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The Journal of Neuroscience, November 21, 2007, 27(47):12916-12923; doi:10.1523/JNEUROSCI.4036-07.2007

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Cellular/Molecular
Thr³³⁹-to-Serine Substitution in Rat P2X₂ Receptor Second Transmembrane Domain Causes Constitutive Opening and Indicates a Gating Role for Lys³⁰⁸

Lishuang Cao, Mark T. Young, Helen E. Broomhead, Samuel J. Fountain, and R. Alan North

Faculty of Life Sciences, University of Manchester, Manchester M13 9PL, United Kingdom

Correspondence should be addressed to Lishuang Cao, Michael Smith Building, Faculty of Life Sciences, University of Manchester, Manchester M13 9PL, UK. Email: lishuang.cao{at}manchester.ac.uk

P2X₂ receptors are ATP-gated ion channels widely expressed by neurons. Thr³³⁹ lies in the second of the two transmembrane domains of the rat P2X₂ receptor protein, and is likely to be close to the narrowest part of the pore. Single-channel and whole-cell recording after expression in human embryonic kidney 293 cells showed that P2X₂[T339S] receptors had pronounced spontaneous channel openings that were never seen in wild-type P2X₂ receptors. P2X₂[T339S] receptors were 10-fold more sensitive than wild type to exogenous ATP, and ßmeATP also increased channel opening. Two conserved ectodomain lysine residues (Lys⁶⁹ and Lys³⁰⁸) are critical for function and have been proposed to contribute to the ATP binding site of P2X receptors. The spontaneous opening of P2X₂[K69A/T339S] receptors was not different than that seen in P2X₂[T339S], but for P2X₂[K308A/T339S] the spontaneous activity was absent. Suramin, which is a noncompetitive antagonist at wild-type P2X₂ receptors, had a pronounced agonist action at both P2X₂[T339S] and P2X₂[K69A/T339S] receptors but not at P2X₂[K308A/T339S]. 2',3'-O-O-(2,4,6-Trinitrophenyl)-ATP (TNP-ATP), which is a competitive agonist at wild-type receptors, was also an agonist at P2X₂[T339S] receptors, but not at either double mutant. The results indicate that the T339S mutation substantially destabilizes the closed channel and suggest an important role in channel gating. The correction of this gating defect, in the absence of any agonist, by the second mutation K308A shows that Lys³⁰⁸ is also involved in channel gating. A similar interpretation can account for the results with suramin and TNP-ATP.

Key words: P2X receptors; ATP; binding site; channel gating; mutations; lysine

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Received Sept. 4, 2007; revised Oct. 8, 2007; accepted Oct. 10, 2007.

Correspondence should be addressed to Lishuang Cao, Michael Smith Building, Faculty of Life Sciences, University of Manchester, Manchester M13 9PL, UK. Email: lishuang.cao{at}manchester.ac.uk

This article has been cited by other articles:

				J. A. Roberts, H. R. Digby, M. Kara, S. E. Ajouz, M. J. Sutcliffe, and R. J. Evans Cysteine Substitution Mutagenesis and the Effects of Methanethiosulfonate Reagents at P2X2 and P2X4 Receptors Support a Core Common Mode of ATP Action at P2X Receptors J. Biol. Chem., July 18, 2008; 283(29): 20126 - 20136. [Abstract] [Full Text] [PDF]

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