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Review
. 2014 Aug 20;21(6):953-70.
doi: 10.1089/ars.2013.5549. Epub 2013 Sep 25.

Regulation of ATP-gated P2X channels: from redox signaling to interactions with other proteins

Stanko S Stojilkovic  1 , Elías Leiva-Salcedo, Milos B Rokic, Claudio Coddou
Affiliations
Review

Regulation of ATP-gated P2X channels: from redox signaling to interactions with other proteins

Stanko S Stojilkovic et al. Antioxid Redox Signal. .

Abstract

Significance: The family of purinergic P2X receptors (P2XRs) is a part of ligand-gated superfamily of channels activated by extracellular adenosine-5'-triphosphate. P2XRs are present in virtually all mammalian tissues as well as in tissues of other vertebrate and nonvertebrate species and mediate a large variety of functions, including fast transmission at central synapses, contraction of smooth muscle cells, platelet aggregation, and macrophage activation to proliferation and cell death.

Recent advances: The recent solving of crystal structure of the zebrafish P2X4.1R is a major advance in the understanding of structural correlates of channel activation and regulation. Combined with growing information obtained in the post-structure era and the reinterpretation of previous work within the context of the tridimensional structure, these data provide a better understanding of how the channel operates at the molecular levels.

Critical issues: This review focuses on the relationship between redox signaling and P2XR function. We also discuss other allosteric modulation of P2XR gating in the physiological/pathophysiological context. This includes the summary of extracellular actions of trace metals, which can be released to the synaptic cleft, pH decrease that happens during ischemia and inflammation, and calcium, an extracellular and intracellular messenger.

Future directions: Our evolving understanding of activation and regulation of P2XRs is helpful in clarifying the mechanism by which these channels trigger and modulate cellular functions. Further research is required to identify the signaling pathways contributing to the regulation of the receptor activity and to develop novel and receptor-specific allosteric modulators, which could be used in vivo with therapeutic potential.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Activation properties of rP2XRs. (A) Concentration–response relationships of five homomeric P2XRs expressed in HEK293 cells. Whole-cell currents were gated with the concentrations of ATP indicated in each case, and currents were normalized against the concentration of ATP that elicited the maximal response. (B) Desensitization profiles of homomeric P2XRs; superimposed recordings showing the different desensitization rates (τdes) derived from monoexponential fitting. Currents were gated with 10 μM (P2X1R and P2X3R), 100 μM (P2X2R and P2X4R), or 3 mM (P2X7R) ATP. P2XRs, purinergic P2×receptors; ATP, adenosine-5′-triphosphate.
<b>FIG. 2.</b>
FIG. 2.
Schematic representation of P2X7R-mediated ROS production by NADPH oxidase. Activation of the P2X7R can promote ROS production by NADPH oxidases, a cell membrane complex consisting of several subunits. On assembly of these subunits in the membrane, this enzyme via its NOX2 (gp91phox) catalytic subunit generates a burst of ROS and activation of ASK1-p38 pathway. Activated P2X7Rs trigger NADPH oxidase activity by the translocation of NOX2 to plasma membrane, which is a critical step in the activation of the NADPH oxidase. This represents the major pathway for P2X7R-induced ROS formation. Calcium influx through P2X7R pore and mitochondrial overload could also mediate ROS production. Calcium can also stimulate NO synthase to generate NO, which enhances ROS production by mitochondria. It also appears that P2X7R activates ERK1/2/JNK1 pathways. These pathways trigger activation of various caspases, leading to changes in cellular functions. The best-characterized signaling downstream event of P2X7R activation is mediating the processing and release of IL-1β and IL-18. The P2X7R-induced and caspase-mediated cell apoptosis is also well established. ROS, reactive oxygen species; IL, interleukin.
<b>FIG. 3.</b>
FIG. 3.
The P2X2R activity is regulated by ROS. (A) Representative recordings from HEK293 cells expressing the P2X2aR, P2X2bR, or the P2X2aR-C430S mutant. The potentiation induced by 1 mM H2O2 is observed only in cells expressing the P2X2aR. C, control; W, washout. (B) Effects of H2O2 on 3 μM ATP-evoked currents during repetitive 5 s agonist applications for 10 min in cells expressing P2X2aR (top) or P2X2bR (bottom). (C) Possible mechanisms of P2X2aR modulation by ROS. Cys430 residue could be oxidized indirectly and/or directly by ROS. Top, Indirect effects of H2O2, mercury and the oxidative stress inducers myxothiazol and rotenone could be mediated by ROS and free radicals (FR), including the hydroxyl radical (OH·) produced by mitochondria. H2O2 could also generate OH·through the Fenton reaction. Bottom, ROS could directly oxidize the SH group of Cys430, forming a mercaptide product CH2–S–Hg2+ or a sulfonic acid derivative CH2–SO2−. Endogenous antioxidants such as glutathione (GSH) could partially revert these reactions, resulting in the reduction of the Cys430 residue and the formation of glutathione/glutathione disulfide (GS-SG).
<b>FIG. 4.</b>
FIG. 4.
Schematic representation of Zn2+ and Cu2+ allosteric effects. The upper part represents positive allosteric regulation, which results in potentiation of ATP currents in some P2XRs that is exerted by both Zn2+ (left) and Cu2+ (right). The lower part represents negative allosteric regulation, which results in an inhibition of the ATP-currents. Gray traces represent the currents obtained with ATP alone, whereas black traces represent the currents after the joint application of ATP plus Zn2+ or Cu2+.
<b>FIG. 5.</b>
FIG. 5.
The P2X2R homology model in open and closed state. The model represents the spatial proximity of the ATP-binding site of the crucial Zn2+ and Cu2+-binding histidine residues (blue). The distances between the particular histidine residues (blue) are depicted in a straight line. ATP is depicted in yellow, and particular subunits are indicated in pink and pale green. The particular regions of the molecule are pointed in the miniatures in the inset. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 6.</b>
FIG. 6.
Calcium antagonism on the P2X7R is allosteric. (A) Concentration–response curves to ATP and BzATP in P2X7R-expressing HEK293 cells. The experiments were performed in the absence (open circles) or in the presence (closed circles) of 2 mM extracellular Ca2+. (B) Concentration-dependent effects of ATP and BzATP on free Ca2+ concentration measured with a calcium ion selective electrode. The calculated free Ca2+ concentrations were obtained using the web Maxc program. (C) A comparable free BzATP concentration of 1.6 μM was achieved with variable total BzATP and variable Ca2+ concentrations. (D) Current amplitudes of the different media shown in (C), containing identical free BzATP concentrations (1.6 μM) but increasing Ca2+ concentrations, demonstrating that Ca2+ itself and not its effects on free BzATP are responsible for current inhibition.
<b>FIG. 7.</b>
FIG. 7.
Calcium use-dependent desensitization on the P2X2R. (A) Recordings from the same HEK293 cell expressing the P2X2aR and stimulated with 10 μM ATP for 30 s and washout periods of 3 min in the continuous presence of 2 mM extracellular Ca2+. Note the increase in receptor desensitization after four ATP stimulations. (B) The same protocols as in (A), but using an extracellular media without Ca2+; no use-dependent desensitization is observed under these conditions.
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