Intracellular Calcium Activates TRPM2 and Its Alternative Spliced Isoforms

Intracellular calcium activates TRPM2 and its alternative spliced isoforms

Jianyang Du, Jia Xie, and Lixia Yue1

Department of Cell Biology, Calhoun Cardiology Center, University of Connecticut Health Center, Farmington, CT 06030

Edited by David E. Clapham, Harvard Medical School, Boston, MA, and approved February 26, 2009 (received for review November 18, 2008) Melastatin-related transient receptor potential channel 2 (TRPM2) ADPR in Jurkat T cells and heterologously expressed HEK-293 isaCa2؉-permeable, nonselective cation channel that is involved in cells (16, 17). However, another study demonstrated that cADPR oxidative stress-induced cell death and inflammation processes. was incapable of TRPM2 activation in neutrophil granulocytes (21). Although TRPM2 can be activated by ADP-ribose (ADPR) in vitro, it These discrepancies about mechanisms of TRPM2 activation may was unknown how TRPM2 is gated in vivo. Moreover, several be attributable to differences in expression systems or cell lines. alternative spliced isoforms of TRPM2 identified recently are in- Moreover, different intracellular Ca2ϩ concentrations used in dif- sensitive to ADPR, and their gating mechanisms remain unclear. ferent studies also may have contributed to the controversial results. 2؉ 2؉ 2ϩ 2ϩ Here, we report that intracellular Ca ([Ca ]i) can activate TRPM2 Intracellular Ca ([Ca ]i) is involved in a variety of cellular 2ϩ as well as its spliced isoforms. We demonstrate that TRPM2 functions. It has been suggested that [Ca ]i is a modulator for mutants with disrupted ADPR-binding sites can be activated ADPR- and cADPR-mediated TRPM2 activation (6, 22). An 2؉ 2؉ 2ϩ readily by [Ca ]i, indicating that [Ca ]i gating of TRPM2 is increase in [Ca ]i level significantly reduces the ADPR concen- 2؉ ϩ independent of ADPR. The mechanism by which [Ca ]i activates tration required for TRPM2 activation (6). External Ca2 also has TRPM2 is via a calmodulin (CaM)-binding domain in the N terminus been shown to influence ADPR-mediated TRPM2 gating (22, 23). ؉ of TRPM2. Whereas Ca2 -mediated TRPM2 activation is indepen- However, the detailed mechanisms by which Ca2ϩ synergizes with 2؉ dent of ADPR and ADPR-binding sites, both [Ca ]i and the CaM- ADPR in activating TRPM2 remain unknown. binding motif are required for ADPR-mediated TRPM2 gating. Whereas the gating mechanism and physiological functions of the ؉ Importantly, we demonstrate that intracellular Ca2 release acti- full-length TRPM2 have been studied extensively, information vates both recombinant and endogenous TRPM2 in intact cells. pertaining to TRPM2 alternative spliced isoforms is largely un- ؉ Moreover, receptor activation-induced Ca2 release is capable of available (24). Several splice variants of TRPM2 have been iden- 2؉ activating TRPM2. These results indicate that [Ca ]i is a key tified, including a shorter form (SSF-TRPM2) in which the N- activator of TRPM2 and the only known activator of the spliced terminal 214-aa residues are removed (25), a C-terminal truncation 2؉ isoforms of TRPM2. Our findings suggest that [Ca ]i-mediated (TRPM2-⌬C) lacking exon 27, and an N-terminal truncation activation of TRPM2 and its alternative spliced isoforms may (TRPM2-⌬N) lacking a portion of exon 11 (13, 15). Although the represent a major gating mechanism in vivo, therefore conferring ϩ full-length TRPM2 can be activated by ADPR, NAD , and H2O2, important physiological and pathological functions of TRPM2 and it appears that the spliced isoforms cannot be activated by the PHYSIOLOGY 2؉ its spliced isoforms in response to elevation of [Ca ]i. known activators for the full-length TRPM2 (18, 24, 26). Therefore, it was unclear whether the spliced isoforms can form functional 2ϩ ͉ ͉ ͉ Ca signaling gating mechanism ADP-ribose channels (18, 24, 26). Insufficient knowledge about the gating ͉ calmodulin-binding domain oxidative stress mechanism of the alternative spliced isoforms of TRPM2 largely hampered the investigation of their physiological and/or patholog- ransient receptor potential (TRP) channels have been shown to ical functions. Tplay important roles under physiological and pathological con- A better understanding of TRPM2 gating mechanism as well as ditions (1–3). TRPM2, also referred to as TRPC7 (4) or LTRPC2 how TRPM2 alternative spliced isoforms can be activated is essen- (5–7), is a member of the melastatin-related (TRPM) TRP channel tial for uncovering physiological functions of TRPM2 and its spliced subfamily, which possesses both ion-channel and ADP-ribose isoforms. Here, we report that [Ca2ϩ] alone can activate both ϩ i (ADPR) hydrolase functions (5–7). TRPM2 is a Ca2 -permeable, full-length TRPM2 and its spliced isoforms. Importantly, Ca2ϩ nonselective cation channel that is predominantly expressed in release from intracellular Ca2ϩ stores is able to activate TRPM2 in various regions of the brain and is also expressed in other tissues, intact cells. Given that endogenous ADPR, cADPR, NADϩ, and including spleen, heart, liver, lung, and bone marrow (4–6). Studies NAADP concentrations are much lower than those required for 2ϩ at cellular levels have implicated that TRPM2 is involved in TRPM2 activation (16), [Ca ]i-mediated gating of TRPM2 and its oxidative stress-mediated cortical and striatal neuronal cell death spliced isoforms may represent one of the major gating mechanisms (8, 9), hematopoietic cell death (5, 8, 10), and insulin secretion (11). in vivo, and therefore may confer a variety of physiological and A recent report demonstrated that TRPM2 regulates reactive pathological functions of TRPM2 and its spliced isoforms. oxygen species-induced chemokine production in monocytes, thereby aggravating inflammation (12). Results TRPM2 has been shown to be activated by ADPR (6, 7), 2؉ ϩ [Ca ]i Alone Is Sufficient for TRPM2 Activation. To investigate oxidative stress (5, 13, 14), NAD (5, 7, 15), cADPR, and nicotinic 2ϩ whether [Ca ]i can activate TRPM2, ADPR was excluded from acid adenine dinucleotide phosphate (NAADP) (16, 17). ADPR the internal pipette solution for whole-cell current recordings. As 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 Author contributions: J.D., J.X., and L.Y. designed research; J.D. and J.X. performed research; J.D. and J.X. analyzed data; and J.D., J.X., and L.Y. wrote the paper. that H2O2 activates TRPM2 via intracellular release of ADPR (19) ϩ or by converting NADH to NAD (5), whereas other studies The authors declare no conflict of interest. support a direct activation mechanism (20), as evidenced by H2O2- This article is a PNAS Direct Submission. mediated activation of whole-cell TRPM2 currents (13) and single- 1To whom correspondence should be addressed. E-mail: [email protected]. channel currents (14). Further, NAADP and cADPR have been This article contains supporting information online at www.pnas.org/cgi/content/full/ reported to activate TRPM2 either directly or in synergy with 0811725106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811725106 PNAS ͉ April 28, 2009 ͉ vol. 106 ͉ no. 17 ͉ 7239–7244 Downloaded by guest on September 23, 2021 2+ 2+ AD100 µM [Ca ]i ADPR/Ca G 8000 20000 20 µMACA 4000 10000 20 µM ACA +100 mV µ +100 mV 10 M ADPR w/o ADPR 0 ± µ ± µ pA pA 0 EC =0.49 1.1 M EC =16.9 1.4 M -100 mV 50 50 -100 mV 1.0 -4000 -10000 NMDG-Cl NMDG-Cl -8000 -20000 0 100 200 300 400 500 0 100 200 300 400 Time (s) Time (s)

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2ϩ 2ϩ Fig. 1. Activation of TRPM2 by intracellular Ca .(A and D) Time-dependent changes of inward (blue) and outward (red) currents elicited by 100 ␮M [Ca ]i and ADPR/Ca2ϩ (200 ␮M ADPR/100 nM Ca2ϩ), respectively, under the indicated conditions. No current was observed in mock-transfected cells (green). Note that ACA (20 ␮M) blocked TRPM2 in a reversible manner. NMDG-Cl was applied to exclude leak current. (B and E) Representative currents elicited by voltage ramps 2ϩ 2ϩ ranging from Ϫ100 to ϩ100 mV in cells activated by 100 ␮M [Ca ]i (B) and ADPR/Ca (E). The current amplitudes at ϩ100 mV were 272.4 Ϯ 23.7 pA/pF (mean Ϯ 2ϩ SEM, n ϭ 9) and 936.9 Ϯ 130.4 pA/pF (n ϭ 10) in B and E, respectively. (C and F) Single-channel currents activated by 100 ␮M [Ca ]i (C) and 100 ␮M ADPR/100 2ϩ 2ϩ 2ϩ nM Ca (F) in inside-out patches. [Ca ]i in the cytosal side in C was left unbuffered. (G) Concentration-dependent effects of Ca on TRPM2 whole-cell currents 2ϩ 2ϩ in the absence and presence of 10 ␮M ADPR. The pipette Ca concentrations were titrated by 1 mM EGTA. EC50sofCa obtained by best-fit of dose–response curves were 16.9 Ϯ 1.4 ␮M(nH ϭ 1.3, n ϭ 5–9) in the absence of ADPR and 0.49 Ϯ 1.1 ␮M(nH ϭ 1.7, n ϭ 4–11) in the presence of 10 ␮M 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 Ϯ 1.8 pS (n ϭ 8) for Ca2ϩ-gated TRPM2 (C) and 75.4 Ϯ 1.2 pS (n ϭ 11) for ADPR/Ca2ϩ-activated TRPM2 (F).

2ϩ 2؉ shown in Fig. 1 A and B, TRPM2 was robustly activated by [Ca ]i [Ca ]i Activates TRPM2 Mutants with Disrupted ADPR-Binding Sites. alone. Like ADPR/Ca2ϩ-mediated TRPM2 activation (Fig. 1 D and ADPR activates TRPM2 via binding to the ADPR-binding cleft at E), which can be blocked by N-(p-amylcinnamoyl)anthranilic acid the C terminus of TRPM2 (19, 28). Mutations of TRPM2, N1326D, 2ϩ (ACA) (27), [Ca ]i-activated TRPM2 was completely and revers- and I1405E/L1406F result in nonfunctional channels that cannot be 2ϩ ibly blocked by 20 ␮M ACA (Fig. 1 A and B). Moreover, [Ca ]i activated by ADPR, NAD, or other known activators (18). Because 2ϩ activated TRPM2 in a concentration-dependent manner, with an we have shown that [Ca ]i-mediated TRPM2 activation is inde- EC50 of 16.9 ␮M (Fig. 1G). The EC50 was decreased to 0.49 ␮Mby pendent of ADPR (Fig. 1), we then investigated whether the 2ϩ 2ϩ 10 ␮M ADPR, suggesting a synergistic effect of ADPR and Ca . ADPR-insensitive mutants can be activated by [Ca ]i. Although The current amplitude of TRPM2 was about 2.5-fold greater when the ADPR-containing pipette solution (100 nM Ca2ϩ/200 ␮M 10 ␮M ADPR was included in the pipette solution (Fig. 1G Inset). ADPR) used for activating WT TRPM2 failed to activate N1326D 2ϩ And the time required for TRPM2 activation was significantly and I1405E/L1406F mutants (Fig. S2C), [Ca ]i alone (100 ␮M) 2ϩ shortened by ADPR (Fig. S1). These results indicate that [Ca ]i is activated both N1326D (Fig. 2 A and C) and I1405E/L1406F sufficient for TRPM2 activation, and the effect of Ca2ϩ can be mutants (Fig. S2 A and B). Immunostaining demonstrated that synergized by ADPR. In agreement with this notion, application of TRPM2 proteins were expressed in WT TRPM2-transfected and Ca2ϩ alone in the cytosolic side was able to activate single-channel N1326D-transfected cells but not in mock-transfected cells (Fig. openings of TRPM2 in inside-out patches (Fig. 1C). The single- 2B). ADPR (200 ␮M) failed to influence the current amplitude of 2ϩ 2ϩ channel currents of TRPM2 elicited by Ca (Fig. 1C) were similar N1326D activated by [Ca ]i, although it significantly increased WT 2ϩ 2ϩ to those activated by ADPR/Ca (Fig. 1F). Neither [Ca ]i nor TRPM2 current amplitude (Fig. 2D), indicating that N1326D is ADPR/Ca2ϩ elicited any channel opening in mock-transfected indeed a mutant insensitive to ADPR. Similar results were obtained 2ϩ cells. The single-channel conductance of [Ca ]i-activated TRPM2 for the I1405E/L1406F mutant (Fig. S2). Like WT TRPM2, 2ϩ 2ϩ (77.0 pS) was indistinguishable from that of ADPR/Ca -activated N1326D was activated by [Ca ]i in a concentration-dependent TRPM2 (75.4 pS) (Fig. 1H). These results strongly indicate that manner (Fig. 2C). Moreover, Ca2ϩ was able to elicit single-channel 2ϩ [Ca ]i alone is sufficient for TRPM2 activation. Taken together, opening of N1326D in inside-out patches. Single-channel properties 2ϩ our results indicate that [Ca ]i-mediated TRPM2 activation is and conductance (Fig. 2 E and F) of N1326D were similar to those 2ϩ independent of ADPR, and that ADPR can synergize with [Ca ]i of WT TRPM2 (Fig. 1 F and H). In mock-transfected cells, no 2ϩ 2ϩ in gating of TRPM2 channels. channel opening was elicited by Ca . The ability of [Ca ]i to

7240 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811725106 Du et al. Downloaded by guest on September 23, 2021 pA A D p< 0.001 5000 1500 (7) 100 µM [Ca2+] WT TRPM2 i 100 µM Ca2+/ N1326D 2500 1000 200 µM ADPR Mock p> 0.05 pA/pF 500 -100 -50 50 100 mV (9) (6)(10) (9) (9) 0 -2500 ock M RPM2 1326D T N -5000 WT B GFP Mock cell E +80 mV

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WT TRPM2 N1326D 2ϩ -40 mV Fig. 3. [Ca ]i activates TRPM2 via CaM-binding domain. (A) Mutated resi- µ 20 m dues in the IQ-like motif (406 to 416) within the CaM-binding site located in -80 mV 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 biotiny- C WT TRPM2 F pA EC =16.9 ± 1.4 µM 8 lation. Similar results were obtained in 3 separate experiments. (D) Represen- 1.00 50 N1326D tative recordings of WT TRPM2, IQ-mut, and mock-transfected HEK-293 cells EC =14.5 ± 2.1 µM N1326D 50 2ϩ 2ϩ SC=70.0 ± 1.6 pS with a pipette solution containing 100 ␮MCa . [Ca ]i (100 ␮M) failed to 0.75 4 activate IQ-mut. (E) Current amplitude of WT TRPM2, IQ-mut, and mock- 2ϩ WT TRPM2 transfected cells in the presence of 100 ␮M [Ca ] with or without 200 ␮M 0.50 N1326D i 300 I/Imax ADPR. Note that ADPR was ineffective on IQ-mut.

pA/pF -100 -50 50 100 mV 0.25 150

0 -1.0 1.5 -4 0.00 2+ µ Log [Ca ]i M with WT TRPM2 and N1326D (Fig. 3C). And the ratio of -1 0 1 2 3 membrane versus cytosol protein was also similar among WT 2+ µ -8 Log [Ca ]i M TRPM2, IQ-mut, and N1326D. Thus, the nonfunctional IQ-mut 2ϩ 2ϩ indicates that the CaM-binding site is essential for [Ca ]i activation Fig. 2. [Ca ]i activates TRPM2 mutants carrying disrupted ADPR-binding 2ϩ of TRPM2 (Fig. 3E). Moreover, the IQ-mut could not be activated sites. (A) Representative currents activated by 100 ␮M [Ca ]i in WT TRPM2, N1326D, and mock-transfected cells. (B) TRPM2 expression detected by im- by addition of ADRP (200 ␮M) in the pipette solution (Fig. 3E), 2ϩ PHYSIOLOGY munostaining with anti-FLAG in WT TRPM2- and N1326D-transfected cells, suggesting that the IQ-like motif is essential not only for [Ca ]i- but not in mock-transfected cells. (C) Concentration-dependent effects of mediated TRPM2 activation, but also for ADPR/Ca2ϩ-elicited 2ϩ 2ϩ [Ca ]i on N1326D and TRPM2. The EC50sofCa for N1326D and TRPM2 were TRPM2 activation. 14.5 Ϯ 2.1 ␮M and 16.9 Ϯ 1.4 ␮M, respectively (mean Ϯ SEM, n ϭ 5–9). (Inset) 2ϩ Mean current amplitude at indicated [Ca ]i.(D) The average current ampli- 2؉ [Ca ]i Can Activate Alternative Spliced Isoforms of TRPM2. Although tude of N1326D was similar to that of WT TRPM2 with the pipette solution of several alternative spliced isoforms of human TRPM2 have been 100 ␮MCa2ϩ. ADPR (200 ␮M) did not produce synergistic effect with 100 ␮M [Ca2ϩ] in N1326D. (E)Ca2ϩ-activated single-channel currents of N1326D in inside- identified, their gating mechanism and activators have remained i 2ϩ out patches. (F) Single-channel conductance of N1326D (70 Ϯ 1.6 pS, n ϭ 4–7). unknown. Since we have provided compelling evidence that [Ca ]i alone is sufficient to activate WT TRPM2 and the mutants lacking 2ϩ ADPR-binding sites, we investigated whether [Ca ]i was capable activate TRPM2 mutants lacking the ADPR-binding sites further of TRPM2 spliced isoform activation. We created the following 2ϩ indicates that [Ca ]i alone is sufficient to activate TRPM2. truncation mutants to mimic the endogenous spliced isoforms: TRPM2-⌬N, TRPM2-⌬C, and TRPM2-⌬N/⌬C (Fig. 4A). Al- 2؉ 2ϩ Mechanism of [Ca ]i-Mediated TRPM2 Activation. The above results though 100 nM Ca /1 mM ADPR failed to elicit any current (Fig. 2ϩ 2ϩ demonstrated a crucial role for [Ca ]i in activating TRPM2. To 4E), intracellular Ca alone at 10 ␮M remarkably activated 2ϩ investigate the mechanism of [Ca ]i-mediated TRPM2 activation, TRPM2-⌬N, TRPM2-⌬C, and TRPM2-⌬N/⌬C (Fig. 4 B and C). 2ϩ we first tested whether calmodulin (CaM) is involved in [Ca ]i Concentration-dependent activation of TRPM2-⌬N, TRPM2-⌬C, 2ϩ activation of TRPM2. Overexpression of CaM did not significantly and TRPM2-⌬N/⌬Cby[Ca ]i also was observed (Fig. 4D). These 2ϩ alter the current amplitude of TRPM2 (TRPM2, 200.5 Ϯ 25.1 results indicate that [Ca ]i alone is sufficient for activation of the pA/pF, n ϭ 10; TRPM2 and CaM, 244.5 Ϯ 51.0 pA/pF, n ϭ 7; P Ͼ spliced isoforms of TRPM2. Because these spliced isoforms of 0.05), presumably because of sufficient expression of endogenous TRPM2 cannot be activated by other activators, such as ADPR and 2ϩ CaM. However, when the CaM mutant CaM1-4, with mutations at H2O2 (19), our results suggest that [Ca ]i may serve as an in vivo 4 EF hands (CaM1-4) was cotransfected with either WT TRPM2 activator of the alternative spliced isoforms of TRPM2, therefore or N1326D (Fig. 3B), their current amplitude decreased signifi- conferring their physiological functions. 2ϩ cantly, suggesting a potential role for CaM in [Ca ]i-mediated 2؉ TRPM2 activation. We then created a TRPM2 mutant (Fig. 3A)via [Ca ]i Activates TRPM2 Under Physiological Conditions. To study the 2ϩ substitution of the IQ-like motif, based on a previous study sug- physiological relevance of [Ca ]i-mediated activation of TRPM2, gesting that an IQ-like motif in the N terminus of TRPM2 binds to we performed perforated-patch experiments to determine whether 2ϩ 2ϩ CaM and is involved in H2O2-induced Ca influx through TRPM2 intracellular Ca release is sufficient for TRPM2 activation. As (29). Although the IQ-mut was able to express TRPM2 protein as shown in Fig. 5A, under the perforated patch configuration, no detected by immunostaining (Fig. S3), IQ-mut could not be acti- currents were elicited by the voltage-ramp protocol before appli- 2ϩ vated by [Ca ]i at 100 ␮M (Fig. 3D). Examination of surface cation of ionomycin. However, extracellular perfusion of 5 ␮M protein of IQ-mut yielded similar expression levels in comparison ionomycin evoked substantial TRPM2 channel activation (Fig. 5A).

Du et al. PNAS ͉ April 28, 2009 ͉ vol. 106 ͉ no. 17 ͉ 7241 Downloaded by guest on September 23, 2021 A B pA Rupture 15000 NMDG-Cl 15000 Iοno 5 µM c +100 mV 7500

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0 a pA 350 s a -100 -50 50 100 mV -2500 b -100 mV -2500 -5000 2ϩ Fig. 4. [Ca ]i is an activator of TRPM2 alternative spliced isoforms. (A) c Putative structure of TRPM2 illustrating the N and C termini of TRPM2 and -5000 alternative spliced isoforms. TRPM2-⌬N lacks residues 535–555; TRPM2-⌬C 2ϩ lacks residues 1291–1329. TRPM2-⌬N/⌬C carries deletions in both the N and C Fig. 5. Intracellular Ca activates TRPM2 in perforated-patch experiments. (A) termini. (B) [Ca2ϩ] (10 ␮M) robustly activated TRPM2-⌬N, TRPM2-⌬C, and Time-dependent changes of inward and outward currents elicited by application i ␮ TRPM2-⌬N/⌬C with amplitudes similar to WT TRPM2. (C) Time-dependent of 5 M ionomycin (iono), and after cell rupture. The pipette solution contained ␮ ␮ ␮ changes of inward current amplitudes of TRPM2-⌬N, TRPM2-⌬C, and TRPM2- 180 g/mL nystatin, 200 M ADPR, and 100 M Fura-2. (B) Representative 2ϩ recordings under indicated conditions (a, b, c) as shown in A.(C) Simultaneous ⌬N/⌬C. (D) Concentration-dependent effects of [Ca ]i on TRPM2-⌬N, TRPM2- 2ϩ ⌬C, and TRPM2-⌬N/⌬C. (E) Current amplitude of TRPM2-⌬N, TRPM2-⌬C, and recordings of current development and changes of [Ca ]i (red). The left y axis 2ϩ 2ϩ represents current amplitude, and the right y axis represents F340/F380 changes. TRPM2-⌬N/⌬C activated by 10 ␮M [Ca ]i. ADPR (1 mM) with 100 nM [Ca ]i failed to activate TRPM2 spliced isoforms. (D) Mean current amplitude of TRPM2 before and after cell rupture. (E) Time- dependent changes of inward and outward TRPM2 currents elicited by 100 ␮M TBHQ in the absence (perforated-patch configuration) and presence of ADPR After cell rupture, current amplitude was dramatically increased (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) because of the dialysis of ADPR from pipette solution into cytosol as shown in E. The mean current amplitudes of TRPM2 before and after cell 2ϩ (Fig. 5 A and B). To verify that it was the change of [Ca ]i that rupture were 282.9 Ϯ 50.2 pA/pF and 969.2 Ϯ 100.2 pA/pF, respectively. activated TRPM2, we did simultaneous current recording and Ca2ϩ concentration measurement. As illustrated in Fig. 5C, an increase 2ϩ 2ϩ in [Ca ]i led to TRPM2 activation, which in turn increased activation of TRPM2 was through PLC-induced Ca release via 2ϩ intracellular Ca levels. IP3R because CCh-induced TRPM2 was eliminated by PLC and Because ionomycin may mobilize both intracellular and extra- IP3R blockers (Fig. S4 C and D) and largely inhibited by cellular Ca2ϩ, we further investigated whether Ca2ϩ release could EGTA-AM (Fig. S4A). The current was readily blocked by 20 ␮M activate TRPM2 in perforated whole-cell current recordings. Fig. 5 ACA in a reversible manner (Fig. 6 A and B). Currents elicited by E and F show that 100 ␮M tert-butylhydroquinone (TBHQ), a Ca2ϩ voltage steps also displayed the typical instantaneous activation pump blocker, induced intracellular Ca2ϩ release that was sufficient characteristics of TRPM2 (Fig. 6C). Current amplitude was larger to activate TRPM2. The mean current amplitude of TRPM2 when 100 ␮MCa2ϩ or 200 ␮M ADPR/100 nM Ca2ϩ was included elicited by TBHQ was 2,067 Ϯ 357.5 pA (n ϭ 6). Maximal current in the pipette solution for whole-cell current recording (Fig. 6D). amplitude of TRPM2 induced by ADPR after cell rupture was All of the features, including the linear I–V relation, instantaneous 8,333 Ϯ 666.7 pA (n ϭ 6). Although the current amplitude of activation, and blockade by ACA, are typical characteristics of TRPM2 induced by TBHQ was only Ϸ25% of the maximal current TRPM2. To further confirm that there was no contamination from 2ϩ 2ϩ 2ϩ elicited by Ca /ADPR, these results indicate that [Ca ]i release Ca -activated TRPM4, which has been shown to express in beta is capable of activating TRPM2. cell lines, including INS-1 and RINm5F (30), we transfected nonfunctional IQ-mut, which can inhibit heterologously expressed .Receptor Activation-Induced Ca2؉ Release Can Activate TRPM2. Be- TRPM2 but not TRPM4 currents (Fig. 6E), into HIT T15 cells cause passive Ca2ϩ release by TBHQ could activate TRPM2, we Expression of IQ-mut dramatically diminished the current induced investigated whether receptor-mediated Ca2ϩ release was capable by CCh in HIT T15 cells, indicating that the CCh-elicited current of activating TRPM2. As shown in Fig. 6, under the perforated- in HIT T15 cells is TRPM2 current. Importantly, the TRPM2 patch configuration, application of 500 ␮M carbachol (CCh) in- current elicited by CCh in HIT T15 cells was not affected by the duced typical TRPM2 activation with a linear I–V relation (Fig. 6 dominant-negative TRPM4 (DN-TRPM4) transfected into HIT A and B) in a pancreatic beta cell (HIT T15 cell). CCh-mediated T15 cells (Fig. 6F). Furthermore, because TRPM2 is Ca2ϩ-

7242 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811725106 Du et al. Downloaded by guest on September 23, 2021 spliced isoforms, but also provide new insights into their physio- CCh 500 μM A NMDG-Cl B pA μ logical and/or pathological functions. ACA 20 M 0 s 0 140 s 2000 2؉ 190 s [Ca ]i Is an Activator for TRPM2 and Its Alternative Spliced Forms. We NMDG-Cl 2ϩ HIT-T15 20 μM ACA provide several lines of evidence demonstrating that [Ca ]i is an cell line pA -2000 activator of TRPM2. Both whole-cell and single-channel currents -100 100mV 2ϩ w/o CCh can be activated by [Ca ]i in the absence of ADPR (Figs. 1 and 2). 2ϩ 500 μM CCh in HIT T15 cell Moreover, [Ca ] can activate TRPM2 mutants (N1326D and -4000 -2000 i 0 200 400 ϩ I1405E/L1406F) that are insensitive to ADPR, NAD , and H2O2 Time (s) 2ϩ (13, 19). More strikingly, [Ca ]i can activate the alternative spliced isoforms, TRPM2-⌬C, TRPM2-⌬N, and TRPM2-⌬C-⌬N, identi- +100 mV 400 200 μM ADPR/100 nM Ca 2+ CD(6) fied in human neutrophil cells (13, 15), which are insensitive to μ 2+ 100 M [Ca ]i 2ϩ 300 CCh 500 μM ADPR and H2O2 (18, 19) (Fig. 4). These results suggest that [Ca ]i (5) may serve as a major activator in vivo for TRPM2 and its spliced 200

pA/pF (9) isoforms. 2ϩ 100 The EC50 of Ca for WT TRPM2 and N1326D is about 16 ␮M in the presence of 1 mM EGTA. However, this value may have been 1000 pA 50 ms 0 -100 mV overestimated, because we found that EGTA can directly block 2ϩ TRPM2 (Fig. S7). Thus, the EC50 of Ca should be lower than 16 p>0.05 ϩ E p< 0.001 F Control ␮M in the absence of Ca2 chelators. Indeed, in the perforated- (8) (10) DN-TRPM4 ϩ 1500 (4) Control 1500 2 p< 0.001 patch experiments, TRPM2 can be activated by intracellular Ca 1250 IQ-mut ϩ 800 2 1000 p>0.05 (8) release (Figs. 5 and 6). Furthermore, because local Ca concen- 750 p> 0.05 100 trations can readily reach 100 ␮M in nanodomains and 1–5 ␮Min 250 80 (6) p< 0.001 (8) (4) pA/pF pA/pF microdomains (31, 32), it is likely that TRPM2 or its spliced (9) (9) (8) 2ϩ 125 (8) 40 isoforms can be readily activated by elevation of local [Ca ]i in the (6) 0 0 absence of ADPR, thereby conferring physiological functions. TRPM2 HIT-T15 TRPM4 TRPM2 HIT-T15 TRPM4 2؉ 2؉ 2؉ Fig. 6. Receptor activation-induced Ca2ϩ release activates endogenous TRPM2 Ca –CaM Binding to TRPM2 Is Essential for [Ca ]i and ADPR/Ca - current in HIT T15 cells under perforated-patch conditions. (A) Time-dependent Mediated Activation of TRPM2. We demonstrate that CaM-binding 2ϩ changes in inward currents measured at Ϫ100 mV before and after application of domain IQ-like motif is essential for [Ca ]i-mediated TRPM2 500 ␮M CCh in a pancreatic beta cell line (HIT T15). Note that NMDG completely activation. The IQ-like motif is located in the N terminus of TRPM2 eliminated the inward current. ACA (20 ␮M) effectively and reversibly blocked (406–416: IQDIVRRRQLL) (29). Replacement of the motif with TRPM2. (B) Representative recordings of TRPM2 elicited by ramp protocols at AADIVAAAQLA (IQ-mut) disrupted the interaction of CaM and indicated time points after ACA and NMDG-Cl. Note the linear I–V relation of TRPM2 (Fig. S8), therefore abolishing TRPM2 activation evoked TRPM2. (C) Typical TRPM2 current elicited by voltage-step protocol at a 20-mV 2ϩ 2ϩ by [Ca ]i and ADPR/Ca (Fig. 4). These results not only establish increment. (D) The mean current density of TRPM2 induced by CCh (perforated 2ϩ PHYSIOLOGY ␮ 2ϩ ␮ 2ϩ that [Ca ]i is necessary and sufficient for TRPM2 activation, but patch), 100 M [Ca ]i alone (whole cell), and 200 M ADPR/100 nM Ca (whole 2ϩ cell) in HIT T15 cells. (E) The nonfunctional IQ-mut significantly inhibited TRPM2 they also elucidate that the Ca –CaM binding to TRPM2 is 2ϩ 2ϩ current expressed in HEK-293 cells and the CCh-elicited current in HIT T15 cells. required for both [Ca ]i- and ADPR/Ca -mediated TRPM2 However, TRPM4 current expressed in HEK-293 cells was not influenced by activation. ϩ IQ-mut of TRPM2. (F) DN-TRPM4 did not alter CCh-induced currents in HIT T15 The Ca2 –CaM binding to TRPM2 may also have contributed to cells or the TRPM2 currents expressed in HEK-293 cells, although TRPM4 current the activation kinetics of TRPM2. Previous study has observed that was significantly decreased by DN-TRPM4. activation time course of TRPM2 is dependent on the concentra- tions of intracellular activators (17). We demonstrate that the time 2ϩ 2ϩ course of [Ca ]i gating of TRPM2 is also strongly associated with permeable channel, whereas TRPM4 is Ca -impermeable chan- 2ϩ 2ϩ [Ca ]i concentrations (Fig. S1). Moreover, because Ca –CaM nel, we tested current amplitude by perfusing the cells with isotonic binding to various target proteins may have different kinetics (33), 2ϩ Ca solution. The ratio of the ICa/Ityrode inward current amplitude 2ϩ the relatively slow activation by [Ca ]i may be attributable to a slow of the CCh-activated channel in HIT T15 cells was similar to that binding kinetics between Ca2ϩ–CaM and TRPM2. Nonetheless, of TRPM2 expressed in HEK-293 cells, whereas TRPM4 inward further studies are required to understand whether there is a 2ϩ current was virtually eliminated by isotonic Ca solution (Fig. S5). correlation between the kinetics of Ca2ϩ–CaM binding to TRPM2 These results further support that CCh-activated channel in HIT 2ϩ and the activation time course of TRPM2 gated by [Ca ]i. T15 cells is TRPM2. Taken together, our results indicate that Ca2ϩ 2؉ release through receptor activation can induce TRPM2 channel Physiological Relevance of [Ca ]i-Mediated Activation of TRPM2 and activation. This finding implies that TRPM2 may play important Its Alternative Spliced Isoforms. Ca2ϩ is involved in a variety of 2ϩ roles in response to [Ca ]i release under a variety of physiological signaling pathways and diverse cellular functions. Here, we dem- 2ϩ conditions. onstrate for the first time that [Ca ]i is an activator for TRPM2 and its alternative spliced isoforms. Because the full-length TRPM2 is Discussion 2ϩ 2ϩ activated by both [Ca ]i and ADPR/Ca , whereas the spliced 2ϩ 2ϩ Our results reveal that [Ca ]i plays a pivotal role in TRPM2 isoforms can only be activated by [Ca ]i, the differential activation 2ϩ channel gating (Fig. S6): not only is [Ca ]i sufficient to activate mechanisms and specific distribution pattern of these isoforms (13, TRPM2, but it is required for ADPR-mediated TRPM2 activation. 25) suggest that TRPM2 and its spliced isoforms may play distinc- 2ϩ Further, [Ca ]i is capable of activating alternative spliced isoforms tive roles. of TRPM2, which are insensitive to the full-length TRPM2 agonists, TRPM2 has been shown to be involved in oxidative stress- 2ϩ 2ϩ including ADPR. The mechanism of [Ca ]i-mediated TRPM2 induced cell death (5, 8, 10, 34), mainly because of excessive Ca 2ϩ activation is via a CaM-binding domain, an IQ-like motif located in entry via TRPM2 channels. We have demonstrated that [Ca ]i- the N terminus of TRPM2. Importantly, we found Ca2ϩ release via activated TRPM2 is about 20–40% of the maximal TRPM2 current receptor activation can activate TRPM2 in intact cells. Our results amplitude elicited by ADPR/Ca2ϩ. Thus, TRPM2 may have 2 2ϩ not only establish that [Ca ]i is an activator of TRPM2 and its modes in terms of the execution of physiological functions (Fig. S6):

Du et al. PNAS ͉ April 28, 2009 ͉ vol. 106 ͉ no. 17 ͉ 7243 Downloaded by guest on September 23, 2021 In the presence of elevated ADPR concentrations or under oxida- Electrophysiology. Whole-cell and single-channel currents were recorded from tive stress conditions, which stimulate ADPR production (35), HEK-293 cells transfected with full-length TRPM2, TRPM2 mutants, and alterna- TRPM2 can be activated to the maximal degree, which may tive spliced isoforms (SI Materials and Methods). Detailed methods for whole-cell recordings, perforated-patch experiments, and single-channel recordings are produce detrimental effects and lead to cell death, whereas under described in SI Materials and Methods. normal conditions, when intracellular ADPR is less than 5 ␮M (21) 2ϩ or 1 ␮M (23), increases in [Ca ]i levels locally or globally will elicit Ratio Ca2؉ Imaging Experiments. Changes in intracellular Ca2ϩ were measured by moderate TRPM2 activation, thereby conferring physiological ratio Ca2ϩ imaging (IonOptix). Simultaneous measurement of Ca2ϩ signal and functions. Nonetheless, our results provide a novel Ca2ϩ gating TRPM2 activation was conducted by using perforated-patch and ratio imaging on mechanism that may confer novel, yet uncharacterized, physiolog- the same cell (SI Materials and Methods). ical functions of TRPM2 and its alternative spliced isoforms. Immunostaining and Western Blot Experiments. TRPM2-transfected cells were ␣ 2ϩ immunostained with -FLAG antibody. The immunostained cells were analyzed Conclusions. In the present study, we have demonstrated that [Ca ]i by using a Zeiss LSM 510 confocal microscope. Protein expression was detected by can activate TRPM2 and its alternative spliced isoforms, and that Western blot experiments with anti-FLAG antibody. Surface and cytosol proteins 2ϩ 2ϩ the CaM-binding motif confers [Ca ]i- as well as ADPR/Ca - were extracted by using the biotinylation method (SI Materials and Methods). mediated TRPM2 activation. Importantly, Ca2ϩ release from in- tracellular Ca2ϩ stores can activate TRPM2 in intact cells. Our Data Analysis. Pooled data are presented as mean Ϯ SEM. Dose–response curves ϭ ϩ n findings reveal a novel gating mechanism of TRPM2 and its were fitted by an equation: E Emax{1/[1 (EC50/C) ]}, where E is the effect at alternative spliced isoforms that may represent a major gating concentration C,Emax 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 mechanism in vivo, and therefore confer novel, as-yet unknown inhibitory effect. Statistical comparisons were made by using 2-way ANOVA and physiological and/or pathological functions. 2-tailed t test with Bonferroni correction. P Ͻ 0.05 indicated statistical significance. Experimental Procedures Molecular Biology. FLAG-tagged TRPM2 construct was kindly provided by A. ACKNOWLEDGMENTS. We thank Drs. A. Scharenberg (University of Wash- Scharenberg (University of Washington, Seattle). Mutations of TRPM2 were ington, Seattle) and Y. Mori (Kyoto University, Japan) for the TRPM2 con- made by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) fol- structs, Dr. J. Kinet (Harvard Medical School, Boston) for the TRPM4 and DN-TRPM4 constructs, and Dr. D. Yue (The Johns Hopkins University School of lowing the manufacturer’s instruction. Truncation mutations were generated by Medicine, Baltimore) for CaM and CaM1-4 constructs. We thank Drs. Laurinda ⌬ introducing EcoRI sites at the C terminus of TRPM2 for TRPM2- C and by using a Jaffe, Alan Fein, and Dejian Ren for constructive suggestions and comments. primer-loop to delete the 20 residues at the N terminus for generating TRPM2- This work was partially supported by National Institutes of Health Grant ⌬N. The primers will be available upon request. HL078960 and Department of Public Health Grant 2009-0099 (to L.Y.).

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