Mutation of histidine 286 of the human P2X4 purinoceptor removes extracellular pH sensitivity

• Published in: The Journal of physiology Vol. 523; no. 3; pp. 697 - 703
• Main Authors: Clarke, C. E., Benham, C. D., Bridges, A., George, A. R., Meadows, H. J.
• Format: Journal Article
• Language: English
• Published: Oxford, UK The Physiological Society 15.03.2000; Blackwell Science Ltd; Blackwell Science Inc
• Subjects: Adenosine Triphosphate - pharmacology; Amino Acid Sequence - genetics; Amino Acid Substitution - physiology; Animals; Diethyl Pyrocarbonate - pharmacology; Electric Conductivity; Extracellular Space - metabolism; Female; Humans; Hydrogen - metabolism; Hydrogen-Ion Concentration; Mutagenesis, Site-Directed; Mutation - physiology; Oocytes; Original; Receptors, Purinergic P2 - genetics; Receptors, Purinergic P2 - metabolism; Receptors, Purinergic P2 - physiology; Receptors, Purinergic P2X4; Xenopus laevis
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1111/j.1469-7793.2000.00697.x

Effects of external pH on the human P2X 4 purinoceptor, an ATP-activated ion channel, were studied using the Xenopus oocyte expression system. 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. Diethyl pyrocarbonate (DEPC; 10 mM) treatment of P2X 4 -injected oocytes had no effect on the pH sensitivity of the ATP-activated current. 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. 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.