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp³⁵⁵ seems to assist by simultaneously driving intramembrane helix interactions. Overall, these results indicate a complex interplay between topology, helix-helix interactions, and oligomerization to achieve a correctly folded structure.

Received for publication, June 26, 2006 , and in revised form, September 6, 2006.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants Schm 536/6 and Schm536/7 (to G. S.) and by the START-Program of the Faculty of Medicine, RWTH Aachen University (to R. H.). 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: Section on Molecular Neurobiology, Laboratory of Molecular Biology, National Institute of Mental Health, Bldg. 10, Rm. 4D14, 10 Center Dr., Bethesda, MD 20892

² To whom correspondence should be addressed. Tel.: 49-241-8089130; Fax: 49-241-8082433; E-mail: gschmalzing{at}ukaachen.de.

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