Differential role of extracellular histidines in copper, zinc, magnesium and proton modulation of the P2X₇ purinergic receptor

Abstract

The P2X 7 receptor is a non-selective cationic channel activated by extracellular ATP, belonging to the P2X receptor family. To assess the role of extracellular histidines on the allosteric modulation of the rat P2X 7 receptor by divalent metals (copper, zinc and magnesium) and protons, these amino acid residues were singly substituted for corresponding alanines. Wild-type and mutated receptors were injected to Xenopus laevis oocytes; metal-related effects were evaluated by the two-electrode voltage-clamp technique. Copper inhibited the ATP-gated currents with a median inhibitory concentration of 4.4 ± 1.0 lmol/L. The inhibition was noncompetitive and time-dependent; copper was 60-fold more potent than zinc. The mutant H267A, resulted in a copper resistant receptor; mutants H201A and H130A were less sensitive to copper inhibition (p < 0.05). The rest of the mutants examined, H62A, H85A, and H219A, conserved the copper-induced inhibition. Only mutants H267A and H219Awere less sensitive to the modulator action of zinc. Moreover, the magnesium-induced inhibition was abolished exclusively on the H130A and H201A mutants, suggesting that this metal may act at a novel cationic modulator site. Media acidification inhibited the ATP-gated current 87 ± 3%; out of the six mutants examined, only H130A was significantly less sensitive to the change in pH, suggesting that His-130 could be involved as a pH sensor. In conclusion, while His-267 is critically involved in the copper or zinc allosteric modulation, the magnesium inhibitory effects is related to His-130 and His-201, His-130 is involved in proton sensing, highlighting the role of defined extracellular histidines in rat P2X 7 receptor allosteric modulation.

P2X7 receptor activation induces reactive oxygen species formation in erythroid cells

“…This suggests that millimolar (or perhaps micromolar) concentrations of extracellular Ca 2+ and Mg 2+ may act as allosteric inhibitors of murine P2X7 as originally observed for rat P2X7 [46] and established by more recent studies of rat P2X7 [28,29]. The current study also demonstrates that an increase in intracellular Ca 2+ is not essential for P2X7-induced ROS formation (and pore formation) in MEL cells.…”

The P2X7 receptor mediates the uptake of organic cations in canine erythrocytes and mononuclear leukocytes: comparison to equivalent human cell types

“…It has long been considered that Mg 2+ chelates ATP 4− , the active form of the agonist, thereby inhibiting P2X 7 [39]. More recent data, however, demonstrates that Mg 2+ binds to the positively charged residues, histidine 130 and histidine 201 of rat P2X 7 to impair receptor function [40]. In this regard, histidine 201 is present in canine P2X 7 , as well as human P2X 7 , while an uncharged serine exists at position 130 in P2X 7 from these species [23,34].…”

Microglia Proliferation Is Controlled by P2X7 Receptors in a Pannexin-1-Independent Manner during Early Embryonic Spinal Cord Invasion

“…5G), but the Zn 2ϩ effect on P2X7R remains controversial and depends on the preparation used. Indeed, Zn 2ϩ has a low potency on rat P2X7R expressed in oocytes (Coddou et al, 2002;Acuña-Castillo et al, 2007), when compared with rat P2X7R expressed in transfected HEK cells (Liu et al, 2008).…”