Article6 April 2006free access TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion Kazuya Togashi Kazuya Togashi Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki, Japan Search for more papers by this author Yuji Hara Yuji Hara Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan Search for more papers by this author Tomoko Tominaga Tomoko Tominaga Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki, Japan Search for more papers by this author Tomohiro Higashi Tomohiro Higashi Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki, Japan Search for more papers by this author Yasunobu Konishi Yasunobu Konishi Department of Cellular and Molecular Physiology, Mie University School of Medicine, Mie, Japan Search for more papers by this author Yasuo Mori Yasuo Mori Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan Search for more papers by this author Makoto Tominaga Corresponding Author Makoto Tominaga Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki, Japan Search for more papers by this author Kazuya Togashi Kazuya Togashi Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki, Japan Search for more papers by this author Yuji Hara Yuji Hara Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan Search for more papers by this author Tomoko Tominaga Tomoko Tominaga Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki, Japan Search for more papers by this author Tomohiro Higashi Tomohiro Higashi Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki, Japan Search for more papers by this author Yasunobu Konishi Yasunobu Konishi Department of Cellular and Molecular Physiology, Mie University School of Medicine, Mie, Japan Search for more papers by this author Yasuo Mori Yasuo Mori Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan Search for more papers by this author Makoto Tominaga Corresponding Author Makoto Tominaga Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki, Japan Search for more papers by this author Author Information Kazuya Togashi1,2, Yuji Hara3, Tomoko Tominaga1,2, Tomohiro Higashi1,2, Yasunobu Konishi4, Yasuo Mori3 and Makoto Tominaga 1,2 1Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan 2Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki, Japan 3Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan 4Department of Cellular and Molecular Physiology, Mie University School of Medicine, Mie, Japan *Corresponding author. Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji, Okazaki, Aichi 444-8787, Japan. Tel.: +81 564 59 5286; Fax: +81 564 59 5285; E-mail: [email protected] The EMBO Journal (2006)25:1804-1815https://doi.org/10.1038/sj.emboj.7601083 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info There are eight thermosensitive TRP (transient receptor potential) channels in mammals, and there might be other TRP channels sensitive to temperature stimuli. Here, we demonstrate that TRPM2 can be activated by exposure to warm temperatures (>35°C) apparently via direct heat-evoked channel gating. β-NAD+- or ADP-ribose-evoked TRPM2 activity is robustly potentiated at elevated temperatures. We also show that, even though cyclic ADP-ribose (cADPR) does not activate TRPM2 at 25°C, co-application of heat and intracellular cADPR dramatically potentiates TRPM2 activity. Heat and cADPR evoke similar responses in rat insulinoma RIN-5F cells, which express TRPM2 endogenously. In pancreatic islets, TRPM2 is coexpressed with insulin, and mild heating of these cells evokes increases in both cytosolic Ca2+ and insulin release, which is KATP channel-independent and protein kinase A-mediated. Heat-evoked responses in both RIN-5F cells and pancreatic islets are significantly diminished by treatment with TRPM2-specific siRNA. These results identify TRPM2 as a potential molecular target for cADPR, and suggest that TRPM2 regulates Ca2+ entry into pancreatic β-cells at body temperature depending on the production of cADPR-related molecules, thereby regulating insulin secretion. Introduction TRP (transient receptor potential) channels were first described in Drosophila, where photoreceptors carrying trp gene mutations exhibited an abnormal transient responsiveness to continuous light (Montell and Rubin, 1989). In mammals, TRP channels comprise six related protein families (TRPC, TRPV, TRPM, TRPA, TRPML, TRPP) (Minke and Cook, 2002; Clapham, 2003; Montell, 2005). In general, TRP channels are ubiquitously expressed, indicating that most cells have a number of TRP channel proteins. While physiological functions for most TRP channels remain unknown, this wide distribution indicates that biological functions and activation mechanisms for these channels are diverse. Perhaps, TRP channels are best recognized for their contributions to sensory transduction, responding to temperature, nociceptive stimuli, touch, osmolarity, pheromones and other stimuli from both within and outside the cell. In a sense, their role is much broader than classical sensory transduction. Among the huge TRP super-family of ion channels, some have been proven to be involved in thermosensation (Benham et al, 2003; Jordt et al, 2003; Patapoutian et al, 2003; Tominaga and Caterina, 2004). Insight into the molecular nature of temperature-gated ion channels came with the cloning of the capsaicin receptor, TRPV1 (also known as VR1) and the recognition that this ion channel protein could be activated by elevated temperatures with a threshold near 43°C (Caterina et al, 1997; Tominaga et al, 1998; Caterina and Julius, 2001). Three other TRPV channels (TRPV2, TRPV3 and TRPV4) and two TRPM channels (TRPM4 and TRPM5) have been cloned and characterized as thermosensors (Caterina et al, 1999; Guler et al, 2002; Peier et al, 2002b; Smith et al, 2002; Watanabe et al, 2002; Xu et al, 2002; Talavera et al, 2005). The threshold temperatures for the activation of these channels range from relatively warm (TRPV3, TRPV4, TRPM4 and TRPM5) to extremely hot (TRPV2). In contrast to the six heat-sensitive TRPV channels, TRPM8 and TRPA1 have been found to be activated by cold stimuli (McKemy et al, 2002; Peier et al, 2002a; Story et al, 2003). Many of the mammalian thermosensitive TRP channels (thermo-TRPs) identified to date can alternatively be activated by chemical stimuli, such as capsaicin for TRPV1 (Caterina et al, 1997; McKemy et al, 2002; Peier et al, 2002a; Bandell et al, 2004; Jordt et al, 2004; Moqrich et al, 2005), and a shift of voltage dependence by temperature change has been reported to be a fundamental mechanism for thermal activation in some of them (Brauchi et al, 2004; Voets et al, 2004; Nilius et al, 2005). We sought to determine whether additional TRP channels might exhibit thermosensitivity. Among those we focused on TRPM2 (previously named TRPC7 or LTRPC2), a channel known to be activated by nicotinamide adenine dinucleotide (β-NAD+), adenosine 5′-diphosphoribose (ADPR) or hydrogen peroxide (Perraud et al, 2001; Sano et al, 2001; Hara et al, 2002), because it is phylogenetically very close to the cold-gated channel, TRPM8 (Clapham, 2003). While TRPM2 is dominantly expressed in the brain, it is also detected in many other tissues, including bone marrow, spleen, heart, liver and lung. Native TRPM2 currents have been recorded from U937 monocyte cell line (Sano et al, 2001), neutrophil cell line (Heiner et al, 2003), Jurkat T cells (Gasser et al, 2006), microglia (Kraft et al, 2004) and CRI-G1 insulinoma cells (Inamura et al, 2003). We observed that TRPM2 responses were evoked by heat over 35°C and that activation by β-NAD+ or ADPR was greatly potentiated by heat. A related molecule, cyclic ADP-ribose (cADPR) catalyzed from β-NAD+ by ADP-ribosyl cyclase (CD38) is a well-known messenger molecule for Ca2+ signaling in a variety of cells (Guse, 2000; Lee, 2002; Berridge et al, 2003), and has been believed to be important with regard to potential roles in insulin secretion from pancreatic β-sells (Takasawa and Okamoto, 2002), although there is some debate about it. However, cADPR was not found to be an agonist of TRPM2 at room temperature (Perraud et al, 2001; Hara et al, 2002). We found that cADPR activates TRPM2 at body temperature similar to β-NAD+ and ADPR. TRPM2 expression was observed in rat insulinoma RIN-5F cells and rat pancreatic β-cells where heat-evoked responses, including insulin secretion, were observed through endogenous TRPM2 proteins. Thus, TRPM2 activated by cADPR may play an important role in the regulation of insulin secretion in pancreas at body temperature. Results Heat-evoked TRPM2 activation in HEK293 cells We determined the change of cytosolic-free Ca2+ concentration ([Ca2+]i) of the cells expressing human TRPM2 upon cold stimulus with a temperature as low as 5°C using a Ca2+-indicator dye, fura-2, since TRPM2 is phylogenetically very close to the cold stimulus-activated channel, TRPM8. We used CHO-K1 cells for this experiment because some [Ca2+]i increase was observed in HEK293 cells but not in CHO-K1 cells without TRPM2 expression upon cold stimulus. We could not detect any [Ca2+]i changes upon cold stimulus (data not shown). Surprisingly, however, human TRPM2-expressing HEK293 cells (hTRPM2-HEK) but not vector-transfected cells showed significant [Ca2+]i increases upon heating with a threshold of about 40°C (Figures 1A and B), suggesting that TRPM2 is activated by warm temperatures. This [Ca2+]i increase was abolished in the absence of extracellular Ca2+ (Figure 1C), suggesting that Ca2+ influx through TRPM2 channels causes the [Ca2+]i increase. Figure 1.Heat-evoked responses in HEK293 cells expressing TRPM2. (A–C) Representative traces of [Ca2+]i change by heat in cells expressing TRPM2 (A), vector-transfected cells (B) or in the absence of extracellular Ca2+ (C). (D–F) Representative TRPM2-mediated whole-cell current responses by heat without (D) or with β-NAD+ (E) or ADPR (F). (G) Comparison of current densities of responses by the indicated stimuli in cells transfected with vector plasmid (V) or TRPM2 (±s.e.m.). ‘Preheat’ indicates current responses just before heat stimulation. *P<0.01; #P<0.01. Numbers in parenthesis indicate cells tested. Vh: −60 mV. (H) Concentration-dependent profiles of β-NAD+ for TRPM2 activation with (open circle) or without (closed circle) heat (40°C). *P<0.01; **P<0.05. Numbers in parenthesis indicate cells tested. Vh: −60 mV. Download figure Download PowerPoint Next, we measured TRPM2-mediated current responses in hTRPM2-HEK using the patch-clamp technique. We first confirmed TRPM2 activation by its reported stimuli, β-NAD+ and ADPR (Supplementary Figures 1A and B). At a holding potential of −60 mV, a gradual increase in inward current was observed several minutes after the establishment of the whole-cell configuration with a pipette solution containing β-NAD+ or ADPR at room temperature (25°C) (127.7±10.1 pA/pF for β-NAD+, n=20 and 217.1±23.0 pA/pF for ADPR, n=14), although β-NAD+-evoked responses might be induced by ADPR contaminated in the β-NAD+ as reported recently (Kolisek et al, 2005). Heat alone induced significant current responses in hTRPM2-HEK (54.5±6.0 pA/pF, n=14) but not in vector-transfected cells (6.4±0.7 pA/pF, n=6, P<0.01) (Figure 1D and Supplementary Figure 1C), indicating that TRPM2 can be activated by heat. We applied heat to cells already exposed to β-NAD+ (1 mM) or ADPR (100 μM). When temperature ramps were applied to cells showing small inward currents evoked by either β-NAD+ or ADPR, the current responses were immediately observed and robustly potentiated (Figures 1E and F). To facilitate the observation of a maximal TRPM2 response to heat, we applied heat ramps approximately 2–3 min after establishing the whole-cell configuration. Much larger current responses than those induced by heat, β-NAD+ or ADPR alone were detected under these circumstances (7.1±1.2 and 564.5±46.7 pA/pF before and after heat in β-NAD+-exposed cells, respectively, n=15, P<0.01; 36.4±9.5 and 452.2±28.7 pA/pF before and after heat in ADPR-exposed cells, respectively, n=14, P<0.01) (Figure 1G). To examine how heat changes TRPM2 responsiveness, we measured TRPM2 currents in cells by serially applying a range of concentrations of β-NAD+ with or without heat. Substantial inward currents were observed upon heat stimulation at low concentrations of β-NAD+, which induced small currents by itself at 25°C. An increase in heat-evoked TRPM2 responses was detected in cells exposed to 300 μM β-NAD+ and even larger increases to 1 or 3 mM β-NAD+, indicating that the synergistic effects of the two stimuli depend on β-NAD+ concentration (Figure 1H). Whereas maximal current density during heating was 3.7 times bigger than that at 25°C, the EC50 for TRPM2 activation by β-NAD+ was not significantly changed by heat (349±16 and 477±66 μM with and without heat, respectively), suggesting that heat does not alter the kinetics of β-NAD+ binding to TRPM2. TRPM2 activation by cyclic ADP-ribose and heat We next examined whether a related molecule, cADPR, could interact with heat at TRPM2. When cADPR (100 μM) was included in the pipette, little current response was observed at 25°C. However, heat stimulation evoked large inward currents in these cells (Figures 2A and B). cADPR-activated TRPM2 currents showed desensitization upon repeated heat stimuli similar to that observed in β-NAD+-induced TRPM2 responses (Figure 2A). These results indicate that, at mammalian body temperature, TRPM2 functions as a molecular target of cADPR. Only one report (Kolisek et al, 2005), to date, describing the ability of cADPR to activate TRPM2 in heterologous expression systems might therefore be owing to the conduct of experiments at room temperature (Perraud et al, 2001; Hara et al, 2002). The similarities observed between β-NAD+- and cADPR-induced currents suggest that cADPR, like β-NAD+, binds to the Nudix motif of TRPM2 to exert its effects (Hara et al, 2002). Consistent with this hypothesis, a TRPM2 mutant lacking the Nudix motif (ΔNudix) failed to respond to the combination of heat and cADPR (6.8±1.1 pA/pF, n=5) (Figure 2B). The binding of cADPR to the Nudix motif of TRPM2 was further supported biochemically, by the finding that cADPR considerably inhibited binding of [32P]-β-NAD+ to TRPM2 (Figure 2C) and by the cADPR concentration dependence of TRPM2 currents evoked by cADPR with heat (Figure 2D). Figure 2.Heat and cADPR activate TRPM2 through binding to its Nudix motif in HEK293 cells. (A) Heat-evoked activation of TRPM2 by cADPR with repetitive heat stimuli. (B) Comparison of current densities of responses evoked by heat, cADPR (100 μM) or heat with cADPR in HEK293 cells transfected with vector plasmid, TRPM2 or TRPM2 mutant lacking Nudix motif (ΔNudix). ‘Preheat’ indicates current responses just before heat stimulation. *P<0.01, two-tailed unpaired t-test. #P<0.01, two-tailed paired t-test. Numbers in parenthesis indicate cells tested. Vh: −60 mV. (C) β-NAD+ binding to the TRPM2 fusion proteins in the presence (+) or absence (−) of cADPR. *P<0.01; **P<0.05. (D) A dose-dependent profile of cADPR for the activation of TRPM2 at 40°C. Download figure Download PowerPoint Body temperature gates TRPM2 directly We then carried out more detailed analyses of heat-evoked TRPM2 current responses. The temperature-response profile with or without β-NAD+ or cADPR revealed that the temperature threshold was about 35°C with no significant difference in the presence or absence of β-NAD+ or cADPR (33.5±0.3°C, n=8; 35.0±0.8°C, n=10; 35.1±0.9°C, n=9 for heat alone, β-NAD+ plus heat or cADPR plus heat, respectively) (Figures 3A, Supplementary Figure 2A and data not shown). This finding differs from that reported for other thermo-TRPs exposed to simultaneous thermal and chemical stimulation; decreases and increases of the activation temperature threshold in the presence of other effective stimuli, protons and menthol were reported for TRPV1 and TRPM8, respectively (Tominaga et al, 1998; McKemy et al, 2002). To analyze the temperature dependence of TRPM2 activation more precisely, we made Arrhenius plots for the currents activated by heat with ADPR or cADPR at both negative and positive potentials, and found that the plots showed the similar temperature threshold for TRPM2 activation to that obtained in the temperature-response profiles (35.0±0.7°C, n=4; 33.6±1.5°C, n=5; 33.9±0.6°C, n=4 for cADPR plus heat, β-NAD+ plus heat or heat alone). Q10 values were 44.4±5.3 (n=11), 44.1±7.9 (n=12), 32.3±2.9 (n=4), 38.0±3.7 (n=5) and 15.6±2.0 (n=4) for ADPR-activated TRPM2 currents at +60 mV, ADPR-activated currents at −60 mV, cADPR-activated currents at −60 mV, β-NAD+-activated currents at −60 mV and currents activated by heat alone at −60 mV, respectively (Supplementary Figures 2B–D, and data not shown). Linear current–voltage (I–V) relationships were observed for heat-evoked currents both in the presence and absence of each ligand (Figure 3B, Supplementary Figure 2E and data not shown). As the observed reversal potentials were close to 0 mV (Erev=+1.1±2.3 mV, n=3 for heat; Erev=−1.9±1.2 mV, n=3 for heat plus cADPR), the heat-evoked responses most likely involve a nonselective cation channel. Heat-evoked TRPM2 currents exhibited a relatively higher permeability for divalent cations than monovalent cations (PCa/PCs=2.8±0.1, n=3; PMg/PCs=2.2±0.1, n=5; PNa/PCs=0.5±0.1, n=3) (Supplementary Figure 2F). This finding somehow contradicts previous reports in which Ca2+ was shown to be less permeant than Na+ in β-NAD+- or ADPR-induced currents (Perraud et al, 2001; Sano et al, 2001). The relative high permeability of heat-evoked TRPM2 responses to Ca2+ (PCa/PNa=5.83) might explain the robust [Ca2+]i increase upon heat stimulation (Figure 1A). These electrophysiological properties indicate that heat does activate TRPM2 and suggest that heat and ligands share some overlapping mechanism for TRPM2 activation. Temperature-induced change in cADPR-related enzyme activities might also be involved in the TRPM2 activation by heat with cADPR. HEK293 cells expressing mouse TRPM2 (mTRPM2-HEK) exhibited essentially similar current responses to those observed in hTRPM2-HEK (data not shown). Figure 3.Electrophysiological properties of heat-gated current responses in HEK293 cells expressing TRPM2. (A) A representative temperature–response profile of heat-evoked TRPM2 currents in the presence of cADPR (100 μM). Vh: −60 mV. (B) Current–voltage relationship for a heat-evoked current in the presence of cADPR (100 μM) in hTRPM2-HEK. (C) A representative trace of heat-evoked single-channel responses at −60 mV in inside-out configuration. A lower panel shows bath temperature. Broken lines indicate 0, 1, 2 and 3 channel open levels. (D). NP0 values (calculated from data shown in C) plotted against bath temperatures. (E) Traces of heat-evoked TRPM2 single-channel currents in inside-out patches at the indicated holding potentials. Broken lines indicate the closed-channel level. (F) Current–voltage curve of mean single-channel amplitudes (±s.e.m.). (G) Representative current responses to the voltage step-pulses (−160 to +120 mV with 20 mV increment for 100 ms, inset) at 25.4 and 40.0°C (left), and current–voltage relationship (right). Download figure Download PowerPoint How do heat-evoked TRPM2 currents behave at the single-channel level? To address this question, we performed single-channel recordings in inside-out membrane patches excised from hTRPM2-HEK. When we applied heat ramps to these patches, well-resolved single-channel currents were observed with a temperature threshold of about 34°C and maximal activities at about 36°C (Figure 3C). This finding demonstrates the existence of heat-gated ion channels within this patch whose activation does not depend upon soluble cytoplasmic components. Heat-evoked channel activity became relatively diminished when we further increased temperature over 37°C, suggesting that the optimal temperature for TRPM2 activation is near core body temperature. This phenomenon was more clearly recognized when NP0 values were plotted against temperatures (Figure 3D). The I–V relation at the single-channel level was almost identical to that established in the whole-cell configuration (Figures 3E and F). A slope conductance for Na+ as the sole charge carrier was 60.6 pS. These single-channel properties are like those described for β-NAD+- or ADPR-gated TRPM2 currents (Perraud et al, 2001; Sano et al, 2001; Hara et al, 2002). It has been reported that a shift of voltage dependence by temperature is the fundamental mechanism of temperature-evoked activation of TRPV1, TRPM8, TRPM4 or TRPM5 (Brauchi et al, 2004; Voets et al, 2004; Nilius et al, 2005). Therefore, we applied voltage step-pulses to heat-activated TRPM2 currents to examine whether a similar mechanism is involved in TRPM2 activation by warm stimulus. As shown in the Figure 3G, temperature elevation simply increased the slope without changing the linear I–V relationship, suggesting that temperature activation of TRPM2 involves a different mechanism from that reported for TRPV1, TRPM8, TRPM4 or TRPM5. Expression of TRPM2 in pancreatic β-cells To explore the potential physiological significance of heat-evoked activation of TRPM2, we first examined the expression of endogenous TRPM2. An anti-mouse TRPM2 antibody specifically recognized a protein with similar molecular weight (about 171 kDa) in lysates from hTRPM2-HEK or mTRPM2-HEK (Figure 4A). Specificity of the antibody was confirmed in the absorption experiment using the immunogenic peptide (Supplementary Figure 3A). We could not detect TRPM2 expression using this antibody in mouse DRG neurons, where TRPV1 is expressed (Supplementary Figures 3B and C). The fact that TRPM2 can be activated by cADPR with heat prompted us to examine TRPM2 expression in pancreatic β-cells where cADPR is known to be involved in insulin secretion (Takasawa et al, 1993). The antibody recognized TRPM2 in lysates from rat insulinoma RIN-5F cells (Figure 4A). We detected clear TRPM2-like immunoreactivity not only in hTRPM2-HEK or mTRPM2-HEK but also in RIN-5F cells (Figure 4B, and data not shown). TRPM2 was highly coexpressed with insulin, a marker for β-cells, but not with glucagon, a marker for α-cells in mouse pancreatic islets (Figure 4C), suggesting the important and specific function of TRPM2 in pancreatic β-cells. Figure 4.TRPM2 expression in insulin-secreting cells. (A) Immunoblot analysis reveals a specific band (around 171 kDa) in lysates from hTRPM2-HEK, mTRPM2-HEK and RIN-5F cells. (B) TRPM2-like immunoreactivity in RIN-5F cells but not in control cells (right, without anti-TRPM2 antibody). Scale bar, 50 μm. (C) Triple immunofluorescent analysis reveals coexpression of TRPM2 with insulin but not with glucagon in mouse pancreas. Lower panels indicate negative controls without primary antibodies. Scale bar, 50 μm. Download figure Download PowerPoint Heat-evoked responses in RIN-5F cells We next investigated heat-evoked responses in RIN-5F cells, using both Ca2+-imaging and patch-clamp methods. In these cells, as in hTRPM2-HEK, [Ca2+]i was increased upon heat stimulation with a temperature threshold of about 40°C in the presence of extracellular Ca2+ (Supplementary Figures 4A and B). Significant and desensitizing inward currents were observed in the presence or absence of cADPR when temperature ramps were applied at −60 mV (19.3±1.9 pA/pF for heat alone, n=4; 8.1±1.9 and 144.2±26.7 pA/pF before and after heat in cADPR-exposed cells, P<0.05, n=6) (Figure 5A, and data hot shown). The temperature threshold for heat-evoked responses in RIN-5F cells with cADPR was 34.0±1.0°C (n=5) (Figure 5B) and the whole-cell currents showed a linear I–V relationship with a reversal potential near 0 mV (+3.4 mV±1.2, n=3) (Figure 5C). It should be noted that the threshold temperature for heat-evoked activation was not changed, regardless of the heat-stimulus sequence, a quite different phenomenon from those reported for TRPV1 and TRPV2, whose temperature thresholds for activation decrease upon repetitive heat stimulation (Caterina et al, 1999). Heat-evoked currents in the RIN-5F cells decreased upon further temperature increases over 40–42°C, suggesting the existence of an optimal temperature for activation. These electrophysiological properties are almost identical to those obtained from hTRPM2-HEK, suggesting that endogenous TRPM2 functions as a thermosensor and a target of cADPR. To further prove the involvement of endogenous TRPM2 in the heat-evoked responses, we treated RIN-5F cells with a TRPM2-specific siRNA (siTRPM2). This intervention reduced expression of TRPM2 both at protein and mRNA levels, whereas treatment with control siRNA did not (Figure 5D). Heat failed to increase [Ca2+]i upon treatment with siTRPM2, but evoked normal responses with control siRNA (Figures 5E and F), indicating that endogenous TRPM2 is responsible for heat-evoked [Ca2+]i increase in RIN-5F cells. Figure 5.Heat-evoked responses in RIN-5F cells through TRPM2 activation. (A) A representative heat-evoked whole-cell current trace in RIN-5F cells in the presence of cADPR (100 μM). A lower panel shows bath temperature. Vh: −60 mV. (B) Temperature–response profiles of heat-evoked currents in the presence of cADPR (100 μM) shown in (A). Blue, red and green lines indicate the profiles obtained in the first, the second and the third heat stimuli, respectively. Vh: −60 mV. (C) Current–voltage relationship for heat-evoked currents in the presence of cADPR (100 μM). (D) Reduction of TRPM2 protein (upper) and mRNA (lower) expression by treatment with TRPM2-specific siRNA (siTRPM2) but not with control siRNA. (E, F) Increase of [Ca2+]i by heat in RIN-5F cells treated with control siRNA (E) but not with siTRPM2 (F). Lower panels show bath temperature. Download figure Download PowerPoint Heat-evoked responses in pancreatic β-cells To assay for heat-evoked responses in the rat primary pancreatic β-cells, we utilized Ca2+ imaging in dissociated rat pancreatic cells. Isolated cells (49.6%) were insulin positive and 24.5% were glucagon positive. TRPM2 was highly colocalized with insulin but not with glucagon (Figure 6A), a phenomenon similar to that observed in mouse pancreas sections. Almost all TRPM2-positive cells costained with anti-insulin antibody (91.7%). Upon exposure to heat, 55.3% of the isolated pancreatic cells showed a [Ca2+]i increase in the presence of extracellular Ca2+ (Figures 6B–D) like those seen in hTRPM2-HEK and RIN-5F cells. The percentage of heat-sensitive cells in the isolated pancreatic cells (55.3%) was almost equal to that of TRPM2-positive cells (53.1%), suggesting that TRPM2 is involved in the heat-evoked responses. Figure 6.Expression of TRPM2 and heat-evoked responses in isolated pancreatic cells. (A) Expression of TRPM2 (green), insulin (red) and glucagon (blue) in the isolated rat pancreatic cells. An inset indicates the high magnification image of the square box area. (B) Change of cytosolic Ca2+ concentration indicated by the fura-2 ratio with pseudo-color expression in response to heat stimulus. (C, D) Representative traces of [Ca2+]i change by heat in the presence (C) or absence (D) of extracellular Ca2+. Download figure Download PowerPoint Insulin release involving endogenous TRPM2 in pancreatic islets Finally, we examined the temperature effects on insulin release from pancreatic islets. We examined the effects of heat stimulation (40°C, 5 min) on insu
