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The Journal of Neuroscience, November 11, 2009, 29(45):14257-14264; doi:10.1523/JNEUROSCI.4403-09.2009

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Cellular/Molecular
Polar Residues in the Second Transmembrane Domain of the Rat P2X2 Receptor That Affect Spontaneous Gating, Unitary Conductance, and Rectification

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

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

Correspondence should be addressed to R. Alan North, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PL, UK. Email: r.a.north{at}manchester.ac.uk

Membrane ion channels activated by extracellular ATP (P2X receptors) are widely distributed in the nervous system. Their molecular architecture is fundamentally distinct from that of the nicotinic or glutamate receptor families. We have measured single-channel currents, spontaneous gating, and rectification of rat P2X2 receptor in which polar and charged residues of the second transmembrane domain (TM2) were systematically probed by mutagenesis. The results suggest that Asn³³³ and Asp³⁴⁹ lie respectively in external and internal vestibules. Substitutions at Asn³³³, Thr³³⁶, and Ser³⁴⁰ were particularly likely to cause spontaneously active channels. At Thr³³⁶, Thr³³⁹, and Ser³⁴⁰, the introduction of positive charge (Arg, Lys, or His, or Cys followed by treatment with 2-aminoethyl methanethiosulphonate) greatly enhanced outward currents, suggesting that side-chains of these three residues are exposed in the permeation pathway of the open channel. These functional findings are interpreted in the context of the recently reported 3.1 Å crystal structure of the zebrafish P2X4.1 receptor in the closed state. They imply that the gate is formed by residues Asn³³³ to Thr³³⁹ and that channel opening involves a counter-clockwise rotation and separation of the TM2 helices.

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Received Sept. 6, 2009; revised Sept. 29, 2009; accepted Oct. 5, 2009.

Correspondence should be addressed to R. Alan North, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PL, UK. Email: r.a.north{at}manchester.ac.uk

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