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Crystal structure of the ATP-gated P2X4 ion channel in the closed state

Abstract

P2X receptors are cation-selective ion channels gated by extracellular ATP, and are implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. Because P2X receptors are not related to other ion channel proteins of known structure, there is at present no molecular foundation for mechanisms of ligand-gating, allosteric modulation and ion permeation. Here we present crystal structures of the zebrafish P2X4 receptor in its closed, resting state. The chalice-shaped, trimeric receptor is knit together by subunit–subunit contacts implicated in ion channel gating and receptor assembly. Extracellular domains, rich in β-strands, have large acidic patches that may attract cations, through fenestrations, to vestibules near the ion channel. In the transmembrane pore, the ‘gate’ is defined by an 8 Å slab of protein. We define the location of three non-canonical, intersubunit ATP-binding sites, and suggest that ATP binding promotes subunit rearrangement and ion channel opening.

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Figure 1: A functional P2X 4 receptor for structural studies.
Figure 2: The architecture of P2X receptors.
Figure 3: Subunit fold and intersubunit contacts.
Figure 4: Closed, resting conformation.
Figure 5: Gadolinium (Gd 3+ )-binding sites.
Figure 6: ATP-binding site.

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Acknowledgements

We thank the personnel at beamlines 5.0.2, 8.2.1 and 8.2.2 of the Advanced Light Source and at beamline 24-ID-E of the Advanced Photon Source. We also thank M. Voigt for zebrafish P2X receptor DNA, T. Homrichhausen for help with cloning and FSEC screening, J. Berriman for help with electron microscopy, L. Vaskalis for assistance with illustrations, and Gouaux laboratory members for discussions. This work was supported by the National Institutes of Health (NIH) and the American Asthma Foundation. E.G. is an investigator with the Howard Hughes Medical Institute.

Author Contributions E.G. and T.K. designed the project. T.K. performed cloning, cell culture, FSEC screening, purification, characterization, electron microscopy and crystallography. J.C.M. performed cloning, cell culture, FSEC screening, purification and crystallization. W.T.B. carried out the electrophysiology. All authors contributed to writing the manuscript.

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Correspondence to Eric Gouaux.

Additional information

Atomic coordinates and structure factors have been deposited with the Protein Data Bank under codes 3I5D and 3H9V for the ΔP2X4-A and ΔP2X4-B constructs, respectively.

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This file contains Supplementary Tables 1-2 and Supplementary Figures 1-13 with Legends. (PDF 1847 kb)

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Kawate, T., Michel, J., Birdsong, W. et al. Crystal structure of the ATP-gated P2X4 ion channel in the closed state. Nature 460, 592–598 (2009). https://doi.org/10.1038/nature08198

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