Identification of Direct and Indirect Effectors of the Transient Receptor Potential Melastatin 2 (TRPM2) Cation Channel*

Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel involved in physiological and pathophysiological processes linked to oxidative stress. TRPM2 channels are co-activated by intracellular Ca2+ and ADP-ribose (ADPR) but also modulated in intact cells by several additional factors. Superfusion of TRPM2-expressing cells with H2O2 or intracellular dialysis of cyclic ADPR (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) activates, whereas dialysis of AMP inhibits, TRPM2 whole-cell currents. Additionally, H2O2, cADPR, and NAADP enhance ADPR sensitivity of TRPM2 currents in intact cells. Because in whole-cell recordings the entire cellular machinery for nucleotide and Ca2+ homeostasis is intact, modulators might affect TRPM2 activity either directly, by binding to TRPM2, or indirectly, by altering the local concentrations of the primary ligands ADPR and Ca2+. To identify direct modulators of TRPM2, we have studied the effects of H2O2, AMP, cADPR, NAADP, and nicotinic acid adenine dinucleotide in inside-out patches from Xenopus oocytes expressing human TRPM2, by directly exposing the cytosolic faces of the patches to these compounds. H2O2 (1 mm) and enzymatically purified cADPR (10 μm) failed to activate, whereas AMP (200 μm) failed to inhibit TRPM2 currents. NAADP was a partial agonist (maximal efficacy, ∼50%), and nicotinic acid adenine dinucleotide was a full agonist, but both had very low affinities (K0.5 = 104 and 35 μm). H2O2, cADPR, and NAADP did not enhance activation by ADPR. Considering intracellular concentrations of these compounds, none of them are likely to directly affect the TRPM2 channel protein in a physiological context.

TRPM2 2 is a member of the transient receptor potential family of proteins and forms a nonselective cation channel that is permeable to Ca 2ϩ (1). TRPM2 channels are abundantly expressed in the brain, in hematopoietic tissues, and in leukocytes (1)(2)(3)(4) and activate under conditions of oxidative stress to cause elevations in intracellular [Ca 2ϩ ] ([Ca 2ϩ ] i ) that contrib-ute to chemotactic responses (5) and chemokine production (6) of immune cells, as well as to neuronal cell death following ischemia (4,7,8).
The primary activators of TRPM2 are ADP-ribose (ADPR) and Ca 2ϩ (2)(3)(4); simultaneous binding of both agonists is required for channel opening (9). TRPM2 channels are homotetramers (10), and ADPR binds to the NUDT9-H domains located at the cytosolic C termini of the subunits, named based on sequence homology to the mitochondrial enzyme NUDT9 (2). Both NUDT9 and isolated NUDT9-H bind ADPR and convert it to AMP and ribose-5-phosphate (2,11), but the role in TRPM2 channel gating of the very slow ADPR hydrolase (ADPRase) activity of NUDT9-H is unknown. In intact cells ADPR activates TRPM2 only in the presence of either intra-or extracellular Ca 2ϩ (4,12,13); biophysical studies in inside-out patches have shown that the activatory Ca 2ϩbinding sites are located intracellularly of the gate, such that extracellular Ca 2ϩ needs to permeate through the pore before reaching its binding sites (4,9). Nevertheless, the vicinity of these sites to the pore suggests that in intact cells extracellular Ca 2ϩ is the primary source of this activating ligand (9).
What are the signals that initiate TRPM2 channel activity in a cellular context? Early on TRPM2 currents have been linked to the redox status of the cell (4), and several studies have pinpointed oxidative stress, such as that inducible by H 2 O 2 , as a causative agent in TRPM2 activation. One early report of H 2 O 2 -activated whole-cell currents from a truncated TRPM2 construct no longer responsive to ADPR (14) could not be reproduced (15). On the other hand, several mutations and deletions in the conserved Nudix motif (RILRQE) of the NUDT9-H domain that abrogated ADPR binding yielded channels that also failed to activate in response to H 2 O 2 (4,15), leading to the conclusion that H 2 O 2 acts indirectly by increasing intracellular [ADPR]. Intriguingly, recent reports of synergistic effects between subthreshold concentrations of ADPR and H 2 O 2 and of differential inhibitory profiles of ADPR-and H 2 O 2 -induced whole-cell TRPM2 currents again raised the possibility that H 2 O 2 might directly affect TRPM2 activity by a mechanism independent of ADPR (16,17).
Several additional compounds, mostly adenine nucleotides, have been found to modulate TRPM2 whole-cell currents in different cell types, but there is also substantial disagreement between the various reports. Cyclic ADPR (cADPR) fully stimulated TRPM2 currents in some studies (17,18), whereas it had little (16) or no (2,4,5) effect in others. In addition, small (subthreshold) concentrations of cADPR resulted in large, up to 100-fold, sensitization toward activation by ADPR (16 -18), whereas others have found no such synergy (5). More recently, nicotinic acid adenine dinucleotide phosphate (NAADP) was reported to fully activate endogenous TRPM2 currents in Jurkat T-lymphocytes (18) and neutrophil granulocytes (17) and to potentiate ADPR-mediated channel activation just as reported for cADPR (17,18). Finally, ADPR-activated whole-cell TRPM2 currents are inhibited by intracellular dialysis of AMP but are not sensitive to the cADPR analog 8-Br-cADPR (16). In contrast, at least some fraction of cADPR-, NAADP-, and H 2 O 2induced currents seems AMP-resistant but sensitive to inhibition by 8-Br-cADPR (16 -18). Low affinity activation of TRPM2 currents by NAD (3,4) is likely due to contamination by ADPR (16,19), and the significance of TRPM2 activation by O-acetylated ADPR remains to be elucidated (19).
Although the above whole cell patch-clamp studies have identified several interesting compounds as candidates that might directly affect TRPM2 channels, the conclusions of these reports are limited by the fact that in whole-cell recordings the entire cellular machinery involved in nucleotide and Ca 2ϩ homeostasis is in place. Thus, intracellular dialysis of a compound might affect TRPM2 activity either directly, by binding to the TRPM2 protein, or indirectly, by altering the local concentrations of the primary ligands ADPR and Ca 2ϩ ; possible perturbations of other signaling pathways that ultimately affect TRPM2 activity also have to be considered. Regulation of cytosolic [ADPR] is the result of a complex interplay between several cytosolic, nuclear, and mitochondrial enzymes that produce and break down ADPR or that are involved in its translocation between compartments (reviewed in Refs. 20 and 21). It is likely that intracellular dialysis of high concentrations of adenine nucleotides, which are substrates and/or products of several of these enzymes, will ultimately affect cytosolic [ADPR]; just as a bulk overload of intact cells with Ca 2ϩ is likely to cause generation of reactive oxygen species and consequent ADPR production, as suggested by gradual activation of wholecell TRPM2 currents under such conditions (22). Likewise, intracellular [Ca 2ϩ ] is dynamically regulated by Ca 2ϩ release from intracellular stores and plasma membrane Ca 2ϩ influx, counteracted by transporters that extrude Ca 2ϩ from the cytosol. Acting on ryanodine receptors and on a distinct class of receptors, respectively, both cADPR and NAADP are potent Ca 2ϩ mobilizing agents (reviewed in Refs. 23 and 24), which likely alter both bulk intracellular [Ca 2ϩ ] and its microdomains when dialyzed into cells. Therefore, from whole-cell experiments a direct action of these compounds on the TRPM2 protein cannot be concluded.
To differentiate between direct and indirect effects on TRPM2, we have evaluated these compounds in cell-free inside-out patches using direct ligand superfusion. Our results indicate that H 2 O 2 , AMP, and cADPR in their pure forms do not directly affect TRPM2 gating. NAADP is a partial and nicotinic acid adenine dinucleotide (NAAD) a full agonist, both capable of directly activating TRPM2, but based on their low apparent affinities it seems unlikely that they should act as direct modulators of TRPM2 currents in vivo. These results single out the primary ligands ADPR and Ca 2ϩ as the final common pathway into which multiple metabolic routes converge to modulate TRPM2 activity in intact cells.

EXPERIMENTAL PROCEDURES
Molecular Biology-hTRPM2/pGEMHE was constructed as described (9). For in vitro transcription plasmid DNA was linearized by NheI; cRNA was prepared using a T7 mMessage mMachine kit (Ambion) and quantified on denaturing gels.
Isolation and Injection of Xenopus Oocytes-Xenopus oocytes were isolated and injected with cRNA as previously described (9). Isolated oocytes were stored at 18°C in a Ringer's solution supplemented with 1.8 mM CaCl 2 and 50 g/ml gentamycin. Macroscopic current recordings were obtained 2-3 days after injection of 10 ng of hTRPM2 cRNA.
Excised Patch Recordings-Inside-out patch recordings were done at 25°C in a symmetrical sodium gluconate-based solution at a pipette holding potential of ϩ20 mV (V m ϭ Ϫ20 mV) as described (9). Briefly, the tip of the patch pipette (to ϳ1-cm height) contained 140 mM sodium gluconate, 2 mM magnesium gluconate 2 , 10 mM HEPES, pH 7.4, with NaOH. The pipette electrode was placed into a 140 mM NaCl-based solution carefully layered on top to avoid mixing. Bath solution contained 140 mM sodium gluconate, 2 mM magnesium gluconate 2 , 10 mM HEPES, and either 1 mM EGTA (to obtain zero (ϳ8 nM) Ca 2ϩ ) or 1 mM EGTA ϩ 760 M calcium gluconate 2 (to obtain 1 M free [Ca 2ϩ ]), or 0.1-1 mM calcium gluconate 2 (to obtain 15-125 M free [Ca 2ϩ ]); pH was adjusted to 7.1 with NaOH. Na 2 -ADPR, cADPR, Na 2 -NAADP, sodium NAAD, and Na 2 -AMP (Sigma) were added from aqueous stock solutions. Bovine calmodulin (Calbiochem) was added from a 400 M stock solution.
To compensate for Ca 2ϩ binding by the protein and EDTA content of this preparation, 2 M calmodulin was supplemented with 25 M calcium gluconate 2 ; fluorescent measurements using Ca-Green 5N (9) confirmed that free [Ca 2ϩ ] remained unchanged using this protocol. The continuously flowing bath solution was exchanged using computer-driven electronic valves (HEKA); solution exchange was complete within Ͻ1 s. Membrane currents were amplified, filtered at 2 kHz (Axopatch-200B), digitized at 10 kHz (Digidata 1440A), and saved to disk (pCLAMP 10; Molecular Devices, Inc.).
Verification of H 2 O 2 Activity-A 10 M aqueous H 2 O 2 stock solution was stored at ϩ4°C, and the activity was periodically verified by permanganometry. In brief, 1 ml of 20% (v/v) H 2 SO 4 was added to 1 ml of 100-fold diluted H 2 O 2 stock solution and titrated with 0.1 N (0.02 M) KMnO 4 (factor determined using 0.1 N oxalic acid). No measurable loss of H 2 O 2 activity was detected during the time frame of this study.
Data Analysis-To obtain fractional activation by some test concentration of a given nucleotide, quasi-steady macroscopic TRPM2 current in the test segment was normalized to the maximal activity estimated for the same segment. Because after patch excision TRPM2 currents continuously decline (due to a progressive loss of active channels in the patch (9)), the maximal activity used for normalization was obtained by linear interpolation of the activities in the presence of saturating (32 M) ADPR, obtained in bracketing segments of record in the same patch. For test applications that preceded exposure to 32 M ADPR, maximal activity for the test segment was estimated by linear extrapolation of the activities obtained subsequently, in the same patch, upon repeated exposures to 32 M ADPR.
This rundown correction protocol is illustrated in supplemental Fig. S1. Dose-response curves were fitted to the Hill equation by nonlinear least squares methods (SigmaPlot 10; SPSS Inc.). The error bars represent S.E.
Enzymatic Purification of cADPR-cADPR was purified from its ADPR contamination using nucleotide pyrophosphatase type I (P7383; Sigma), following a protocol modified from that described in Ref. 5. In brief, 1 unit of the enzyme was added to a 400-l volume of 2 mM cADPR in the presence of 2 mM MgCl 2 and incubated at 37°C for 60 min. Following incubation the enzyme (molecular mass, ϳ24 kDa by SDS-PAGE) was removed by 2-fold filtering through 3-kDa molecular mass cut-off filter units (Z629367; Sigma). Analysis of the resulting filtrate by TLC (see below) revealed complete conversion of contaminating ADPR into AMP plus ribose-5-phosphate (see Fig. 5A; ribose-5-phosphate shows no fluorescence) but nearly complete recovery of cADPR. At the same time, functional testing confirmed complete removal of the enzyme by the 2-fold filtering procedure (see Fig. 5, D and E).

RESULTS
Human TRPM2 is efficiently expressed in Xenopus oocytes and forms channels with properties similar to those observed in mammalian expression systems (9). In inside-out patches excised from TRPM2-expressing oocytes, macroscopic currents can be routinely obtained. Ligands are applied directly to the cytosolic surface of the membrane; a continuously flowing bath solution affords perfect control over local concentrations of all agonists, whereas the composition of this cytosolic solution is completely exchanged within Ͻ1 s. These properties render our system ideal for addressing whether a compound that affects TRPM2 activity in intact cells exerts its effect by directly targeting the TRPM2 protein.
Earlier work has demonstrated that the Ca 2ϩ -binding sites responsible for TRPM2 activation are found in a restricted space intracellularly, but in the immediate vicinity of the channel pore, such that the primary source for activating Ca 2ϩ is likely extracellular. Even in the face of extremely rapid vectorial rinsing with a buffered zero Ca 2ϩ solution, these activating sites experience [Ca 2ϩ ] in the tens of micromolar range once a channel has opened (9), so this is all the more likely to be true in the static environment of an intact cell, regardless of the bulk [Ca 2ϩ ] i achieved using Ca 2ϩ buffers. Because only open channels contribute to the measured currents, it follows that during whole-cell experiments the observed currents are due to TRPM2 channels that experience high micromolar [Ca 2ϩ ] at their activating sites. Thus, in the present study assaying nucleotide-mediated regulation of TRPM2 in inside-out patches under rapid cytosolic perfusion, we used a saturating [Ca 2ϩ ] i as our reference condition to achieve a local environment comparable with that experienced by TRPM2 channels in whole-cell studies or in a physiological context (26).
Intracellular [Ca 2ϩ ] Little Affects Apparent Affinity for Activation of TRPM2 by ADPR-To obtain adequate controls for our experiments testing the effects of various potential modulators both at high and low [Ca 2ϩ ] i , we first examined to what extent the apparent affinity of the primary ligand ADPR is sensitive to [Ca 2ϩ ] i . To this end, we assayed macroscopic current responses of patches containing hundreds of TRPM2 channels to increasing concentrations of ADPR while maintaining intracellular free [Ca 2ϩ ] at a saturating (125 M) (Fig. 1A) or a subsaturating (15 M; cf. K1 ⁄ 2 for Ca 2ϩ activation is ϳ20 M in saturating [ADPR] (9)) level (Fig. 1B). Although the maximally In Inside-out Patches Calmodulin and Even Millimolar ADPR Fail to Activate TRPM2 Channels at Submicromolar [Ca 2ϩ ]-Because ADPR affinities reported in previous studies were measured in intact cells in which bulk cytosolic Ca 2ϩ levels are submicromolar (2,16,18), we evaluated the possibility of measuring TRPM2 sensitivity to ADPR at submicromolar Ca 2ϩ levels in our cell-free system. In principle, the low affinity of the channels for Ca 2ϩ in inside-out patches (9) might be due to the loss of a high affinity Ca 2ϩ -binding site upon patch excision. A prime candidate for such a binding site could be calmodulin, which has an intrinsically high Ca 2ϩ affinity and has been shown to associate with, and modulate the activity of, TRPM2 (13,27). However, in the presence of 32 M ADPR, fractional current activation by low micromolar Ca 2ϩ was not affected by superfusion of the cytosolic faces of the patches with 2 M calmodulin (Fig. 1D); a 100-fold lower concentration of biotinylated calmodulin was shown to be sufficient to bind TRPM2 protein in an overlay assay (27). The lack of effect of calmodulin on the apparent Ca 2ϩ affinity of TRPM2 in excised patches (Fig. 1E, compare gray versus black bars) suggests either that calmodulin is not involved in Ca 2ϩ activation of TRPM2 or that endogenous calmodulin remains tightly bound to the channels following patch excision.
Because in whole-cell studies apparent Ca 2ϩ affinity of TRPM2 was tested at much higher, millimolar, ADPR concentrations ( 1, A and C) to allow estimation of the number of activatable TRPM2 channels in the same patch ( Fig. 2A, left trace). No TRPM2 channel currents were observed even for incubation times in H 2 O 2 lasting longer than 1 min, whereas subsequent exposure to ADPR typically revealed the presence of several hundred activatable TRPM2 channels ( Fig. 2A, left trace; note that because of the typically observed rundown, channel numbers must have been even larger during the periods of exposure to H 2 O 2 ). Fractional currents in H 2 O 2 from seven similar experiments were not significantly different from zero (Fig. 2B, leftmost bar). Thus, TRPM2 is not activated by H 2 O 2 -mediated oxidation of amino acid side chains on the cytosolic surface of the channel. We next tested whether a potential side chain oxidation by H 2 O 2 might perhaps inhibit rather than stimulate channel activity. However, the activity of channels that had been opened by 32 M ADPR was not affected by exposure for up to tens of seconds to 1 mM H 2 O 2 ( Fig. 2A, right trace); fractional current remained 0.99 Ϯ 0.01 (n ϭ 9) in ADPR ϩ H 2 O 2 (Fig. 2B, rightmost black bar).
We also examined whether H 2 O 2 exposure might enhance activation by ADPR, as suggested in recent whole-cell studies (16,17). However, superfusion of inside-out patches with 0.1 and 1 M ADPR in the continued presence of 1 mM H 2 O 2 activated fractions of 0.026 Ϯ 0.023 (n ϭ 7) and 0.55 Ϯ 0.06 (n ϭ 7) (Fig. 2B, black bars), respectively, of maximal current (in 32 M ADPR), indicating no change in apparent affinity for ADPR in the presence of H 2 O 2 (K1 ⁄ 2 ϳ 1 M; Fig. 1, A and C).
Finally, we considered the possibility that the reported facilitatory effect of H 2 O 2 might happen through the same or a similar mechanism by which Ca 2ϩ potentiates TRPM2 activity. In this case such an effect of H 2 O 2 might be masked by our use of a saturating [Ca 2ϩ ] i . We therefore repeated experiments similar to those summarized in Fig. 2 (A and B) Fig. 1C). Taken together, these results indicate either that no amino acid side chains on the cytosolic face of TRPM2 are oxidized by H 2 O 2 or that such oxidation has no functional consequences for TRPM2 channel activity, at least for H 2 O 2 concentrations up to 1 mM.
AMP Does Not Affect ADPR-induced TRPM2 Currents in Inside-out Patches-Intracellular perfusion of AMP suppresses ADPR-induced whole-cell TRPM2 currents in a dose-dependent manner (16). Because AMP is one of the cleavage products of the ADPRase reaction reportedly catalyzed by the NUDT9-H domain (2,11), AMP might inhibit TRPM2 channel activity by competing with ADPR for binding to NUDT9-H. On the other hand, TRPM2 homolog TRPM4 (28) (and native TRPM4-like (29)) cation channels that lack the C-terminal NUDT9-H domain are also inhibited by AMP with high affinity. An alternative possibility therefore is that AMP binds to the large cytosolic N-terminal TRPM homology region present in all TRPM family channels, resulting in noncompetitive inhibition of ADPR-induced TRPM2 activity.
To evaluate these possibilities, we set out to study the kinetics of AMP-mediated inhibition of TRPM2 reported to occur with an IC 50 of ϳ70 M in whole cells (16). To our surprise, TRPM2 currents activated by saturating concentrations of Ca 2ϩ and ADPR (125 and 32 M, respectively) were barely affected by superfusion of the cytosolic face of the channels with 100 M AMP (Fig. 3A), yielding a fractional current of 0.95 Ϯ 0.04 (n ϭ 6) in the presence of AMP (Fig. 3B, left panel, black bar). Because in another whole-cell study conducted at a lower [ADPR] AMP inhibited with even higher apparent affinity (IC 50 ϭ 10 M) (17), we also tested inhibition by AMP at a 10-fold lower [ADPR] (3. To minimize rundown, in the above experiments (Fig. 3, A and B) AMP was applied for relatively short time intervals, bracketed by segments of record in AMP-free solution. However, if AMP competes with ADPR for the same binding site, then such brief exposures might not allow AMP to displace  SEPTEMBER 24, 2010 • VOLUME 285 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 30095 ADPR, because the binding sites have already been preloaded with ADPR at the time of AMP application, and the kinetics of ADPR unbinding might be slow. We therefore tested whether a pre-exposure to AMP might prevent subsequent activation by ADPR (Fig. 3C). However, pre-equilibration with 200 M AMP did not affect subsequent activation (in the continued presence of AMP) by 3.2 M ADPR (Fig. 3C); thus, comparison of current amplitudes (corrected for rundown; see "Experimental Procedures") revealed nearly maximal activation by 3.2 M ADPR in the presence of 200 M AMP (Fig. 3D, gray bar; compare with white bar) and a lack of current increase following AMP removal (Fig. 3D, black bar).

Direct Effectors of TRPM2 Channels
Finally, to examine whether a possible inhibitory effect of AMP might be masked by the facilitatory effect of our saturating [Ca 2ϩ ] i , we repeated experiments similar to those shown in Fig. 3 (C and D) at a suboptimal [Ca 2ϩ ] i of 15 M (Fig. 3, E and  F) Fig. 1C), and no further current increase was observed upon AMP removal (Fig. 3, E and F, black bar). These results suggest that AMP-mediated inhibition of TRPM2 whole-cell currents is not a direct effect of AMP on the TRPM2 protein.
Effects of Commercial cADPR on TRPM2 Currents in Cell-free Patches-Given the large scatter in the potency of cADPR to activate TRPM2 currents in intact cells of various sources (4, 5, 16 -18), we tested whether direct superfusion of inside-out patches with a solution containing 10 M cADPR and saturating Ca 2ϩ would elicit any TRPM2 currents. To our surprise, 10 M cADPR maximally stimulated TRPM2 channels in patches, as witnessed by a lack of further current increase upon the addition of a saturating dose (10 M) of ADPR (Fig. 4A). As little as 1 M cADPR elicited sizeable TRPM2 currents; a dose-response curve of fractional activation by cADPR yielded a K1 ⁄ 2 of 1.6 Ϯ 0.3 M (Fig. 4B), only ϳ2-fold larger than the K1 ⁄ 2 for ADPR measured under identical conditions (Fig. 1, A and C). We next examined whether a synergy between submaximal concentrations of cADPR and ADPR, as observed in intact cells (16 -18), could be reproduced in a cell-free system by first applying 1 M cADPR in saturating Ca 2ϩ and then adding increasing concentrations of ADPR to this background (Fig.  4C). At this concentration cADPR alone elicited ϳ30% of maximal activity, which was further increased in a dose-dependent manner by subsequently applied ADPR. The dose-response curve for activation by ADPR in the presence of a fixed concentration of 1 M cADPR (Fig. 4D, black symbols) was fitted to a modified Hill equation to yield a K1 ⁄ 2 of 0.8 Ϯ 0.1 M for ADPR. Thus, the presence of a submaximal concentration (1 M) of cADPR did not significantly affect the apparent affinity for ADPR activation (Fig. 1, A and C). The high affinity potent stimulation by cADPR alone, together with the lack of synergy with ADPR, raised the possibility of a substantial contamination of our cADPR stock by ADPR, as had been observed in another study (5). Indeed, mass spectrometric analysis (not shown) of an aliquot of cADPR from the same batch revealed an estimated ϳ50% of contaminant ADPR, which, on its own, could account for the apparent affinity observed for stimulation by our cADPR solutions (Fig. 4B).
Refined Purification Procedure Yields Functionally Pure cADPR-To obtain a cADPR solution devoid of contaminating ADPR, cADPR stock solution was treated with nucleotide pyrophosphatase (P7383; Sigma), which degrades ADPR (but not cADPR) into AMP plus ribose-5-phosphate, followed by removal of the enzyme by filtering (5). To minimize potential artifacts, we verified three aspects of this procedure.
First, the efficiency of the enzymatic digestion was monitored by TLC ( Fig. 5A; see "Experimental Procedures"). Loading a sample of untreated cADPR onto the TLC plate resulted in two distinct spots, the faster one co-migrating with ADPR and accounting for ϳ20% of total density in this batch (Fig. 5A, compare two right lanes). In contrast, in a sample of enzymatically treated cADPR, the spot corresponding to ADPR was absent, replaced by a slow spot of comparable density co-migrating with AMP (Fig. 5A, compare two left lanes), confirming enzymatic conversion of contaminant ADPR into AMP and ribose-5-phosphate. (Ribose-5-phosphate elicits no fluorescence and is therefore not visible on the TLC.) Second, our enzymatically purified cADPR solution contains AMP and ribose-5-phosphate (ϳ2-5 M each for 10 M cADPR). We therefore tested whether these two breakdown products could affect TRPM2 channel activity. However, in control experiments application of AMP (100 M) or ribose-5phosphate (100 M) alone or co-application of 100 M AMP ϩ 100 M ribose-5-phosphate did not affect TRPM2 current activation by 1 or 32 M ADPR in saturating Ca 2ϩ .
Third, although a filter with a 10-kDa molecular mass cut-off, as described in Ref. 5, seems adequate for removal of the 24-kDa nucleotide pyrophosphatase, we wanted to functionally verify the efficacy of this crucial step. This is because in experiments in which purified cADPR is co-applied with ADPR, any residual enzymatic activity might result in slow degradation of ADPR, yielding decreased channel activity. Because TRPM2 activity is most sensitive to [ADPR] in a concentration range near ϳ1 M, fractional TRPM2 current in nominally 1 M ADPR is a sensitive measure of any remaining ADPRase activity. We therefore filtered an aliquot of nucleotide pyrophosphatase equivalent to that used for treatment of one batch of cADPR through a 10-kDa molecular mass cut-off filter column. An aliquot of this filtrate equivalent to that used for obtaining a final concentration of 10 M purified cADPR was added to a bath solution containing 1 M ADPR. Fractional TRPM2 current activation by this test solution, relative to that obtained with 1 M untreated ADPR, was assayed in excised patches at various times (0 -7 h) following the addition of the filtrate to the test solution. To our surprise, the ability of the test solution to activate TRPM2 channels was virtually abolished within 2-3 h of the addition of the filtrate; Fig.  5B shows a current trace from a patch obtained ϳ4 h after the test solution had been prepared. The time-dependent decline of fractional TRPM2 activity supported by this test solution (Fig. 5E, black  squares) is a measure of the rate at which ADPR is slowly degraded by residual pyrophosphatase activity in the filtrate. To reduce this enzymatic contamination, we passed a similar aliquot of the pyrophosphatase through a 3-kDa molecular mass cut-off filter column. Adding an equivalent amount of this filtrate to a 1 M ADPR solution yielded a test solution that still showed a timedependent decline in its efficiency to activate TRPM2, but the rate of this decline was dramatically reduced (Fig. 5C, current trace obtained ϳ5 h after the addition of the filtrate; and Fig. 5E, white circles, time course of fractional current activation). Finally, sequential filtering of the pyrophosphatase through two 3-kDa molecular mass cut-off columns resulted in a filtrate that, when added to a 1 M ADPR solution, did not decrease the efficiency of this solution to activate TRPM2 currents even after incubation times of ϳ9 h (Fig.  5, D and E, black circles), signaling complete removal of the pyrophosphatase activity by this filtering protocol.
These results establish that treatment of cADPR with the nucleotide pyrophosphatase, followed by 2-fold filtering through 3-kDa molecular mass cut-off filter columns, yields a purified cADPR stock solution devoid of any factors that could distort the effect of cADPR on TRPM2 activity. All of the experiments in the following section in which cADPR and ADPR were co-applied were performed within 3 h of adding purified cADPR to the bath solutions.
Pure cADPR Neither Activates TRPM2 nor Sensitizes TRPM2 to Activation by ADPR in Cell-free Patches-We next tested enzymatically purified cADPR for its ability to activate TRPM2. However, 10 M purified cADPR applied in the presence of saturating Ca 2ϩ elicited no channel activity even in patches in which subsequent application of saturating ADPR could activate hundreds of TRPM2 channels (Fig. 6A); fractional currents in 10 M pure cADPR, normalized to the currents obtained in ADPR in the same patch, averaged 0.012 Ϯ 0.003 (n ϭ 23) (Fig.  6C, leftmost bar). We also verified whether pure cADPR could FIGURE 5. Testing of the cADPR purification procedure. A, TLC analysis of (from left to right) AMP, enzymatically purified cADPR, ADPR, and nontreated cADPR. For all samples 20 nmol of nucleotide were loaded, followed by drying. Note conversion of the ADPR contamination of nontreated cADPR into AMP in the enzymatically purified cADPR sample. B-D, activation of TRPM2 currents by 1 M ADPR solutions containing an aliquot of nucleotide pyrophosphatase filtrate and by pure 1 M ADPR (bars). 10-and 3-kDa molecular mass cut-off filter columns were used to produce the filtrates for the experiments in B and C, respectively. For the experiment in D, the filtrate was produced by sequential passages through two 3-kDa molecular mass cut-off filter columns. Approximate timing of the start of each experiment relative to addition of the filtrate to the test solution is indicated above each panel. E, fractional current activated by test solutions containing 1 M ADPR ϩ nucleotide pyrophosphatase filtrate, normalized to the current supported by 1 M ADPR in the same patch, as a function of time following the addition of the filtrate to the test solution. The symbols with error bars represent the values averaged over 2-10 experiments with similar timing. The three time courses represent three protocols for producing the filtrate (panels B-D): 1ϫ passage through 10-kDa molecular mass cut-off filter (black squares), 1ϫ passage through 3-kDa molecular mass cut-off filter (white circles), and 2ϫ passage through 3-kDa molecular mass cut-off filters (black circles). SEPTEMBER 24, 2010 • VOLUME 285 • NUMBER 39 enhance currents activated by a subthreshold concentration of 0.1 M or by a half-saturating concentration of 1 M, ADPR (Fig. 6B). However, the currents elicited by these submaximal concentrations of ADPR were not affected by the addition of 10 M purified cADPR (Fig. 6C, black and gray bars). This lack of a potentiating effect of purified cADPR was not due to our use of saturating [Ca 2ϩ ] i , because even at a subsaturating (15 M) [Ca 2ϩ ] i , 10 M purified cADPR failed to activate TRPM2 currents when applied on its own (Fig. 6, D and F, leftmost bar) and failed to affect TRPM2 currents elicited by 0.1 or 1 M ADPR (Fig. 6, E and F, black and gray bars).

Direct Effectors of TRPM2 Channels
In principle, the lack of effect of cADPR on TRPM2 activity in excised patches might be due to the loss of some cellular factor required for a direct action of cADPR on TRPM2. Because calmodulin has been implicated in the regulation of TRPM2 activity (13,27), we tested whether the whole-cell effects of cADPR could be reproduced in a cell-free system continuously supplied with calmodulin. However, even in the presence of 2 M calmodulin (and subsaturating Ca 2ϩ ), purified cADPR (10 M) failed to elicit TRPM2 currents (Fig. 6, G and I, leftmost bar) or to enhance TRPM2 activity in 1 M ADPR (Fig. 6, H and I, black  and gray bars).
In Cell-free Patches NAADP Is a Low Affinity Partial Agonist and Does Not Sensitize TRPM2 to Activation by ADPR-The recently identified stimulation of TRPM2 activity by the Ca 2ϩ mobilizing second messenger NAADP, both on its own and in synergy with ADPR, prompted us to test the effect of this nucleotide in our cell-free system. Although in the presence of saturating Ca 2ϩ ADPR activates TRPM2 currents with a K1 ⁄ 2 of ϳ1 M (Fig. 1, A and C), for NAADP we had to apply concentrations in the tens of micromolar range to elicit any currents (Fig.  7A). Moreover, even at a saturating concentration (1 mM), NAADP could only partially stimulate TRPM2 activity, as reflected by an approximate doubling of the current by subsequently applied 32 M ADPR (Fig. 7A). The dose-response curve for fractional current activation by NAADP (Fig. 7E, black  squares) identifies this compound as a low affinity partial agonist; a fit to the Hill equation yielded K1 ⁄2 ϭ 104 Ϯ 16 M and a maximal fractional current of 0.49 Ϯ 0.04 relative to that obtained in saturating ADPR in the same patch. This effect of NAADP seems safely attributable to the compound itself, because TLC analysis of our NAADP sample did not reveal any detectable contamination by ADPR (Fig. 7F).
We also examined a possible synergy by submaximal concentrations of NAADP and ADPR. To test whether NAADP could enhance the sensitivity for ADPR activation, we first exposed TRPM2 channels to 50 M NAADP followed by stepwise addition of increasing concentrations of ADPR (Fig. 7B). However, the midpoint of the resulting dose-response curve (Fig. 7E, white circles) was not different from that obtained for ADPR alone (Fig. 7E, black circles, replotted from Fig. 1C); fitted K1 ⁄ 2 for ADPR activation in the presence of 50 M NAADP was 1.2 Ϯ 0.1 M. Conversely, stepwise activation of TRPM2 currents by increasing concentrations of NAADP, all in the presence of 0.1 M ADPR (Fig. 7C), yielded a dose-response curve (Fig. 7E, white squares) that was only slightly shifted (fitted K1 ⁄ 2 ϭ 48 Ϯ 10 M) relative to the one obtained in the absence of ADPR (Fig.  7E, black squares).
Because a potential NAADP effect could be masked at our saturating [Ca 2ϩ ] i , we also measured fractional current activation (relative to that obtained in 32 M ADPR in the same patch) by 50 M and 1 mM NAADP, all in the presence of subsaturating [Ca 2ϩ ] i (15 M). However, this lowered [Ca 2ϩ ] i did not increase the apparent affinity for activation by NAADP, because 50 M NAADP again elicited only ϳ10% of the current obtained with 1 mM NAADP (Fig. 7H, black bars; compare with Fig. 7A and black squares in Fig. 7E). Moreover, the efficacy of NAADP actually seemed to decrease at this lower [Ca 2ϩ ] i , because the maximal current evoked by 1 mM NAADP was only ϳ20% of that supported by 32 M ADPR (Fig. 7H, right black  bar). Finally, even at 15 M [Ca 2ϩ ] i , the presence of 0.1 M ADPR did not affect fractional current activation by 50 M and 1 mM NAADP (Fig. 7H, white versus black bars). Thus, the low affinity and low efficacy of NAADP activation (Fig. 7, A and E) and the lack of synergy between NAADP and ADPR (Fig. 7, B, C, and E) in inside-out patches are not attributable to our use of a saturating [Ca 2ϩ ] i .
NAAD Is a Low Affinity Direct Activator of TRPM2 Channels-NAAD, the dephospho-form of NAADP, has not yet been tested for its ability to modulate TRPM2 currents. Because NAAD is structurally more similar to ADPR than NAADP, we extended our study to include this nucleotide. NAAD resembled NAADP in that it could stimulate TRPM2 currents at high concentrations but seemed more efficient than NAADP in opening the channels at saturating concentrations, because subsequent application of 32 M ADPR caused little further current enhancement (Fig. 7D). Fitting the NAAD dose-response curve (Fig. 7E, black diamonds) yielded K1 ⁄ 2 ϭ 35 Ϯ 4 M and a maximal fractional current of 0.77 Ϯ 0.04. Based on these fit parameters and the lack of detectable ADPR contamination in our NAAD stock (Fig. 7F), this compound can be classified as a low affinity full agonist of TRPM2. In contrast, we could not address the controversial activation of TRPM2 by NAD (3,4,16,19), because TLC analysis revealed a substantial contamination of our NAD stock solution by ADPR (Fig. 7F).

DISCUSSION
Since the initial identification of ADPR and Ca 2ϩ as the primary agonists of TRPM2 (2, 3), a cation channel that becomes activated in cells subjected to oxidative stress (4), several studies have undertaken to elucidate the connection between reactive oxygen species-and ADPR-mediated TRPM2 activation (4,14,15). At the same time, the search for potential additional TRPM2 modulators has identified several adenine nucleotides as potential candidates for affecting TRPM2 gating, including cADPR, AMP, NAADP, and O-acetylated ADPR (4, 5, 16 -19). However, many of the described effects, particularly those of H 2 O 2 and cADPR, have remained controversial, and the reported apparent affinities for all of the above compounds scatter over several orders of magnitude. The most likely reason for these discrepancies is that all of the above studies have been  . 4D). F, TLC analysis of 1-l aliquots of (from left to right) 10 mM ADPR, 100 mM NAAD, 100 mM NAADP, 100 mM NAD, and 32 mM ADPR. Note ADPR contamination in NAD, and a rapidly migrating contaminant in ADPR (also visible in Fig. 5A). G, TLC analysis of 1-l aliquots of (from left to right) 2 mM adenine, 2 mM adenosine, and 10 mM ADPR. The rapidly migrating contaminant in ADPR is fluorescent and roughly co-migrates with adenine. conducted in intact cells with limited control over the intracellular concentrations of metabolites, which are rapidly formed, degraded, and/or compartmentalized by an intact cellular machinery. For the same reason, these studies have not allowed any conclusions regarding whether the newly identified modulators act directly, by binding to the TRPM2 protein, or indirectly, by altering the local concentrations of the primary agonists in the vicinity of the channels. Further uncertainty was provided by reported impurities in commercially available nucleotide preparations (5,19). In the present study we have examined for the first time the effects of the above compounds in cell-free inside-out patches of membrane, which allowed us to identify direct effectors of TRPM2 channel activity.
Direct superfusion of the cytosolic face of the TRPM2 protein by a large concentration (1 mM) of H 2 O 2 for periods up to 60 s did not result in channel activation (Fig. 2, A, left panel, and  B), precluding the possibility that H 2 O 2 -induced activation of TRPM2 currents might reflect oxidation of some residues on the cytosolic surface of the channel. Because H 2 O 2 exposure also failed to inhibit maximal TRPM2 currents induced by saturating ADPR ( Fig. 2A, right panel, and B) and did not affect the apparent affinity for ADPR (Fig. 2B), we conclude that any potential oxidation of TRPM2 residues by H 2 O 2 is unlikely to have a functional consequence. These results are congruent with previous interpretations that oxidative stress leads to TRPM2 activation in cells by promoting accumulation of free ADPR (15) and/or Ca 2ϩ in the vicinity of the channels (also see Ref. 4) and suggest that the recently described synergism between subthreshold concentrations of H 2 O 2 and ADPR for activation of TRPM2 whole-cell currents (16,17) is not due to a direct effect of H 2 O 2 on the TRPM2 channel.
In our cell-free system we did not observe inhibition of ADPR-induced TRPM2 currents by AMP, reported to occur in intact cells with IC 50 values between 10 and 70 M (16, 17), even when AMP was applied at a 60-fold excess relative to ADPR (Fig. 3). This finding is in contrast with TRPM4 channels (28) and TRPM4-like cation channels in brain endothelium (30), which are inhibited with high affinity by AMP in inside-out patches. Thus, although both the C-terminal NUDT9-H domain and the N-terminal TRPM homology region seemed to be potential candidates for binding inhibitory AMP, in the case of TRPM2 the AMP effect must be indirect. In intact cells AMP is a strong metabolic signal that regulates cellular energy production through several signaling pathways including AMP kinase (reviewed in Ref. 31). Therefore, intracellular dialysis of large concentrations of AMP likely causes drastic changes in cellular metabolism that are expected to be cell type-specific. Elucidating how these changes ultimately result in TRPM2 inhibition will be a target of future investigations.
Activation of TRPM2 current by commercial cADPR (Fig. 4) was entirely due to heavy contamination of our cADPR preparation by ADPR (Fig. 5A, right lane), because enzymatic removal of ADPR abolished activation of TRPM2 by cADPR (Fig. 6A). Further, we could observe no sign of sensitization toward ADPR stimulation by 10 M pure cADPR in our cellfree system (Fig. 6, B and C), in contrast to a reported 100-fold lowering of K1 ⁄ 2 for ADPR by dialysis of 10 M cADPR into cells (16). A result similar to ours, obtained with enzymatically treated cADPR in intact cells (5), was argued to reflect clouding of the stimulatory cADPR effect by inhibitory AMP, which is produced by the nucleotide pyrophosphatase treatment (17). In the present experiments such an antagonizing effect can be excluded, because in our cell-free system AMP had no effect on TRPM2 activity even at a concentration of 200 M (Fig. 3, B-F), whereas our test solutions with 10 M purified cADPR contained only an estimated 2-5 M AMP (Fig. 5A). Similarly, ribose-5-phosphate, the other breakdown product present in our purified cADPR solution, neither stimulated nor inhibited TRPM2 activity even at a concentration of 100 M (2, 16), whether applied alone or in combination with AMP. Thus, we conclude that cADPR, at least at concentrations up to 10 M, does not directly stimulate or potentiate stimulation of TRPM2 activity. Taking into account that resting intracellular ADPR concentrations are low micromolar (5), whereas cADPR concentrations were found to be in the nanomolar range (e.g. ϳ200 nM in brain (32), ϳ50 nM in HL-60 leukemia cells (32), ϳ500 nM in Jurkat T-cells (33), and ϳ200 nM in neutrophil granulocytes (5)) and remained Ͻ2 M following cell stimulation by various activating signals (5,32,33), any possible direct effects on TRPM2 of cADPR concentrations higher than 10 M would hardly have physiological relevance. One possible explanation for the observed apparent synergism between low concentrations of cADPR and ADPR in cells (16 -18) might be mobilization of Ca 2ϩ by cADPR acting on ryanodine receptors (24). Although in one study that reported ADPR-cADPR synergism bulk intracellular [Ca 2ϩ ] was not elevated by cADPR dialysis, elevation of [Ca 2ϩ ] in microdomains in the vicinity of TRPM2 could not be excluded (16).
Consistent with whole-cell reports (17,18), we confirm low affinity activation of TRPM2 by NAADP in inside-out patches (Fig. 7A). Because by TLC we could not detect any significant ADPR contamination in our NAADP stock (Fig. 7F), we conclude that this activating effect is a consequence of direct binding of NAADP to TRPM2. However, a direct comparison of TRPM2 currents activated by saturating concentrations of NAADP and ADPR in the same patch revealed only ϳ50% activation by NAADP, identifying the latter as a partial agonist (Fig.  7, A and E). Given the 100-fold lower apparent affinity for NAADP (K1 ⁄ 2 ϭ ϳ100 M; Fig. 7E) (17,18) as compared with ADPR and the low nanomolar concentrations of this nucleotide in living cells (ϳ60 nM in red blood cells (34), ϳ20 nM in hepatocytes, (34), ϳ4 nM in resting, and ϳ30 nM in stimulated Jurkat T-cells (24)), this partial agonist mechanism cannot be considered physiologically relevant. On the other hand, the more promising strong synergism between low concentrations of NAADP and ADPR observed in living cells (i.e. full activation by a combination of subthreshold concentrations of ADPR and NAADP (18)) is not observed in a cell-free system (Fig. 7, B, C, and E) and must therefore reflect some indirect effect of NAADP in intact cells, such as an influence on local [Ca 2ϩ ] and/or on local [ADPR] in the vicinity of TRPM2.
Because our results suggested that NAADP can bind, albeit with low affinity, to the ADPR-binding site on TRPM2, we reasoned that the dephosphorylated form NAAD might be a better agonist given its closer structural resemblance to ADPR. Our data indeed confirmed that removal of the phosphate group from NAADP increases the apparent affinity by ϳ3-fold and also enhances the efficacy for channel opening (Fig. 7, D and E).
Whether NAD is a true activator of TRPM2 is still an open question. Early studies suggested NAD as the primary activator of TRPM2 (3,4), but the very low affinity of this activation (K1 ⁄ 2 Ͼ 1 mM (4,16)), together with demonstrated significant contamination of commercial NAD preparations by ADPR (16,19), has cast doubt on this original interpretation. Our demonstration of a low affinity direct activation of TRPM2 by NAAD (K1 ⁄ 2 ϭ 35 M), together with the structural resemblance of this nucleotide to NAD, raises the possibility that NAD might also act as a low affinity direct agonist of TRPM2, and this might even bear physiological relevance given the high cytosolic NAD concentrations in living cells (4). Unfortunately, the substantial contamination of commercially available NAD by ADPR, also confirmed by our TLC analysis (Fig. 7F), prevents us from addressing this question. An important corollary of our studies is that the purity of nucleotide preparations must be seriously considered when studying TRPM2 regulation. Indeed, even in our stock of ADPR, our TLC studies detected a small (ϳ10%) contamination by a hydrophobic compound that runs almost at the solution front (Figs. 5A and 7, F and G), roughly co-migrating with adenine ( Fig. 7G; note that unlike ribose-5-phosphate, the contaminant in ADPR is fluorescent, suggesting the presence of the adenine base). In control experiments (not shown) adenosine and adenine neither activated nor inhibited TRPM2 currents at concentrations of 10 M, ϳ3-fold higher than we would expect them to be present in our saturating 32 M ADPR solution.
Because we were unable to reproduce in our cell-free system several of the effects reported in intact cells for the compounds under study, we wondered whether this discrepancy might be attributable to some specific environmental parameter in our experiments. Along these lines, we considered the possibility that the reported stimulation by H 2 O 2 (4,14,16) and cADPR (17,18), inhibition by AMP (16,17), or sensitization toward ADPR activation by H 2 O 2 (16,18), cADPR (16 -18), and NAADP (18) might require these compounds to bind to the same site(s) to which activating Ca 2ϩ binds (in this case saturation by Ca 2ϩ might make these sites unavailable for binding the agonist/antagonist of interest) or that they might share a common stimulatory mechanism. In both cases the modulatory effect of the agonist of interest could be masked at our saturating [Ca 2ϩ ] i . Although even in intact cells open channels likely experience saturating, high micromolar, local [Ca 2ϩ ] i (9, 26), we extended our studies to a [Ca 2ϩ ] i of 15 M, which is lower than the K1 ⁄ 2 for Ca 2ϩ activation (ϳ20 M) (9) and is expected to leave more than half of the Ca 2ϩ -binding sites free at any time. However, even at this suboptimal [Ca 2ϩ ] i , which resulted in a moderate shift in the dose-response curve for ADPR activation (Fig. 1, B and C), we could not detect any activation by H 2 O 2 (Fig. 2, C and D), any inhibition by AMP (Fig. 3, E and F), or any synergistic effects when co-applying H 2 O 2 ϩ ADPR (Fig. 2, C  and D), cADPR ϩ ADPR (Fig. 6, E and F), or NAADP ϩ ADPR (Fig. 7H). Thus, the reported whole-cell effects of H 2 O 2 , AMP, cADPR, and NAADP do not involve binding of these compounds to the activating Ca 2ϩ -binding sites on the TRPM2 pro-tein and are mechanistically distinct from the activating effect of Ca 2ϩ .
We also considered the possibility that the use of different intracellular cations might be responsible for the discrepancies between our data and various whole-cell studies; to minimize cell type-specific endogenous currents, the physiological intracellular K ϩ ion used in some of those studies (16 -18) was replaced by Cs ϩ in others (4,14,18) and Na ϩ in the present study. For instance, voltage-dependent inactivation of TRPM2 whole-cell currents was observed in HEK-293 cells loaded with Na ϩ (2), and in the same cell line cADPR activated significant whole-cell currents only when intracellular K ϩ was replaced by Cs ϩ (18), although the latter phenomenon was attributed to some indirect effect of Cs ϩ on ADPR metabolism. Even though in an earlier study we have shown that in inside-out patches intracellular Na ϩ does not confer voltage dependence to TRPM2 gating and that the apparent affinities of ADPR and Ca 2ϩ for activating TRPM2 are the same using intracellular Na ϩ or K ϩ (9), in that study ADPR activation was studied only at saturating [Ca 2ϩ ] i , and synergism between potential co-activators was not addressed. We therefore also investigated the dose dependence for ADPR activation in inside-out patches using a K ϩ -gluconate based bath solution and a subsaturating (15 M) Ca 2ϩ concentration. However, this dose-response relationship (supplemental Fig. S2A) was again identical to that obtained in symmetrical Na ϩ solution (Fig. 1C, white symbols). Moreover, substituting K ϩ for intracellular Na ϩ affected neither the potency nor the efficacy of NAADP to activate TRPM2 currents in subsaturating (15 M) Ca 2ϩ ; as reflected by fractional currents evoked by 50 M and 1 mM NAADP (supplemental Fig. S2B, black bars), which remained identical to those measured using Na ϩ (Fig. 7H, black bars). Finally, even in a K ϩ -based bath solution and subsaturating Ca 2ϩ , we could detect no sign of synergism when co-applying low concentrations of NAADP and ADPR (supplemental Fig. S2B, white bars). Thus, neither intracellular Na ϩ nor saturating Ca 2ϩ used in most of our experiments are responsible for the absence in a cell-free system of the modulatory effects observed in intact cells. Taken together, the most likely explanation is that those whole-cell effects are indirect, although it remains a formal possibility that H 2 O 2 , AMP, cADPR, or NAADP might bind directly to the TRPM2 protein but require the presence of a factor, lost during patch excision, to exert their effects on TRPM2 activity. We have ruled out the possibility that the loss of calmodulin in excised patches might be the culprit for the observed low Ca 2ϩ affinity (Fig. 1, D-F) or the lack of effect of cADPR (Fig. 6, G-I) but cannot exclude the role of some other, as yet unidentified, cellular factor.
In conclusion, we have shown that in inside-out patches H 2 O 2 , AMP, and pure cADPR do not directly affect TRPM2 channel activity, whereas NAADP is a low affinity partial agonist but does not directly potentiate ADPR-mediated activation. Finally, we have also identified NAAD as a low affinity full agonist. Our work suggests that the local concentrations of the primary ligands, ADPR and Ca 2ϩ , should be regarded as a final common pathway through which other signaling molecules can indirectly modulate TRPM2 channel activity in living cells.