Mutation of histidine 286 of the human P2X 4 purinoceptor removes extracellular pH sensitivity

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.