Intracellular calcium activates TRPM2 and its alternative spliced isoforms

Abstract

Melastatin-related transient receptor potential channel 2 (TRPM2) is a Ca²⁺-permeable, nonselective cation channel that is involved in oxidative stress-induced cell death and inflammation processes. Although TRPM2 can be activated by ADP-ribose (ADPR) in vitro, it was unknown how TRPM2 is gated in vivo. Moreover, several alternative spliced isoforms of TRPM2 identified recently are insensitive to ADPR, and their gating mechanisms remain unclear. Here, we report that intracellular Ca²⁺ ([Ca²⁺]_i) can activate TRPM2 as well as its spliced isoforms. We demonstrate that TRPM2 mutants with disrupted ADPR-binding sites can be activated readily by [Ca²⁺]_i, indicating that [Ca²⁺]_i gating of TRPM2 is independent of ADPR. The mechanism by which [Ca²⁺]_i activates TRPM2 is via a calmodulin (CaM)-binding domain in the N terminus of TRPM2. Whereas Ca²⁺-mediated TRPM2 activation is independent of ADPR and ADPR-binding sites, both [Ca²⁺]_i and the CaM-binding motif are required for ADPR-mediated TRPM2 gating. Importantly, we demonstrate that intracellular Ca²⁺ release activates both recombinant and endogenous TRPM2 in intact cells. Moreover, receptor activation-induced Ca²⁺ release is capable of activating TRPM2. These results indicate that [Ca²⁺]_i is a key activator of TRPM2 and the only known activator of the spliced isoforms of TRPM2. Our findings suggest that [Ca²⁺]_i-mediated activation of TRPM2 and its alternative spliced isoforms may represent a major gating mechanism in vivo, therefore conferring important physiological and pathological functions of TRPM2 and its spliced isoforms in response to elevation of [Ca²⁺]_i.

Transient receptor potential (TRP) channels have been shown to play important roles under physiological and pathological conditions (1–3). TRPM2, also referred to as TRPC7 (4) or LTRPC2 (5–7), is a member of the melastatin-related (TRPM) TRP channel subfamily, which possesses both ion-channel and ADP-ribose (ADPR) hydrolase functions (5–7). TRPM2 is a Ca²⁺-permeable, nonselective cation channel that is predominantly expressed in various regions of the brain and is also expressed in other tissues, including spleen, heart, liver, lung, and bone marrow (4–6). Studies at cellular levels have implicated that TRPM2 is involved in oxidative stress-mediated cortical and striatal neuronal cell death (8, 9), hematopoietic cell death (5, 8, 10), and insulin secretion (11). A recent report demonstrated that TRPM2 regulates reactive oxygen species-induced chemokine production in monocytes, thereby aggravating inflammation (12).

TRPM2 has been shown to be activated by ADPR (6, 7), oxidative stress (5, 13, 14), NAD⁺ (5, 7, 15), cADPR, and nicotinic acid adenine dinucleotide phosphate (NAADP) (16, 17). ADPR activates TRPM2 by directly binding to the channel's enzymatic NUDT9-H domain (18, 19). Different mechanisms have been proposed for H₂O₂-mediated TRPM2 gating. Some studies suggest that H₂O₂ activates TRPM2 via intracellular release of ADPR (19) or by converting NADH to NAD⁺ (5), whereas other studies support a direct activation mechanism (20), as evidenced by H₂O₂-mediated activation of whole-cell TRPM2 currents (13) and single-channel currents (14). Further, NAADP and cADPR have been reported to activate TRPM2 either directly or in synergy with ADPR in Jurkat T cells and heterologously expressed HEK-293 cells (16, 17). However, another study demonstrated that cADPR was incapable of TRPM2 activation in neutrophil granulocytes (21). These discrepancies about mechanisms of TRPM2 activation may be attributable to differences in expression systems or cell lines. Moreover, different intracellular Ca²⁺ concentrations used in different studies also may have contributed to the controversial results.

Intracellular Ca²⁺ ([Ca²⁺]_i) is involved in a variety of cellular functions. It has been suggested that [Ca²⁺]_i is a modulator for ADPR- and cADPR-mediated TRPM2 activation (6, 22). An increase in [Ca²⁺]_i level significantly reduces the ADPR concentration required for TRPM2 activation (6). External Ca²⁺ also has been shown to influence ADPR-mediated TRPM2 gating (22, 23). However, the detailed mechanisms by which Ca²⁺ synergizes with ADPR in activating TRPM2 remain unknown.

Whereas the gating mechanism and physiological functions of the full-length TRPM2 have been studied extensively, information pertaining to TRPM2 alternative spliced isoforms is largely unavailable (24). Several splice variants of TRPM2 have been identified, including a shorter form (SSF-TRPM2) in which the N-terminal 214-aa residues are removed (25), a C-terminal truncation (TRPM2-ΔC) lacking exon 27, and an N-terminal truncation (TRPM2-ΔN) lacking a portion of exon 11 (13, 15). Although the full-length TRPM2 can be activated by ADPR, NAD⁺, and H₂O₂, it appears that the spliced isoforms cannot be activated by the known activators for the full-length TRPM2 (18, 24, 26). Therefore, it was unclear whether the spliced isoforms can form functional channels (18, 24, 26). Insufficient knowledge about the gating mechanism of the alternative spliced isoforms of TRPM2 largely hampered the investigation of their physiological and/or pathological functions.

A better understanding of TRPM2 gating mechanism as well as how TRPM2 alternative spliced isoforms can be activated is essential for uncovering physiological functions of TRPM2 and its spliced isoforms. Here, we report that [Ca²⁺]_i alone can activate both full-length TRPM2 and its spliced isoforms. Importantly, Ca²⁺ release from intracellular Ca²⁺ stores is able to activate TRPM2 in intact cells. Given that endogenous ADPR, cADPR, NAD⁺, and NAADP concentrations are much lower than those required for TRPM2 activation (16), [Ca²⁺]_i-mediated gating of TRPM2 and its spliced isoforms may represent one of the major gating mechanisms in vivo, and therefore may confer a variety of physiological and pathological functions of TRPM2 and its spliced isoforms.

Results

[Ca²⁺]_i Alone Is Sufficient for TRPM2 Activation.

To investigate whether [Ca²⁺]_i can activate TRPM2, ADPR was excluded from the internal pipette solution for whole-cell current recordings. As shown in Fig. 1 A and B, TRPM2 was robustly activated by [Ca²⁺]_i alone. Like ADPR/Ca²⁺-mediated TRPM2 activation (Fig. 1 D and E), which can be blocked by N-(p-amylcinnamoyl)anthranilic acid (ACA) (27), [Ca²⁺]_i-activated TRPM2 was completely and reversibly blocked by 20 μM ACA (Fig. 1 A and B). Moreover, [Ca²⁺]_i activated TRPM2 in a concentration-dependent manner, with an EC₅₀ of 16.9 μM (Fig. 1G). The EC₅₀ was decreased to 0.49 μM by 10 μM ADPR, suggesting a synergistic effect of ADPR and Ca²⁺. The current amplitude of TRPM2 was about 2.5-fold greater when 10 μM ADPR was included in the pipette solution (Fig. 1G Inset). And the time required for TRPM2 activation was significantly shortened by ADPR (Fig. S1). These results indicate that [Ca²⁺]_i is sufficient for TRPM2 activation, and the effect of Ca²⁺ can be synergized by ADPR. In agreement with this notion, application of Ca²⁺ alone in the cytosolic side was able to activate single-channel openings of TRPM2 in inside-out patches (Fig. 1C). The single-channel currents of TRPM2 elicited by Ca²⁺ (Fig. 1C) were similar to those activated by ADPR/Ca²⁺ (Fig. 1F). Neither [Ca²⁺]_i nor ADPR/Ca²⁺ elicited any channel opening in mock-transfected cells. The single-channel conductance of [Ca²⁺]_i-activated TRPM2 (77.0 pS) was indistinguishable from that of ADPR/Ca²⁺-activated TRPM2 (75.4 pS) (Fig. 1H). These results strongly indicate that [Ca²⁺]_i alone is sufficient for TRPM2 activation. Taken together, our results indicate that [Ca²⁺]_i-mediated TRPM2 activation is independent of ADPR, and that ADPR can synergize with [Ca²⁺]_i in gating of TRPM2 channels.

Fig. 1.

Activation of TRPM2 by intracellular Ca²⁺. (A and D) Time-dependent changes of inward (blue) and outward (red) currents elicited by 100 μM [Ca²⁺]_i and ADPR/Ca²⁺ (200 μM ADPR/100 nM Ca²⁺), respectively, under the indicated conditions. No current was observed in mock-transfected cells (green). Note that ACA (20 μM) blocked TRPM2 in a reversible manner. NMDG-Cl was applied to exclude leak current. (B and E) Representative currents elicited by voltage ramps ranging from −100 to +100 mV in cells activated by 100 μM [Ca²⁺]_i (B) and ADPR/Ca²⁺ (E). The current amplitudes at +100 mV were 272.4 ± 23.7 pA/pF (mean ± SEM, n = 9) and 936.9 ± 130.4 pA/pF (n = 10) in B and E, respectively. (C and F) Single-channel currents activated by 100 μM [Ca²⁺]_i (C) and 100 μM ADPR/100 nM Ca²⁺ (F) in inside-out patches. [Ca²⁺]_i in the cytosal side in C was left unbuffered. (G) Concentration-dependent effects of Ca²⁺ on TRPM2 whole-cell currents in the absence and presence of 10 μM ADPR. The pipette Ca²⁺ concentrations were titrated by 1 mM EGTA. EC₅₀s of Ca²⁺ obtained by best-fit of dose–response curves were 16.9 ± 1.4 μM (n_H = 1.3, n = 5–9) in the absence of ADPR and 0.49 ± 1.1 μM (n_H = 1.7, n = 4–11) in the presence of 10 μM ADPR. (Inset) The averaged current amplitude. (H) A linear regression fit of the single-channel current at indicated potentials (C and F) yielded unitary conductance of 77.4 ± 1.8 pS (n = 8) for Ca²⁺-gated TRPM2 (C) and 75.4 ± 1.2 pS (n = 11) for ADPR/Ca²⁺-activated TRPM2 (F).

[Ca²⁺]_i Activates TRPM2 Mutants with Disrupted ADPR-Binding Sites.

ADPR activates TRPM2 via binding to the ADPR-binding cleft at the C terminus of TRPM2 (19, 28). Mutations of TRPM2, N1326D, and I1405E/L1406F result in nonfunctional channels that cannot be activated by ADPR, NAD, or other known activators (18). Because we have shown that [Ca²⁺]_i-mediated TRPM2 activation is independent of ADPR (Fig. 1), we then investigated whether the ADPR-insensitive mutants can be activated by [Ca²⁺]_i. Although the ADPR-containing pipette solution (100 nM Ca²⁺/200 μM ADPR) used for activating WT TRPM2 failed to activate N1326D and I1405E/L1406F mutants (Fig. S2C), [Ca²⁺]_i alone (100 μM) activated both N1326D (Fig. 2 A and C) and I1405E/L1406F mutants (Fig. S2 A and B). Immunostaining demonstrated that TRPM2 proteins were expressed in WT TRPM2-transfected and N1326D-transfected cells but not in mock-transfected cells (Fig. 2B). ADPR (200 μM) failed to influence the current amplitude of N1326D activated by [Ca²⁺]_i, although it significantly increased WT TRPM2 current amplitude (Fig. 2D), indicating that N1326D is indeed a mutant insensitive to ADPR. Similar results were obtained for the I1405E/L1406F mutant (Fig. S2). Like WT TRPM2, N1326D was activated by [Ca²⁺]_i in a concentration-dependent manner (Fig. 2C). Moreover, Ca²⁺ was able to elicit single-channel opening of N1326D in inside-out patches. Single-channel properties and conductance (Fig. 2 E and F) of N1326D were similar to those of WT TRPM2 (Fig. 1 F and H). In mock-transfected cells, no channel opening was elicited by Ca²⁺. The ability of [Ca²⁺]_i to activate TRPM2 mutants lacking the ADPR-binding sites further indicates that [Ca²⁺]_i alone is sufficient to activate TRPM2.

Fig. 2.

[Ca²⁺]_i activates TRPM2 mutants carrying disrupted ADPR-binding sites. (A) Representative currents activated by 100 μM [Ca²⁺]_i in WT TRPM2, N1326D, and mock-transfected cells. (B) TRPM2 expression detected by immunostaining with anti-FLAG in WT TRPM2- and N1326D-transfected cells, but not in mock-transfected cells. (C) Concentration-dependent effects of [Ca²⁺]_i on N1326D and TRPM2. The EC₅₀s of Ca²⁺ for N1326D and TRPM2 were 14.5 ± 2.1 μM and 16.9 ± 1.4 μM, respectively (mean ± SEM, n = 5–9). (Inset) Mean current amplitude at indicated [Ca²⁺]_i. (D) The average current amplitude of N1326D was similar to that of WT TRPM2 with the pipette solution of 100 μM Ca²⁺. ADPR (200 μM) did not produce synergistic effect with 100 μM [Ca²⁺]_i in N1326D. (E) Ca²⁺-activated single-channel currents of N1326D in inside-out patches. (F) Single-channel conductance of N1326D (70 ± 1.6 pS, n = 4–7).

Mechanism of [Ca²⁺]_i-Mediated TRPM2 Activation.

The above results demonstrated a crucial role for [Ca²⁺]_i in activating TRPM2. To investigate the mechanism of [Ca²⁺]_i-mediated TRPM2 activation, we first tested whether calmodulin (CaM) is involved in [Ca²⁺]_i activation of TRPM2. Overexpression of CaM did not significantly alter the current amplitude of TRPM2 (TRPM2, 200.5 ± 25.1 pA/pF, n = 10; TRPM2 and CaM, 244.5 ± 51.0 pA/pF, n = 7; P > 0.05), presumably because of sufficient expression of endogenous CaM. However, when the CaM mutant CaM1-4, with mutations at 4 EF hands (CaM1-4) was cotransfected with either WT TRPM2 or N1326D (Fig. 3B), their current amplitude decreased significantly, suggesting a potential role for CaM in [Ca²⁺]_i-mediated TRPM2 activation. We then created a TRPM2 mutant (Fig. 3A) via substitution of the IQ-like motif, based on a previous study suggesting that an IQ-like motif in the N terminus of TRPM2 binds to CaM and is involved in H₂O₂-induced Ca²⁺ influx through TRPM2 (29). Although the IQ-mut was able to express TRPM2 protein as detected by immunostaining (Fig. S3), IQ-mut could not be activated by [Ca²⁺]_i at 100 μM (Fig. 3D). Examination of surface protein of IQ-mut yielded similar expression levels in comparison with WT TRPM2 and N1326D (Fig. 3C). And the ratio of membrane versus cytosol protein was also similar among WT TRPM2, IQ-mut, and N1326D. Thus, the nonfunctional IQ-mut indicates that the CaM-binding site is essential for [Ca²⁺]_i activation of TRPM2 (Fig. 3E). Moreover, the IQ-mut could not be activated by addition of ADRP (200 μM) in the pipette solution (Fig. 3E), suggesting that the IQ-like motif is essential not only for [Ca²⁺]_i-mediated TRPM2 activation, but also for ADPR/Ca²⁺-elicited TRPM2 activation.

Fig. 3.

[Ca²⁺]_i activates TRPM2 via CaM-binding domain. (A) Mutated residues in the IQ-like motif (406 to 416) within the CaM-binding site located in the N terminus of TRPM2. (B) CaM mutant (CaM1-4) significantly decreased current amplitude of WT TRPM2 and N1326D. (C) Membrane protein versus cytosol protein expression of WT TRPM2, IQ-mut, and N1326D. GAPDH was used as loading control. Membrane protein was obtained by surface biotinylation. Similar results were obtained in 3 separate experiments. (D) Representative recordings of WT TRPM2, IQ-mut, and mock-transfected HEK-293 cells with a pipette solution containing 100 μM Ca²⁺. [Ca²⁺]_i (100 μM) failed to activate IQ-mut. (E) Current amplitude of WT TRPM2, IQ-mut, and mock-transfected cells in the presence of 100 μM [Ca²⁺]_i with or without 200 μM ADPR. Note that ADPR was ineffective on IQ-mut.

[Ca²⁺]_i Can Activate Alternative Spliced Isoforms of TRPM2.

Although several alternative spliced isoforms of human TRPM2 have been identified, their gating mechanism and activators have remained unknown. Since we have provided compelling evidence that [Ca²⁺]_i alone is sufficient to activate WT TRPM2 and the mutants lacking ADPR-binding sites, we investigated whether [Ca²⁺]_i was capable of TRPM2 spliced isoform activation. We created the following truncation mutants to mimic the endogenous spliced isoforms: TRPM2-ΔN, TRPM2-ΔC, and TRPM2-ΔN/ΔC (Fig. 4A). Although 100 nM Ca²⁺/1 mM ADPR failed to elicit any current (Fig. 4E), intracellular Ca²⁺ alone at 10 μM remarkably activated TRPM2-ΔN, TRPM2-ΔC, and TRPM2-ΔN/ΔC (Fig. 4 B and C). Concentration-dependent activation of TRPM2-ΔN, TRPM2-ΔC, and TRPM2-ΔN/ΔC by [Ca²⁺]_i also was observed (Fig. 4D). These results indicate that [Ca²⁺]_i alone is sufficient for activation of the spliced isoforms of TRPM2. Because these spliced isoforms of TRPM2 cannot be activated by other activators, such as ADPR and H₂O₂ (19), our results suggest that [Ca²⁺]_i may serve as an in vivo activator of the alternative spliced isoforms of TRPM2, therefore conferring their physiological functions.

Fig. 4.

[Ca²⁺]_i is an activator of TRPM2 alternative spliced isoforms. (A) Putative structure of TRPM2 illustrating the N and C termini of TRPM2 and alternative spliced isoforms. TRPM2-ΔN lacks residues 535–555; TRPM2-ΔC lacks residues 1291–1329. TRPM2-ΔN/ΔC carries deletions in both the N and C termini. (B) [Ca²⁺]_i (10 μM) robustly activated TRPM2-ΔN, TRPM2-ΔC, and TRPM2-ΔN/ΔC with amplitudes similar to WT TRPM2. (C) Time-dependent changes of inward current amplitudes of TRPM2-ΔN, TRPM2-ΔC, and TRPM2-ΔN/ΔC. (D) Concentration-dependent effects of [Ca²⁺]_i on TRPM2-ΔN, TRPM2-ΔC, and TRPM2-ΔN/ΔC. (E) Current amplitude of TRPM2-ΔN, TRPM2-ΔC, and TRPM2-ΔN/ΔC activated by 10 μM [Ca²⁺]_i. ADPR (1 mM) with 100 nM [Ca²⁺]_i failed to activate TRPM2 spliced isoforms.

[Ca²⁺]_i Activates TRPM2 Under Physiological Conditions.

To study the physiological relevance of [Ca²⁺]_i-mediated activation of TRPM2, we performed perforated-patch experiments to determine whether intracellular Ca²⁺ release is sufficient for TRPM2 activation. As shown in Fig. 5A, under the perforated patch configuration, no currents were elicited by the voltage-ramp protocol before application of ionomycin. However, extracellular perfusion of 5 μM ionomycin evoked substantial TRPM2 channel activation (Fig. 5A). After cell rupture, current amplitude was dramatically increased because of the dialysis of ADPR from pipette solution into cytosol (Fig. 5 A and B). To verify that it was the change of [Ca²⁺]_i that activated TRPM2, we did simultaneous current recording and Ca²⁺ concentration measurement. As illustrated in Fig. 5C, an increase in [Ca²⁺]_i led to TRPM2 activation, which in turn increased intracellular Ca²⁺ levels.

Fig. 5.

Intracellular Ca²⁺ activates TRPM2 in perforated-patch experiments. (A) Time-dependent changes of inward and outward currents elicited by application of 5 μM ionomycin (iono), and after cell rupture. The pipette solution contained 180 μg/mL nystatin, 200 μM ADPR, and 100 μM Fura-2. (B) Representative recordings under indicated conditions (a, b, c) as shown in A. (C) Simultaneous recordings of current development and changes of [Ca²⁺]_i (red). The left y axis represents current amplitude, and the right y axis represents F340/F380 changes. (D) Mean current amplitude of TRPM2 before and after cell rupture. (E) Time-dependent changes of inward and outward TRPM2 currents elicited by 100 μM TBHQ in the absence (perforated-patch configuration) and presence of ADPR (after cell rupture). NMDG solution was used to test leak current. (F) Typical recordings of TRPM2 elicited by TBHQ and ADPR at indicated time points (a, b, c) as shown in E. The mean current amplitudes of TRPM2 before and after cell rupture were 282.9 ± 50.2 pA/pF and 969.2 ± 100.2 pA/pF, respectively.

Because ionomycin may mobilize both intracellular and extracellular Ca²⁺, we further investigated whether Ca²⁺ release could activate TRPM2 in perforated whole-cell current recordings. Fig. 5 E and F show that 100 μM tert-butylhydroquinone (TBHQ), a Ca²⁺ pump blocker, induced intracellular Ca²⁺ release that was sufficient to activate TRPM2. The mean current amplitude of TRPM2 elicited by TBHQ was 2,067 ± 357.5 pA (n = 6). Maximal current amplitude of TRPM2 induced by ADPR after cell rupture was 8,333 ± 666.7 pA (n = 6). Although the current amplitude of TRPM2 induced by TBHQ was only ≈25% of the maximal current elicited by Ca²⁺/ADPR, these results indicate that [Ca²⁺]_i release is capable of activating TRPM2.

Receptor Activation-Induced Ca²⁺ Release Can Activate TRPM2.

Because passive Ca²⁺ release by TBHQ could activate TRPM2, we investigated whether receptor-mediated Ca²⁺ release was capable of activating TRPM2. As shown in Fig. 6, under the perforated-patch configuration, application of 500 μM carbachol (CCh) induced typical TRPM2 activation with a linear I–V relation (Fig. 6 A and B) in a pancreatic beta cell (HIT T15 cell). CCh-mediated activation of TRPM2 was through PLC-induced Ca²⁺ release via IP₃R because CCh-induced TRPM2 was eliminated by PLC and IP₃R blockers (Fig. S4 C and D) and largely inhibited by EGTA-AM (Fig. S4A). The current was readily blocked by 20 μM ACA in a reversible manner (Fig. 6 A and B). Currents elicited by voltage steps also displayed the typical instantaneous activation characteristics of TRPM2 (Fig. 6C). Current amplitude was larger when 100 μM Ca²⁺ or 200 μM ADPR/100 nM Ca²⁺ was included in the pipette solution for whole-cell current recording (Fig. 6D). All of the features, including the linear I–V relation, instantaneous activation, and blockade by ACA, are typical characteristics of TRPM2. To further confirm that there was no contamination from Ca²⁺-activated TRPM4, which has been shown to express in beta cell lines, including INS-1 and RINm5F (30), we transfected nonfunctional IQ-mut, which can inhibit heterologously expressed TRPM2 but not TRPM4 currents (Fig. 6E), into HIT T15 cells. Expression of IQ-mut dramatically diminished the current induced by CCh in HIT T15 cells, indicating that the CCh-elicited current in HIT T15 cells is TRPM2 current. Importantly, the TRPM2 current elicited by CCh in HIT T15 cells was not affected by the dominant-negative TRPM4 (DN-TRPM4) transfected into HIT T15 cells (Fig. 6F). Furthermore, because TRPM2 is Ca²⁺-permeable channel, whereas TRPM4 is Ca²⁺-impermeable channel, we tested current amplitude by perfusing the cells with isotonic Ca²⁺ solution. The ratio of the I_Ca/I_tyrode inward current amplitude of the CCh-activated channel in HIT T15 cells was similar to that of TRPM2 expressed in HEK-293 cells, whereas TRPM4 inward current was virtually eliminated by isotonic Ca²⁺ solution (Fig. S5). These results further support that CCh-activated channel in HIT T15 cells is TRPM2. Taken together, our results indicate that Ca²⁺ release through receptor activation can induce TRPM2 channel activation. This finding implies that TRPM2 may play important roles in response to [Ca²⁺]_i release under a variety of physiological conditions.

Fig. 6.

Receptor activation-induced Ca²⁺ release activates endogenous TRPM2 current in HIT T15 cells under perforated-patch conditions. (A) Time-dependent changes in inward currents measured at −100 mV before and after application of 500 μM CCh in a pancreatic beta cell line (HIT T15). Note that NMDG completely eliminated the inward current. ACA (20 μM) effectively and reversibly blocked TRPM2. (B) Representative recordings of TRPM2 elicited by ramp protocols at indicated time points after ACA and NMDG-Cl. Note the linear I–V relation of TRPM2. (C) Typical TRPM2 current elicited by voltage-step protocol at a 20-mV increment. (D) The mean current density of TRPM2 induced by CCh (perforated patch), 100 μM [Ca²⁺]_i alone (whole cell), and 200 μM ADPR/100 nM Ca²⁺ (whole cell) in HIT T15 cells. (E) The nonfunctional IQ-mut significantly inhibited TRPM2 current expressed in HEK-293 cells and the CCh-elicited current in HIT T15 cells. However, TRPM4 current expressed in HEK-293 cells was not influenced by IQ-mut of TRPM2. (F) DN-TRPM4 did not alter CCh-induced currents in HIT T15 cells or the TRPM2 currents expressed in HEK-293 cells, although TRPM4 current was significantly decreased by DN-TRPM4.

Discussion

Our results reveal that [Ca²⁺]_i plays a pivotal role in TRPM2 channel gating (Fig. S6): not only is [Ca²⁺]_i sufficient to activate TRPM2, but it is required for ADPR-mediated TRPM2 activation. Further, [Ca²⁺]_i is capable of activating alternative spliced isoforms of TRPM2, which are insensitive to the full-length TRPM2 agonists, including ADPR. The mechanism of [Ca²⁺]_i-mediated TRPM2 activation is via a CaM-binding domain, an IQ-like motif located in the N terminus of TRPM2. Importantly, we found Ca²⁺ release via receptor activation can activate TRPM2 in intact cells. Our results not only establish that [Ca²⁺]_i is an activator of TRPM2 and its spliced isoforms, but also provide new insights into their physiological and/or pathological functions.

[Ca²⁺]_i Is an Activator for TRPM2 and Its Alternative Spliced Forms.

We provide several lines of evidence demonstrating that [Ca²⁺]_i is an activator of TRPM2. Both whole-cell and single-channel currents can be activated by [Ca²⁺]_i in the absence of ADPR (Figs. 1 and 2). Moreover, [Ca²⁺]_i can activate TRPM2 mutants (N1326D and I1405E/L1406F) that are insensitive to ADPR, NAD⁺, and H₂O₂ (13, 19). More strikingly, [Ca²⁺]_i can activate the alternative spliced isoforms, TRPM2-ΔC, TRPM2-ΔN, and TRPM2-ΔC-ΔN, identified in human neutrophil cells (13, 15), which are insensitive to ADPR and H₂O₂ (18, 19) (Fig. 4). These results suggest that [Ca²⁺]_i may serve as a major activator in vivo for TRPM2 and its spliced isoforms.

The EC₅₀ of Ca²⁺ for WT TRPM2 and N1326D is about 16 μM in the presence of 1 mM EGTA. However, this value may have been overestimated, because we found that EGTA can directly block TRPM2 (Fig. S7). Thus, the EC₅₀ of Ca²⁺ should be lower than 16 μM in the absence of Ca²⁺ chelators. Indeed, in the perforated-patch experiments, TRPM2 can be activated by intracellular Ca²⁺ release (Figs. 5 and 6). Furthermore, because local Ca²⁺ concentrations can readily reach 100 μM in nanodomains and 1–5 μM in microdomains (31, 32), it is likely that TRPM2 or its spliced isoforms can be readily activated by elevation of local [Ca²⁺]_i in the absence of ADPR, thereby conferring physiological functions.

Ca²⁺–CaM Binding to TRPM2 Is Essential for [Ca²⁺]_i and ADPR/Ca²⁺-Mediated Activation of TRPM2.

We demonstrate that CaM-binding domain IQ-like motif is essential for [Ca²⁺]_i-mediated TRPM2 activation. The IQ-like motif is located in the N terminus of TRPM2 (406–416: IQDIVRRRQLL) (29). Replacement of the motif with AADIVAAAQLA (IQ-mut) disrupted the interaction of CaM and TRPM2 (Fig. S8), therefore abolishing TRPM2 activation evoked by [Ca²⁺]_i and ADPR/Ca²⁺ (Fig. 4). These results not only establish that [Ca²⁺]_i is necessary and sufficient for TRPM2 activation, but they also elucidate that the Ca²⁺–CaM binding to TRPM2 is required for both [Ca²⁺]_i- and ADPR/Ca²⁺-mediated TRPM2 activation.

The Ca²⁺–CaM binding to TRPM2 may also have contributed to the activation kinetics of TRPM2. Previous study has observed that activation time course of TRPM2 is dependent on the concentrations of intracellular activators (17). We demonstrate that the time course of [Ca²⁺]_i gating of TRPM2 is also strongly associated with [Ca²⁺]_i concentrations (Fig. S1). Moreover, because Ca²⁺–CaM binding to various target proteins may have different kinetics (33), the relatively slow activation by [Ca²⁺]_i may be attributable to a slow binding kinetics between Ca²⁺–CaM and TRPM2. Nonetheless, further studies are required to understand whether there is a correlation between the kinetics of Ca²⁺–CaM binding to TRPM2 and the activation time course of TRPM2 gated by [Ca²⁺]_i.

Physiological Relevance of [Ca²⁺]_i-Mediated Activation of TRPM2 and Its Alternative Spliced Isoforms.

Ca²⁺ is involved in a variety of signaling pathways and diverse cellular functions. Here, we demonstrate for the first time that [Ca²⁺]_i is an activator for TRPM2 and its alternative spliced isoforms. Because the full-length TRPM2 is activated by both [Ca²⁺]_i and ADPR/Ca²⁺, whereas the spliced isoforms can only be activated by [Ca²⁺]_i, the differential activation mechanisms and specific distribution pattern of these isoforms (13, 25) suggest that TRPM2 and its spliced isoforms may play distinctive roles.

TRPM2 has been shown to be involved in oxidative stress-induced cell death (5, 8, 10, 34), mainly because of excessive Ca²⁺ entry via TRPM2 channels. We have demonstrated that [Ca²⁺]_i- activated TRPM2 is about 20–40% of the maximal TRPM2 current amplitude elicited by ADPR/Ca²⁺. Thus, TRPM2 may have 2 modes in terms of the execution of physiological functions (Fig. S6): In the presence of elevated ADPR concentrations or under oxidative stress conditions, which stimulate ADPR production (35), TRPM2 can be activated to the maximal degree, which may produce detrimental effects and lead to cell death, whereas under normal conditions, when intracellular ADPR is less than 5 μM (21) or 1 μM (23), increases in [Ca²⁺]_i levels locally or globally will elicit moderate TRPM2 activation, thereby conferring physiological functions. Nonetheless, our results provide a novel Ca²⁺ gating mechanism that may confer novel, yet uncharacterized, physiological functions of TRPM2 and its alternative spliced isoforms.

Conclusions.

In the present study, we have demonstrated that [Ca²⁺]_i can activate TRPM2 and its alternative spliced isoforms, and that the CaM-binding motif confers [Ca²⁺]_i- as well as ADPR/Ca²⁺-mediated TRPM2 activation. Importantly, Ca²⁺ release from intracellular Ca²⁺ stores can activate TRPM2 in intact cells. Our findings reveal a novel gating mechanism of TRPM2 and its alternative spliced isoforms that may represent a major gating mechanism in vivo, and therefore confer novel, as-yet unknown physiological and/or pathological functions.

Experimental Procedures

Molecular Biology.

FLAG-tagged TRPM2 construct was kindly provided by A. Scharenberg (University of Washington, Seattle). Mutations of TRPM2 were made by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) following the manufacturer's instruction. Truncation mutations were generated by introducing EcoRI sites at the C terminus of TRPM2 for TRPM2-ΔC and by using a primer-loop to delete the 20 residues at the N terminus for generating TRPM2-ΔN. The primers will be available upon request.

Electrophysiology.

Whole-cell and single-channel currents were recorded from HEK-293 cells transfected with full-length TRPM2, TRPM2 mutants, and alternative spliced isoforms (SI Materials and Methods). Detailed methods for whole-cell recordings, perforated-patch experiments, and single-channel recordings are described in SI Materials and Methods.

Ratio Ca²⁺ Imaging Experiments.

Changes in intracellular Ca²⁺ were measured by ratio Ca²⁺ imaging (IonOptix). Simultaneous measurement of Ca²⁺ signal and TRPM2 activation was conducted by using perforated-patch and ratio imaging on the same cell (SI Materials and Methods).

Immunostaining and Western Blot Experiments.

TRPM2-transfected cells were immunostained with α-FLAG antibody. The immunostained cells were analyzed by using a Zeiss LSM 510 confocal microscope. Protein expression was detected by Western blot experiments with anti-FLAG antibody. Surface and cytosol proteins were extracted by using the biotinylation method (SI Materials and Methods).

Data Analysis.

Pooled data are presented as mean ± SEM. Dose–response curves were fitted by an equation: E = E_max{1/[1+(EC₅₀/C)ⁿ]}, where E is the effect at concentration C, E_max is maximal effect, EC₅₀ is the concentration for half-maximal effect, and n is the Hill coefficient. EC₅₀ is replaced with IC₅₀ if the effect is an inhibitory effect. Statistical comparisons were made by using 2-way ANOVA and 2-tailed t test with Bonferroni correction. P < 0.05 indicated statistical significance.