TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion

Abstract

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 degrees C) apparently via direct heat-evoked channel gating. beta-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 degrees 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 Ca(2+) and insulin release, which is K(ATP) 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 Ca(2+) entry into pancreatic beta-cells at body temperature depending on the production of cADPR-related molecules, thereby regulating insulin secretion.

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. V_h: −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. V_h: −60 mV.

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. V_h: −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.

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). V_h: −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).

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.

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. V_h: −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. V_h: −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.

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⁺.

Figure 7

Body temperature-evoked insulin release from pancreatic islets. (A) Heat stimulus (40°C for 5 min) causes insulin release from the islets. Data from the islets incubated at 37 or 29°C (1 h) with (+) or without (−) heat stimulation (±s.e.m.) in the presence of 3.3 mM (G3.3) or 16.7 mM (G16.7) glucose after culture at 33°C (16 h). ^*P<0.01; ^**P<0.05. Numbers in parenthesis indicate samples tested. (B) Reduction of insulin release by Eco (10 μM) or FFA (200 μM) at 37°C. ^*P<0.01. (C) Insulin release by heat stimulus (+) was inhibited by treatment with siTRPM2 but not with control siRNA (±s.e.m.). ^*P<0.01; ^**P<0.05. An inset indicates amounts of RT–PCR products from the pancreatic islets treated with control siRNA or siTRPM2. (D) cADPR-induced TRPM2-mediated whole-cell currents were potentiated by FSK (2 μM), and the potentiation was inhibited by H-89 (1 μM). ^*P<0.01. (E) Increase in insulin release by FSK (2 μM) in the presence of nimodipine (NDP, 2 μM, +) was significantly reduced in the islets treated with siTRPM2 but not with control siRNA. ^*P<0.01; ^**P<0.05. Numbers in parenthesis indicate samples tested. (F) Increase in insulin release by exendin-4 (10 nM) was significantly reduced in the islets treated with siTRPM2 but not with control siRNA. ^*P<0.01; ^**P<0.05.