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

Abstract

To elucidate the role of extracellular histidines in the modulation of the rat P2X 4 receptor by trace metals, we generated single, double, and triple histidine mutants for residues 140, 241, and 286, replacing them with alanines. cDNAs for the wild-type and receptor mutants were expressed in Xenopus laevis oocytes and in human embryonic kidney 293 cells and examined by the two electrode and patch clamp techniques, respectively. Whereas copper inhibited concentration-dependently the ATP-gated currents in the wild-type and in the single or double H241A and H286A receptor mutants, all receptors containing H140A were insensitive to copper in both cell systems. The characteristic bell-shaped concentration-response curve of zinc observed in the wildtype receptor became sigmoid in both oocytes and human embryonic kidney cells expressing the H140A mutant; in these mutants, the zinc potentiation was 2.5-4-fold larger than in the wild-type. Results with the H140T and H140R mutants further support the importance of a histidine residue at this position. We conclude that His-140 is critical for the action of copper, indicating that this histidine residue, but not His-241 or His-286, forms part of the inhibitory allosteric metal-binding site of the P2X 4 receptor, which is distinct from the putative zinc facilitator binding site.The notion that trace metals such as zinc or copper are atypical brain messengers has attracted much attention in view of their emerging role in the modulation of brain excitability (1). The importance of trace metals in synaptic activity is highlighted by the description that both copper and zinc are stored in synaptic vesicles from where they are released by electrical depolarization, reaching a high micromolar concentration at the synaptic cleft (2-4). Trace metals are known to modulate a wide variety of brain ionotropic receptors such as glycine, N-methyl-D-aspartate, ␥-aminobutyric acid, nicotinic, and the novel nucleotide receptors (P2X) family (5-9). The P2X purinoceptors are homomeric or heteromeric membrane channels gated by extracellular ATP and related synthetic nucleotides; North (10) recently reviewed the principles of the molecular physiology of this family of receptors. Within the P2X receptor family, the P2X 4 is the most widely distributed in the central nervous system, including the cerebellum and the CA1 region of the hippocampus, where it has been proposed to play a role in glutamatergic synapses (11).The P2X 4 receptor is an interesting model of an ionic channel differentially modulated by trace metals. Acuñ a-Castillo et al. (12) and Coddou et al. (13) reported that zinc potentiates the ATP-evoked currents whereas copper has an inhibitory effect on the activity of this receptor. Based on these findings, Acuñ aCastillo et al. (12) proposed that trace metals modulate the activity of the P2X 4 receptor via two separate metal-binding sites. One of these sites has a preferential selectivity for copper and is characterized by a non-competitive inhibition of the ATP-gated channel acti...

Dissecting the Facilitator and Inhibitor Allosteric Metal Sites of the P2X4 Receptor Channel

“…We demonstrate that Cys 132 , but not Cys 126 , is critical for the modulator role of zinc but not copper, identifying the first critical amino acid residue necessary for zinc potentiation in this receptor channel. The C132A mutant proved not only resistant to the zinc-induced potentiation, but this metal inhibited ATP-gated currents, in full support that large zinc concentrations may also interact with the copper inhibitory site (13). In addition, we determined the key role of the carboxylic acid group of Asp 138 as a second residue critically involved in copper inhibition, establishing a possible metal coordination binding pocket in the extracellular receptor domain region surrounding His 140 .…”

“…Site-directed mutagenesis has provided pivotal information about specific P2X properties: the channel pore, agonist binding residues, receptor desensitization and allosteric modulation (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). As with other ligand-gated ionic channels, the P2X receptors are modulated by divalent metals including trace metals although the nature of the modulation and the magnitude of these effects vary among the different P2X subunits (12)(13)(14)(15)(16)(17). The role of divalent trace metals as neuromodulators is of interest as zinc and copper are both novel and atypical brain transmitters (18,19) and novel intracellular second messengers (20).…”

“…In a series of studies, Acuña-Castillo et al (16) and Coddou et al (23,24) reported that zinc potentiated the ATP-evoked currents, whereas copper exerts an inhibitory effect on the activity of this receptor. Furthermore, single site-directed mutagenesis of each of the three extracellular histidine residues of the P2X 4 receptor revealed that only histidine 140 plays a key role in the inhibitory modulation by copper and high zinc concentrations (13). The replacement of His 140 by an alanine (H140A mutant) was not only resistant to the copper-induced inhibition of the ATPgated receptor activity but evidenced a dramatic increase in the zinc-induced potentiation.…”

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).…”

Molecular Determinants of the Agonist Binding Domain of a P2X Receptor Channel

“…However, it is well established that the constituent components of ATP show no agonist or antagonist actions at P2XRs, indicating that both the adenine ring and the triphosphate chain are critical for high-affinity binding (North, 2002). It is also established that P2XRs are regulated allosterically by protons, divalent cations, and metals (Li et al, 1997;Wildman et al, 1999;Clarke et al, 2000;Negulyaev and Markwardt, 2000;Clyne et al, 2002a;Coddou et al, 2003).…”