**3. Activation mechanism of TRPM2 by redox signaling**

**Figure 1.** Chemical structure of TRPA1-activating reactive compounds (A) and reported target amino acid residues in

TRPA1 (B).

206 Redox - Principles and Advanced Applications

TRPM2 channels are activated by H2 O2 [17], and this activation could be enhanced by pretreatment with Fe2+ to promote formation of hydroxyl radicals generated by the Fenton reaction that are also thought to have an important role in TRPM2 activation [38]. Indirect TRPM2 activation through the production of intracellular ADPR and direct action independent of ADPR production have also been reported (**Figure 2A**). One ADPR production pathway involves nuclear poly(ADPR) polymerases (PARPs) and poly(ADPR) glycohydrolases (PARGs). PARP is activated by DNA damage upon oxidative stress and has DNA repair functions [39]. Meanwhile, PARG hydrolyzes poly-ADPR chains to generate free ADPR. The finding that treatment of cells with a PARP inhibitor decreased H2 O2 -mediated TRPM2 activation supports a role for ADPR in activation of this channel [40]. Another possible source of ADPR is mitochondria, where oxidative stress could induce the production of free ADPR [41]. Indeed, reduced mitochondrial ADPR concentrations suppressed H2 O2 -mediated TRPM2 currents [42]. ADPR can also be produced by plasma membrane/intracellular organelle CD38, which has implications for immunocyte function [43, 44]. However, the mechanism by which extracellular ADPR generated by the CD38 ecto-enzyme enters the cells is unclear [45]. In contrast, an ADPR-independent TRPM2 activation pathway may also exist based on the finding that a TRPM2 variant lacking the Nudix domain that contains the ADPR-binding domain still responded to H2 O2 [46]. In addition, our recent results showed that H2 O2 clearly enhanced heat-evoked responses of TRPM2 in inside-out single channel recordings in which intracellular components were completely absent (**Figure 2B**) [19]. This action was accompanied by a reduction in the temperature threshold for TRPM2 heat activation (**Figure 2C**) and was mimicked by an oxidant that reacts with methionine residues. Taken together, these results suggest that ADPR-independent actions could also be involved in TRPM2 activation; however, because H2 O2 -evoked reduction in the temperature threshold for TRPM2 activation was affected by a PARP inhibitor, there could be both direct and indirect actions in H<sup>2</sup> O2 -mediated effects.

**Figure 2.** (A) Scheme showing the activation cascade of TRPM2 by reactive oxygen species (ROS). (B) H2 O2 -enhanced heat-evoked single channel opening observed in inside-out single channel recordings. (a,b) Magnified traces at the time points indicated in the upper column. (C) H2 O2 -treatment lowered the temperature threshold for TRPM2 activation. The temperature-fura2 ratio relationship is plotted for H<sup>2</sup> O2 -untreated and H2 O2 -treated (100 μM, 3 min) cells. Average temperature thresholds for each group (Mean ± SEM) are shown.
