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Mutation of histidine 286 of the human P2X 4 purinoceptor removes extracellular pH sensitivity
Author(s) -
Clarke C. E.,
Benham C. D.,
Bridges A.,
George A. R.,
Meadows H. J.
Publication year - 2000
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.2000.00697.x
Subject(s) - histidine , xenopus , chemistry , agonist , alanine , extracellular , protonation , biophysics , mutagenesis , receptor , ion channel , biochemistry , purinergic receptor , amino acid , mutation , biology , ion , organic chemistry , gene
1 Effects of external pH on the human P2X 4 purinoceptor, an ATP‐activated ion channel, were studied using the Xenopus oocyte expression system. 2 Changing the external pH from 7·4 to 6·5 significantly reduced, whilst an increase to pH 8 enhanced, maximum ATP‐activated current amplitude, without changing the current‐ voltage relationship of the ATP‐activated current. 3 Diethyl pyrocarbonate (DEPC; 10 mM) treatment of P2X 4 ‐injected oocytes had no effect on the pH sensitivity of the ATP‐activated current. 4 Site‐directed mutagenesis of histidine 286 (H286) to alanine completely abolished the pH sensitivity of the P2X 4 receptor at all agonist concentrations. ATP potency showed a small (fourfold) leftward shift. Mutagenesis of the other three histidines present in the P2X 4 sequence had no effect on pH sensitivity. 5 The results show that pH modulation of P2X 4 in the pathophysiological range is mediated by protonation of H286. This provides direct confirmation that pH sensitivity resides in the P2X 4 channel protein rather than the agonist species.