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Proc Natl Acad Sci U S A. 2016 Apr 26;113(17):4741-6. doi: 10.1073/pnas.1600519113. Epub 2016 Apr 11.

Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region.

Author information

1
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan;
2
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan;
3
Department of Physiology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan;
4
Institute for Innovation, Ajinomoto, Kawasaki 210-8681, Japan;
5
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Yangpu District, Shanghai 200438, China; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075, Japan.
6
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075, Japan.
7
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; shimada@iw-nmr.f.u-tokyo.ac.jp.

Abstract

Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s(-1)), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.

KEYWORDS:

NMR; insect cell expression system; ligand-gated ion channels; membrane proteins; purinergic receptors

PMID:
27071117
PMCID:
PMC4855573
DOI:
10.1073/pnas.1600519113
[Indexed for MEDLINE]
Free PMC Article

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