Critical Intracellular Ca2+ Dependence of Transient Receptor Potential Melastatin 2 (TRPM2) Cation Channel Activation*

Damian McHughDagger , Richard FlemmingDagger , Shang-Zhong XuDagger , Anne-Laure Perraud§, and David J. BeechDagger

From the Dagger  School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom and the § National Jewish Medical and Research Center, Denver, Colorado 80206

Received for publication, October 22, 2002, and in revised form, January 12, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TRPM2 is a member of the melastatin-related TRP (transient receptor potential) subfamily. It is expressed in brain and lymphocytes and forms a cation channel that is activated by intracellular ADP-ribose and associated with cell death. In this study we investigated the calcium dependence of human TRPM2 expressed under a tetracycline-dependent promoter in HEK-293 cells. TRPM2 expression was associated with enhanced hydrogen peroxide-evoked intracellular calcium signals. In whole-cell patch clamp recordings, switching from barium- to calcium-containing extracellular solution markedly activated TRPM2 as long as ADP-ribose was in the patch pipette and exogenous intracellular calcium buffering was minimal. We suggest this effect reveals a critical dependence of TRPM2 channel activity on intracellular calcium. In the absence of extracellular calcium we observed concentration-dependent activation of TRPM2 channels by calcium delivered from the patch pipette (EC50 340 nM, slope 4.9); the maximum effect was at least as large as that evoked by extracellular calcium. Intracellular dialysis of cells with high concentrations of EGTA or 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) strongly reduced the amplitude of the extracellular calcium response, and the residual response was abolished by a mixture of high and low affinity calcium buffers. TRPM2 channel currents in inside-out patches showed a strong requirement for Ca2+ at the intracellular face of the membrane. We suggest that calcium entering via TRPM2 proteins acts at an intracellular calcium sensor closely associated with the channel, providing essential positive feedback for channel activation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The non-voltage-gated TRP Ca2+ channel encoded by the transient receptor potential (trp)1 gene has a major role in the phospholipase C-dependent light response of the Drosophila photoreceptor (1). Since this discovery, many trp-related Ca2+ channels have been discovered in mammals, beginning with TRPC1 (e.g. Ref. 2), which is a subunit of some store-operated Ca2+ channels (e.g. Ref. 3). There are now known to be at least 20 trp-related mammalian genes, all apparently encoding cationic channels, many of which are Ca2+ permeable. They would appear to be the molecular basis of the many non-voltage-gated cationic channels with diverse functions and expression profiles in mammalian systems. On the basis of amino acid sequence the mammalian TRPs are divided into three subgroups, TRPC (C, canonical), TRPV (V, vanilloid receptor), and TRPM (M, melastatin receptor) (4). Increasingly it is becoming apparent that the regulation of these proteins is complex, with gating factors as diverse as temperature, menthol, diacylglycerol, arachidonic acid, and osmotic stress (5, 6).

TRPM2 (also called TRPC7 or LTRPC2) is a recently characterized member of the TRPM family (7-11). It forms a cationic channel activated by intracellular ADP-ribose, beta -NAD+, or arachidonic acid. The sensitivity of the channel to beta -NAD+ is thought to couple TRPM2 to the redox state of the cell (10). These reports also indicate effects of Ca2+ on TRPM2. The aim of this study was to explore the role of Ca2+ in TRPM2 function. We reveal a critical dependence of TRPM2 function on intracellular, but not extracellular, Ca2+. The Ca2+ levels required are high, and the effect does not occur in the absence of ADP-ribose. We suggest a mechanism by which Ca2+ acts as a positive feedback signal for TRPM2 activation.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HEK293 cells, stably expressing tetracycline-regulated cytomegalovirus-driven transcription of FLAG-epitope-tagged TRPM2 (HEK-TRPM2) were used (8). HEK-TRPM2 cells were grown in Dulbecco's modified Eagle's medium-F12 media (Invitrogen) supplemented with 10% fetal bovine serum and penicillin and streptomycin (100 units/ml-1) at 37 °C in a 5% CO2 incubator. Cells were incubated with tetracycline (tet; 1 µg/ml-1) for 24 h before being subcultured onto poly-L-lysine-coated glass coverslips in tetracycline-free supplemented Dulbecco's modified Eagle's medium-F12 media. Cells were left to recover from subculturing for at least 15 h prior to experiments.

For Ca2+ imaging experiments, cells were preincubated with 1 µM fura PE3-AM at 37 °C for 1 h in bath solution, followed by a 0.5-1 h period in bath solution at room temperature. Fluorescence was observed with an inverted microscope (Zeiss, Martinsried, Germany), and a xenon arc lamp provided excitation light, the wavelength of which was selected by a monochromator (Till Photonics, Gräfelfing, Germany). Experiments were performed at room temperature, and emission was collected via a 510-nm filter and sampled by a CCD camera (Orca ER; Hamamatsu, Japan). Images were sampled every 10 s in pairs for the two excitation wavelengths (340 and 380 nm) and analyzed off-line using regions of interest to select individual cells. [Ca2+]i is expressed as the ratio of the emission intensities for 340 and 380 nm. Imaging was controlled by Openlab 2 software (Image Processing & Vision Company Ltd., UK). The bath solution contained (mM): NaCl 107, KCl 6, MgSO4 1.2, glucose 11.5, Hepes 20, mannitol 30, CaCl2 2, pH 7.2, with NaOH.

For Western blotting experiments, HEK-TRPM2 cells were grown on 100-mm tissue culture dishes (Greiner, Frickenhausen, Germany). The cell monolayer was washed once with room-temperature phosphate-buffered saline (PBS) and cells removed using 1 ml of chilled PBS supplemented with a protease inhibitor mixture (Sigma). The cells were pelleted at 4 °C and then lysed using 1 ml of Laemmli sample buffer, supplemented with protease inhibitor mixture. The resulting lysate was sonicated for 3 × 15 s, centrifuged at 21,000 × g for 2 min and the supernatant run on an 8% SDS-PAGE gel before being transferred to nitrocellulose. Immunoblots were probed with monoclonal anti-FLAG M2 antibody (Sigma).

Voltage clamp was performed at room temperature with the whole-cell configuration of the patch-clamp technique, using an Axopatch 200A patch clamp amplifier and pClamp software v 6.0 (Axon). 100-ms ramps from -100 to +100 mV were applied at a frequency of 0.1 Hz from a holding potential of 0 mV. Records were low-pass filtered at 1 kHz (4-pole Bessel filter) and sampled at 2 kHz. Patch pipettes (2-4 MOmega ) were prepared from borosilicate glass (Clark Electromedical, UK). The Cs-glutamate patch pipette solutions (pH 7.2 with CsOH, 295-300 mOsm) were prepared with various Ca2+ concentrations (Table I). Free Ca2+ concentrations were calculated using EQCAL (Biosoft, Cambridge, UK). The bath solution contained (mM): NaCl 145, KCl 2.8, MgCl2 2, Hepes 10, glucose 10, and CaCl2 1 or BaCl2 1, pH 7.2, with NaOH. The N-methyl-D-glucamine (NMDG) bath solution contained (mM): NMDG 150, HEPES 10, MgCl2 2, and CaCl2 1, pH 7.2, with HCl. On a given recording day, recordings were performed alternately between at least two different pipette solutions. For inside-out patch experiments the bath solution contained 0.3 mM ADP-ribose and 1 mM EGTA, with Ca2+ either added or omitted (Table I). The patch pipette solution contained 1 mM Ba2+ in Ca2+-free standard bath solution. A liquid-liquid junction potential (12.6 mV; pipette negative) was measured between bath and 50 µM EGTA pipette solutions, and relevant voltages are corrected for this value.


                              
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Table I
Compositions of intracellular solutions
The top row is the calculated free Ca2+. Below are the concentrations added (mM). The pH of all solutions was 7.2.

The recording chamber had a volume of 150 µl, there was continuous solution flow, and complete solution exchange occurred in < 30 s. Data are given as mean ± S.E. with n indicating the number of individual cells. Data sets were compared using unpaired Student's t test with significance indicated if p < 0.05. Concentration-effect data were fitted to the Hill equation, y = (xs.A)/(xs + EC50s), where s is the slope and A is the maximum value of y. Na4BAPTA and fura PE3-AM were from Calbiochem. K4-difluoro BAPTA was from Molecular Probes. KCl, D-glucose, and CaCl2 were from BDH (British Drug House, Poole, UK). All other salts and reagents were from Fluka and Sigma (UK).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A band of the predicted mass of FLAG-epitope-tagged TRPM2 protein (171 kDa) was identified using Western blotting in lysates from tet-induced HEK-TRPM2 cells but not in control cells (Fig. 1A; n = 6). To test for functional protein we applied H2O2 in Ca2+ imaging experiments (10, 11). The H2O2-induced Ca2+ rise was significantly larger in tet-induced cells, consistent with the functional expression of TRPM2 (Fig. 1, B and C).


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Fig. 1.   Heterologous expression of functional human TRPM2 protein in HEK cells. A, Western blot of whole-cell lysates obtained from tetracycline-induced HEK-TRPM2 cells. The blot was probed with an anti-FLAG epitope antibody. The band in the + tet lane corresponds closely to the expected mass of 171 kDa for FLAG-epitope-tagged TRPM2 channels. No labeling occurred in non-induced cells (n = 6). B and C, H2O2 caused an increase in [Ca2+]i in TRPM2 cells. [Ca2+]i is given as the ratio of 340 and 380 nm fura-PE3 signals (R340/380). H2O2 was applied for the period indicated by the horizontal bar. C, mean ± S.E. changes in fura-PE3 ratio 3 min after the application of H2O2 (1 mM) in control and TRPM2 cells. The number of cells is shown above the columns; the number of individual experiments is given in parentheses. * p < 0.001.

The Ca2+ dependence of TRPM2 was explored by whole-cell patch-clamp with 0.5 mM ADP-ribose in the patch pipette. When recording from HEK-TRPM2 cells with 50 µM EGTA in the pipette (weak Ca2+ buffering), we noticed that replacing Ba2+ in the bath solution with Ca2+ induced striking increases in currents (Fig. 2, A and C). The TRPM2 currents were linear over a wide voltage range (Fig. 2B) and reversed close to 0 mV (0 ± 2 mV, n = 26). This reversal potential is distant from the chloride equilibrium potential (-64 mV), consistent with TRPM2 being a non-selective cation channel. Non-induced HEK-TRPM2 cells and tet-induced HEK-TRPM2 cells in the absence of ADP-ribose showed little current and minimal responsiveness to Ca2+ application (Fig. 2C).


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Fig. 2.   TRPM2 is activated by Ca2+ if ADP-ribose is present. A, plotted is the current measured at -112.6 mV, commencing shortly after breakthrough to the whole cell in a HEK-TRPM2 cell. The holding potential was 0 mV with a ramp change in voltage from -112.6 to +87.4 mV over 100 ms every 10 s. Changing the bath solution from 1 mM Ba2+ to 1 mM Ca2+ induced an inward current of ~-7 nA. The pipette solution contained 0.5 mM ADP-ribose and 50 µM EGTA (see Table I). B, the traces (a + b) are each the average of three original traces taken from the points a and b indicated on the time-series in panel A. C, plotted are the mean ± S.E. of the absolute inward currents at -112.6 mV for three groups of cells with either Ba2+ or Ca2+ present in the extracellular recording solution. The pipette solution contained 50 µM EGTA with or without 0.5 mM ADP-ribose as indicated.

To explore whether the effect of Ca2+ was at an extracellular or intracellular site we used a high concentration of the Ca2+ buffer BAPTA in the patch pipette while maintaining the free Ca2+ concentration at the physiological level of 100 nM. Under this condition the effect of extracellular Ca2+ was about 7 times smaller than in controls (Fig. 2C, compare Fig. 3, A and D). Similarly, with Ca2+ buffered to 100 nM by 40 mM EGTA there was a much attenuated response (ITRPM2 at -100 mV with Ba2+o, -0.17 ± 0.09 nA; [Ca2+]o, -0.63 ± 0.16 nA; n = 6). The use of 20 mM BAPTA alone further reduced the effect of extracellular Ca2+, leaving only a small residual response (Fig. 3, B and D). Therefore, the majority of the effect of extracellular Ca2+ appeared to be because of an effect of Ca2+ at an intracellular site.


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Fig. 3.   Intracellular Ca2+ buffering attenuates the effect of extracellular Ca2+. A, ADP-ribose- and 40 mM BAPTA-based 100 nM [Ca2+]i pipette solution reveals stimulation of TRPM2 caused by extracellular Ca2+. B, ADP-ribose- and 20 mM BAPTA-based zero [Ca2+]i pipette solution reveals stimulation of TRPM2 caused by extracellular Ca2+. C, ADP-ribose-, BAPTA-, and difluoro BAPTA-based 100 nM free [Ca2+]i pipette solution abolished stimulation of TRPM2 caused by extracellular Ca2+. D, mean ± S.E. of the peak inward currents measured at the command voltage of -100 mV with either Ba2+ or Ca2+ present in the extracellular solution for cells in panels A, B, and C. Pipette solution compositions are listed in Table I. The traces (a + b) are each the average of three original traces taken from the points a and b indicated on the time-series.

The residual response to extracellular Ca2+ that occurred despite a high concentration of BAPTA in the pipette may indicate there is also an effect of Ca2+ at a separate external regulatory site. Alternatively it may occur because of inadequacy in the exogenous Ca2+ buffering. As Ca2+ enters through TRPM2 channels there will be large local Ca2+ signals in the immediate vicinity of each TRPM2 protein. These signals may act at a very local Ca2+ sensing domain to stimulate TRPM2, perhaps even at an intracellular vestibule of the channel. In this case high affinity Ca2+ buffers could have a significant probability of being saturated by Ca2+ as they approach the Ca2+ sensing domain, making them ineffective Ca2+ buffers at the active site. We therefore hypothesized that the Ca2+ might be better controlled by a mixture of high and low affinity buffers. To this end, in addition to BAPTA, we included the lower affinity Ca2+ buffer difluoro BAPTA (12) at 10 mM. Again, with [Ca2+]i fixed at 100 nM, extracellular Ca2+ now had no effect (Fig. 3, C and D). Difluoro BAPTA does not block the channels non-specifically because ITRPM2 at -25 mV was -0.99 ± 0.18 nA (n = 6) with 40 mM difluoro BAPTA and 10.99 mM Ca2+ (0.3 µM free Ca2+, see below) in the patch pipette, similar to the amplitude with Ca2+ buffered to 0.3 µM with BAPTA alone (-1.26 ± 0.4 nA, n = 7). These data indicate that an external Ca2+ activation site is unlikely and that the intracellular Ca2+ sensing site might be in a restricted domain close to each TRPM2 channel protein.

Further evidence for an exclusive action of Ca2+ at an intracellular site comes from experiments using a range of free Ca2+ concentrations (0 to 1 µM), with ADP-ribose held constant at 0.5 mM, in the patch pipette solution. Ba2+ substituted for Ca2+ in the extracellular solution because it does not activate the channels. Non-induced HEK-TRPM2 cells were insensitive to 1 µM free Ca2+ in the pipette with ADP-ribose present (Fig. 4A); at -100 mV ITRPM2 was -0.19 ± 0.05 nA (Ba2+) and -0.23 ± 0.79 nA (Ca2+) (n = 3). In contrast, large rapidly developing ITRPM2 currents were recorded from induced HEK-TRPM2 cells using the same pipette solution (Fig. 4A). A small but statistically significant ITRPM2 occurred at 100 nM [Ca2+]i (p < 0.05; Fig. 4B). However, major activation only occurred at high Ca2+ concentrations. A Hill equation fitted to the mean data gave an EC50 of 340 nM [Ca2+]i and revealed steep concentration dependence (Hill coefficient = 4.9) (Fig. 4B).


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Fig. 4.   Intracellular Ca2+ concentrations required for TRPM2 activation. A, plotted are the currents measured at the command voltage of -100 mV from induced (filled circles) or non-induced (open circles) HEK-TRPM2 cells with 0.5 mM ADP-ribose and 1.0 µM Ca2+ in the pipette solution. B, a [Ca2+]i concentration response curve was generated by clamping the free [Ca2+]i at various concentrations using BAPTA in the pipette solutions. ADP-ribose (0.5 mM) was always included. The voltage paradigm adopted was as for Fig. 2A. Plotted are the mean ± S.E. inward currents measured from tetracycline-induced HEK-TRPM2 cells at the command voltage of -100 mV for the different free intracellular Ca2+ concentrations. C and D, inside-out patch recordings from induced HEK-TRPM2 cells with 0.3 mM ADP-ribose continuously present. As indicated, 1 µM Ca2+ (1 mM EGTA solution; Table I) was either present or absent; all other constituents of the solution remained unchanged. The current traces are averages of three original traces taken from the points a and b indicated on the time-series plots.

Intracellular Ca2+ dependence was retained in inside-out membrane patches displaying macroscopic TRPM2 currents. Despite the continuous presence of ADP-ribose, channel activity was lost when Ca2+ was removed from the bath solution at the intracellular face of the membrane (mean ± S.E. inhibition of current at -100 mV was 76.4 ± 13.7%, n = 3; Fig. 4C). TRPM2 channels could also be activated upon readdition of Ca2+ (Fig. 4D). The currents reversed at +1.0 ± 1.6 mV (junction potential corrected), as expected for TRPM2 channels. These data indicate that Ca2+ acts at an intracellular site on the channel or a closely associated intracellular protein.

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ADP-ribose activation of human TRPM2 channels was found to have a critical requirement for intracellular Ca2+. The concentration dependence of the [Ca2+]i effect was steep, with only low-level activation at 100 nM [Ca2+]i but maximal activation at 600 nM. Although application of extracellular Ca2+ caused marked activation of the channels, there was little evidence that this was because of an action of Ca2+ at an external regulatory site. Instead, it occurred because of Ca2+ influx that was difficult to control by intracellular Ca2+ buffering. We suggest from these data that Ca2+ entering via TRPM2 proteins acts at a Ca2+ sensing domain in or near an intracellular vestibule of the TRPM2 channels and that this Ca2+ binding is critical for channel activation.

Ca2+ current occurs through TRPM2 channels in the presence of high external Ca2+ concentrations, and a physiological Ca2+ permeability is demonstrated in Ca2+ imaging experiments (8, 10, 11). Furthermore, Na+ entry through TRPM2 channels would reduce the effectiveness of Na+-Ca2+ exchange, further elevating the intracellular Ca2+ concentration. In practice it would seem that the Ca2+ signal resulting from expressed TRPM2 activity is large enough to saturate substantial amounts of exogenous high affinity Ca2+ buffer.

Other members of the TRP family are also Ca2+-sensitive. TRPV6 is inhibited by intracellular Ca2+ (13). TRPM4b is a Ca2+-activated non-selective cation channel that does not depend on ADP-ribose (14). The Ca2+-sensitive properties of TRPM4b bear a strong similarity to those of TRPM2, with an EC50 value for Ca2+ activation of 320 nM (340 nM for TRPM2) and a Hill coefficient of 6 (4.9 for TRPM2). Similar mechanisms may underlie the Ca2+ sensitivity of both channels.

The mechanism for the sensitivity of TRPM2 to Ca2+ is open to speculation. A direct action of Ca2+ on the channel itself, through interaction with an EF-hand, or via an intermediary Ca2+-sensing protein such as calmodulin (15) are plausible. Ca2+ sensing and the apparent ADP-ribose pyrophosphatase property of TRPM2 may, however, not be related. Studies with other Nudix motif-containing proteins revealed that Mn2+, Zn2+, and Ca2+ are only 15-40% as effective as Mg2+ in supporting enzymatic activity (16, 17).

A system of positive feedback appears to be employed by TRPM2 such that as ADP-ribose levels rise, or the cell's redox state changes, normal physiological levels of Ca2+ can support a limited degree of channel activation. Our data indicate that once the channels are activated, the associated Ca2+ entry leads to a local intracellular accumulation of Ca2+, further stimulating the channels and giving rise to large Na+ and Ca2+ currents, which would appear to be a catalyst for TRPM2-dependent cell death.

    ACKNOWLEDGEMENT

We thank A. M. Scharenberg for helpful provision of TRPM2 cells.

    FOOTNOTES

* This work was supported by the Wellcome Trust and the British Heart Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. E-mail: d.j.beech@leeds.ac.uk.

Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M210810200

    ABBREVIATIONS

The abbreviations used are: TRP, transient receptor potential; TRPM, TRP melastatin receptor; TRPC, TRP canonical; TRPV, TRP vanilloid receptor; BAPTA, 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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