From the 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
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ABSTRACT |
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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.
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, 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 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
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-NAD+, or arachidonic acid.
The sensitivity of the channel to
-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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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.
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 M
)
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.
Compositions of intracellular solutions
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).
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RESULTS |
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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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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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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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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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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.
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DISCUSSION |
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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.
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ACKNOWLEDGEMENT |
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We thank A. M. Scharenberg for helpful provision of TRPM2 cells.
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FOOTNOTES |
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* 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
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ABBREVIATIONS |
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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.
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