Reactive Oxygen Species Potentiate the P2X₂Receptor Activity through Intracellular Cys⁴³⁰

Abstract

P2X receptor channels (P2XRs) are allosterically modulated by several compounds, mainly acting at the ectodomain of the receptor. Like copper, mercury, a metal that induces oxidative stress in cells, also stimulates the activity of P2X 2 R and inhibits the activity of P2X 4 R. However, the mercury modulation is not related to the extracellular residues critical for copper modulation. To identify the site(s) for mercury action, we generated two chimeras using the full size P2X 2 subunit, termed P2X 2a , and a splice variant lacking a 69 residue segment in the C terminal, termed P2X 2b , as the donors for intracellular and transmembrane segments and the P2X 4 subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X 4/2a chimera but was absent in oocytes expressing P2X 4/2b chimera. Site-directed mutagenesis experiments revealed that the Cys 430 residue mediates effects of mercury on the P2X 2a R activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide (H 2 O 2 ) and mitochondrial stress inducers myxothiazol and rotenone mimicked mercury effects. These experiments, done in both oocytes and human embryonic kidney HEK293 cells, revealed that these compounds potentiated the ATPevoked P2X 2a R and P2X 4/2a R currents but not P2X 2b R and P2X 2a -C430A and P2X 2a -C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the H 2 O 2 potentiation. Alkylation of Cys 430 residue with methylmethane-thiosulfonate also abolished the mercury and H 2 O 2 potentiation. Altogether, these results are consistent with the hypothesis that the Cys 430 residue is an intracellular P2X 2a R redox sensor.

Activation and Regulation of Purinergic P2X Receptor Channels

“…Intracellular Cys 430 was identified as a redox sensor for this receptor. It is noteworthy that the ATP-evoked currents in P2X4R were slightly inhibited by hydrogen peroxide, suggesting an opposite redox modulation that remains to be identified (Coddou et al, 2009). These novel findings indicate that P2XRs can modify their activity depending on the redox state of the cell.…”

“…These results suggest that P2X2R and P2X4R may have an allosteric site for CO or that this gas exerts its actions through indirect mechanisms. In this regard, the effects of CO in P2X2R and P2X4R are similar to those of ROS and mitochondrial stress inducers (Coddou et al, 2009). Therefore, it is plausible that CO can bind to mitochondrial complexes and stimulate ROS production, a mechanism that has been shown to modulate L-type Ca v channels (Peers et al, 2009).…”

“…However, this hypothesis was discarded when in was reported that the copper-resistant mutants were still modulated by mercury Lorca et al, 2005). Using chimeric P2X2/X4Rs, we recently found that the primary site of action for mercury is an intracellular cysteine at position 430 (Coddou et al, 2009 P2X7R Virginio et al, 1997). This metal seems to bind to the allosteric sites identified for zinc in the P2X2R and P2X4R.…”

Mitochondrial Superoxide Generation Enhances P2X7R-Mediated Loss of Cell Surface CD62L on Naive Human CD4+ T Lymphocytes

“…This suggests that these compounds require P2X7R activation to alter CD62L surface expression. ROS can affect the function of a number of ion channels including P2X2R, and we postulated that rotenone and antimycin A may be affecting P2X7R sensitivity (44,45). To confirm this, we treated naive CD4 + T lymphocytes with increasing concentrations of ATP following pretreatment with vehicle (DMSO), 5 mM rotenone, or 1 mM antimycin A and measured CD62L expression (Fig.…”

Dynamic Regulation of the GABAA Receptor Function by Redox Mechanisms

“…The damaging effects of ROS can be counteracted by the specific detoxifying enzymes and high levels of antioxidant agents in a sophisticated way such that their local concentration and distribution can be rapidly and precisely controlled to allow dynamic cell signaling (Rhee, 2006;Bao et al, 2009;Woo et al, 2010;Rice, 2011;Finkel, 2011). ROS effects in the nervous system can include transient changes in neuronal activity and synaptic plasticity (Klann, 1998;Bao et al, 2009), as well as diverse actions on both excitatory and inhibitory neurotransmission (Frantseva et al, 1998;Sah and Schwartz-Bloom, 1999;Kamsler and Segal, 2003), including regulatory changes on neurotransmitter receptors (Aizenman et al, 1990;Chu et al, 2006;Coddou et al, 2009;Accardi et al, 2014Accardi et al, , 2015Beltrán González et al, 2014;Penna et al, 2014).…”