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

Abstract

1. Effects of external pH on the human P2X4 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 currentvoltage relationship of the ATP-activated current. 3. Diethyl pyrocarbonate (DEPC; 10 mÒ) treatment of P2XÚ-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 P2X4 receptor at all agonist concentrations. ATP potency showed a small (fourfold) leftward shift. Mutagenesis of the other three histidines present in the P2XÚ sequence had no effect on pH sensitivity. 5. The results show that pH modulation of P2X4 in the pathophysiological range is mediated by protonation of H286. This provides direct confirmation that pH sensitivity resides in the P2X4 channel protein rather than the agonist species.

Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism

“…23 Extracellular histidine residues account for proton sensitivity in several membrane proteins, including the anion exchange protein 2, 24 ovarian cancer G protein-coupled receptor-1 25 and purinergic P234 receptor. 26,27 Despite this, we found no evidence that any of the 16 extracellular histidine residues or the one free-cysteine residue (Cys-482) are individually responsible for CaR pH o sensitivity, at least over the pathophysiologic pH o range tested. Among members of the homologous family C GPCRs, mGluR4 is also inhibited by acidity and activated by alkalinity whereas mGluRs 1, 5 and 8 are pH o insensitive.…”

Histidine 140 Plays a Key Role in the Inhibitory Modulation of the P2X4 Nucleotide Receptor by Copper but Not Zinc

“…DEPC was used to specifically alkylate the imidazole group of histidines (22); this treatment resulted in the loss of the copper inhibition and a reduction in the P2X 4 ATP-evoked currents of the receptor. We are aware that there are controversial results reported in the literature with respect to the use of DEPC, because some authors have successfully modified histidine residues (23)(24)(25), whereas others have failed to detect significant changes (26,27). Considering that the rat P2X 4 receptor has only three histidine residues, it became challenging to establish which of these imidazole-bearing residues is responsible for the negative modulator role of copper, as well as the decreased channel activity.…”

“…In the human P2X 4 receptor, the pH sensor is related to His-286, because the replacement of this residue by an alanine abolishes the pH-associated effect (27). The H286A mutant studied here showed a lower channel conductance, making it likely that this amino acid could be acting as a pH sensor in both rat and human receptors.…”

Identification of Ectodomain Regions Contributing to Gating, Deactivation, and Resensitization of Purinergic P2X Receptors

“…P2XRs differ in their sensitivity for ATP with EC 50 values in the following order: P2X 1 R ϭ P2X 3 R Ͻ P2X 2 R Ͻ P2X 4 R ϭ P2X 5 R Ͻ P2X 6 R Ͻ Ͻ P2X 7 R. There are other pharmacological distinctions among receptors, such as the sensitivity to ␣␤-methylene-ATP and antagonists (Khakh et al, 2001), suggesting the structural specificity of the ligandbinding pocket. The ATP binding site was partially characterized (Jiang et al, 2001;Roberts and Evans, 2004), and P2XR ectodomain also contains sites for antagonists and modulators (GarciaGuzman et al, 1997;Clarke et al, 2000;Coddou et al, 2003). The relevance of residues that line the channel walls was also studied (Rassendren et al, 1997;Egan et al, 1998).…”