Soon after cloning of the TRPM2 channel [25], whole-cell patch-clamp studies showed that the channel is activated by intracellular ADPR and activation requires Ca²⁺ [7,26,27,28,29]. As in whole-cell studies the composition of the cytosolic solution, and particularly that of microdomains, is not strictly controlled, the location of the activating Ca²⁺ binding sites (extra- or intracellular) could not be unambiguously determined [27,29]. In contrast, inside-out patch clamp recordings afford strict control over the composition of the cytosolic solution, and rapid application/removal (on the timescale of tens of milliseconds) of intracellular ligands. In patches excised from Xenopus laevis oocytes injected with hsTRPM2 cRNA, large TRPM2 currents were evoked by simultaneous application of ADPR and Ca²⁺ (Figure 1A) [30,31]. Both ligands were required for channel activity, but interestingly, effective concentrations differed from those published earlier in studies using whole cells [7,24,26,27,28,29]. Whereas in whole-cell recordings EC₅₀ for ADPR and Ca²⁺ were ~100 μM and ~300 nM, respectively [7,27], in inside-out patches, ADPR apparent affinity was two orders of magnitude higher (EC₅₀~1 μM) whilst that of Ca²⁺ was two orders of magnitude lower (EC₅₀~20 μM), and these apparent affinities were little affected by the concentration of the other ligand [30,31]. A possible explanation for a lower Ca²⁺ affinity could have been the loss of calmodulin in the patch (cf [29,32]); however, the addition of external bovine calmodulin had no effect on fractional currents at micromolar Ca²⁺ concentrations [31]. Since even 1 mM ADPR elicited only minimal activation in submicromolar Ca²⁺, the TRPM2 channel seems to be intrinsically set to sense high local Ca²⁺ concentrations.

While no structural data were available at the time, functional observations in inside-out patches allowed rough localization of the Ca²⁺ binding sites. In steady-state single channel recordings, channel closed-time durations were independent of extracellular Ca²⁺ concentration, suggesting that in closed channels the binding sites are shielded from extracellular Ca²⁺ [30]. On the other hand, macroscopic channel closure upon sudden intracellular Ca²⁺ withdrawal could be incrementally slowed by raising extracellular Ca²⁺ [30], suggesting that as long as the pore is open extracellular Ca²⁺ can reach its binding sites despite Ca²⁺-free vectorial rinsing of the cytosolic channel surface. These findings strongly suggested that the Ca²⁺ binding sites are located in an intracellular crevice very near the channel pore.

Besides the two obligate ligands, ADPR and Ca²⁺, the presence of membrane phosphatidylinositol-4,5-bisphosphate (PIP₂) is also a prerequisite for channel activation, as in inside-out patches masking the negatively charged headgroups of endogenous PIP₂ with the polycation polylysine shuts TRPM2 pores, which can then be re-opened only by millimolar amounts of Ca²⁺ [36] or by administration with external PIP₂ [33]. While H₂O₂ potently stimulates TRPM2 currents in intact cells [5,26,37], this effect must be a secondary consequence of ADPR and/or Ca²⁺ release in response to the induced oxidative stress [37], as H₂O₂ itself was ruled out as a direct effector of TRPM2 channel activity in cell-free recordings [31].

In the body, CD38, a multifunctional glycoprotein enzyme present on the surface of immune cells, converts the pyridine nucleotides NAD and NAAD into ADPR, but NADP and NAADP into ADPR-2-phosphate (ADPRP) [38]. These nucleotides and others, the metabolism of which is intertwined with that of ADPR, were tested to address the potential effects on TRPM2. NAD was identified as a channel agonist in early studies [5,28], while no binding to the NUDT9-H domain was later reported [24]. NAAD [31], NAADP [31,39,40] and cADPR [39,40] were reported to act as activators, AMP as an inhibitor [24,26], while NADH and NADP were found to be ineffective [5]. Besides these reported effects on channel activity, cADPR [26,39,40] and NAADP [39,40] were also shown to augment the effects of ADPR in a positively cooperative manner. 8-Br-cADPR, an agent shown to reduce ischæmic acute kidney injury [39], was suggested to act as a partial TRPM2 agonist and a competitive inhibitor of activation by cADPR [26]. Most of the above studies examined the effects of nucleotides in whole-cell systems. In a study in inside-out patches, the effects of cADPR on TRPM2 channel activity were shown to be attributable to ADPR contamination [31]. Decontamination of commercial cADPR stocks with a nucleotide pyrophosphatase which selectively cleaves ADPR but not cADPR eliminated channel stimulation by the compound in excised patches. Furthermore, no cooperative effects of cADPR with ADPR were observed. Recently, Yu et al reported that synthetic pure cADPR directly binds to the human NUDT9-H domain [41]. SPR titration indicated an apparent affinity (K_d) of ~12 μM; however, much higher concentrations of cADPR (EC₅₀ ~250 μM) were required to stimulate macroscopic TRPM2 currents. Considering its submicromolar cellular concentration [42], cADPR does not seem to act as a physiological activator of TRPM2. TRPM2 channel activation by various pyridine dinucleotides is explained by their spontaneous degradation, which also produces ADPR(P). Cleavage of contaminating ADPR in NAD and NAAD stocks, or of contaminating ADPRP in NAADP stocks, by the purified NUDT9 ADPRase abolished the stimulatory effects of the dinucleotides, ruling them out as direct effectors of TRPM2 [43]. Moreover, these experiments also demonstrated that none of the spontaneous and/or enzymatic cleavage products—nicotinamide, nicotinic acid, AMP(P) or ribose-5-phosphate—are TRPM2 channel activators. An apparent stimulatory effect of NAADP prior to decontamination pinpointed its spontaneous degradation product ADPRP as an agonist. Indeed, in inside-out patches, pure ADPRP opened TRPM2 channels, albeit its apparent affinity was lower (EC₅₀~13 μM) and maximal stimulation smaller (~80%) when compared to ADPR. The lower open probability supported by ADPRP was explained by an approximately three-times faster macroscopic closing rate, reflecting a shortened open burst [43]. A similar efficacy but even lower affinity (EC₅₀~110 μM) for ADPRP was reported in a whole-cell study [44] that also showed 2’-deoxy-ADPR (dADPR) to act as a superagonist on hsTRPM2 channels. In the presence of dADPR, whole-cell TRPM2 currents were 37% larger, and following patch excision channels inactivated slower, but the EC₅₀ of dADPR was similar to that of ADPR. Since upon oxidative stress-induced DNA damage, dADPR may accumulate due to CD38 activity, this nucleotide is a likely regulator of TRPM2 function under pathophysiological conditions.

In summary, recordings in cell-free systems demonstrated that (d)ADPR is a physiologically relevant activator of the hsTRPM2 channel and ADPRP is a partial agonist, whereas AMP and pyridine dinucleotides are without effect [31,43] and cADPR is probably a non-physiological low affinity agonist [41]. Note, however, that nucleotide affinity and efficacy profiles differ among channel orthologues.