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J. Biol. Chem., Vol. 283, Issue 38, 26241-26251, September 19, 2008

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Molecular Shape, Architecture, and Size of P2X₄ Receptors Determined Using Fluorescence Resonance Energy Transfer and Electron Microscopy^*

Mark T. Young¹, James A. Fisher, Samuel J. Fountain^¶, Robert C. Ford, R. Alan North^¶, and Baljit S. Khakh^||2

From the Manchester Interdisciplinary Biocentre, University of Manchester, Manchester M1 7DN, United Kingdom, the Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom, the ^¶Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom, and the ^||Departments of Physiology and Neurobiology, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1751

P2X receptors are ATP-gated nonselective cation channels with important physiological roles. However, their structures are poorly understood. Here, we analyzed the architecture of P2X receptors using fluorescence resonance energy transfer (FRET) microscopy and direct structure determination using electron microscopy. FRET efficiency measurements indicated that the distance between the C-terminal tails of P2X₄ receptors was 5.6 nm. Single particle analysis of purified P2X₄ receptors was used to determine the three-dimensional structure at a resolution of 21Å_; the orientation of the particle with respect to the membrane was assigned by labeling the intracellular C termini with 1.8-nm gold particles and the carbohydrate-rich ectodomain with lectin. We found that human P2X₄ is a globular torpedo-like molecule with an approximate volume of 270 nm³ and a compact propeller-shaped ectodomain. In this structure, the distance between the centers of the gold particles was 6.1 nm, which closely matches FRET data. Thus, our data provide the first views of the architecture, shape, and size of single P2X receptors, furthering our understanding of this important family of ligand-gated ion channels.

Received for publication, June 10, 2008 , and in revised form, July 14, 2008.

^* This work was supported, in whole or in part, by the National Institutes of Health (to B. S. K., subcontract on Grant GM070925). This work was also supported by the Wellcome Trust (to R. A. N.), the Medical Research Council (to J. A. F. and B. S. K.), and the Human Frontier Science Program (to B. S. K.). 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 Recipient of a Wellcome Trust advanced training fellowship.

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² To whom correspondence should be addressed: Depts. of Physiology and Neurobiology, David Geffen School of Medicine, UCLA, 10833 Le Conte Ave., Los Angeles, CA 90095-1751. E-mail: bkhakh{at}mednet.ucla.edu.

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