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INTRODUCTION |
Regulation of the intracellular calcium concentration
[Ca2+]i is of critical importance in
determination of cell fate. In many cell types, oxidative stress,
through the production of oxygen metabolites including
H2O2, causes an increase in
[Ca2+]i, which results in cell injury, apoptosis,
or necrosis (1, 2). One mechanism through which
H2O2 may disrupt calcium homeostasis has
recently been identified. A widely expressed Ca2+-permeable
cation channel, TRPM2, can be activated by micromolar levels of
H2O2 and other agents that produce reactive
oxygen species (3, 4). This channel is part of a physiological pathway through which H2O2 and tumor necrosis factor
may induce cell death (3).
TRPM2, also called LTRPC-2 or TRPC7, is a member of the
transient receptor potential
(TRP)1 protein superfamily.
This is a diverse group of calcium-permeable cation channels expressed
on nonexcitable cells, related to the archetypal TRP,
Drosophila Trp (5-7). The TRP superfamily, conserved from
Caenorhabditis elegans to humans, has been divided into six subfamilies. Mammalian isoforms share six putative transmembrane domains similar to the core structure of many pore-forming subunits of
voltage-gated channels except that they lack positively charged residues necessary for the voltage sensor. One subfamily of TRP channels are referred to as TRPMs (3, 4, 8-14), because the first
described member was melastatin (MLSN), a putative tumor suppressor
protein (6, 7). Prior to implementation of a unified nomenclature for
the TRP superfamily, this subfamily was also known as LTRPC, named
because of longer open reading frames of ~1600 amino acids (5).
Although the mechanisms of activation of specific TRPM are not known,
some TRPM appear to have important roles in cell proliferation. For
example, TRPM1 (MLSN) is expressed in melanocytes, and its level of
expression correlates inversely with melanoma aggressiveness and the
potential for melanoma metastasis (8, 9). TRPM5 (MTR1 and LTRPC5) is
located in the Beckwith-Wiedemann syndrome critical region of human
chromosome 11, although its function in cell growth is not known (12,
15). TRPM8 (Trp-p8) has significant homology to human melastatin, but
it is up-regulated in prostate cancer and a number of nonprostatic
neoplastic tumors (13).
TRPM2 has been cloned from human brain, lymphocytes, and monocytes (11,
16, 17). TRPM2 is activated by H2O2 and other agents that produce reactive oxygen species, resulting in an increase in the intracellular free calcium concentration
([Ca2+]i) (3, 4). Heterologous expression of
TRPM2 in 293 cells conferred susceptibility to
H2O2-induced cell death, which correlated with
the elevation in [Ca2+]i. Furthermore,
suppression of endogenous TRPM2 expression in rat insulinoma RIN-5F or
monocyte U937 cells resulted in significantly diminished
Ca2+ influx and cell death induced by
H2O2 or tumor necrosis factor
(3). These
data strongly support the physiologic role of TRPM2 as an endogenous
H2O2-activated calcium-permeable channel that
mediates cell death following oxidative stress.
In this report, a truncated isoform of TRPM2 was identified in human
hematopoietic cells that lacks four of the six predicted C-terminal
transmembrane domains and the putative pore region permeable to
calcium. The short form of TRPM2 (TRPM2-S) was determined to interact
directly with full-length (TRPM2-L). Using a digital video imaging
system in which single 293T cells that express transfected TRPM2-S were
identified by detection of green fluorescent protein (GFP), cells that
express transfected TRPM2-L were identified by detection of blue
fluorescent protein (BFP), and [Ca2+]i was
simultaneously measured by Fura Red fluorescence, the ability of
TRPM2-S to suppress H2O2-induced calcium influx through TRPM2-L was demonstrated. In addition, the short form of TRPM2
inhibited susceptibility to cell death induced by
H2O2 through full-length TRPM2. These data
suggest that the interaction between TRPM2-S and TRPM2-L is an
important mechanism for regulating channel activity as well as the
cellular response to oxidative stress.
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EXPERIMENTAL PROCEDURES |
Culture of Cell Lines and Human BFU-E-derived Cells--
Jurkat
cells, K562, AML-193, and TF-1 cells were obtained from the American
Type Culture Collection (Manassas, VA). Jurkat cells were cultured in
RPMI 1640 medium with 10% fetal bovine serum (FBS). K562 cells were
cultured in Iscove's modified Dulbecco's medium with 10% FBS.
AML-193 cells were cultured in Iscove's modified Dulbecco's medium
with 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 5 ng/ml
granulocyte-macrophage colony-stimulating factor, and 5% FBS. TF-1
cells were cultured in RMPI 1640 medium with 1 ng/ml granulocyte-macrophage colony-stimulating factor. HEK 293 cells were
obtained from the American Type Culture Collection, and 293T cells were
obtained from Dr. Dwayne Barber (Ontario Cancer Institute, Toronto,
Canada). Both were cultured in Dulbecco's modified Eagle's medium
with 10% FBS. Peripheral blood from volunteer donors was obtained
under protocols approved by the Geisinger Institutional Review Board.
Human BFU-E were cultured in methyl cellulose medium, and BFU-E-derived
cells were harvested at day 10 as described previously (18).
RT-PCR of TRPM2 in Human Primary Cells and Cell Lines--
RNA
was prepared from human 293T, Jurkat, K562, AML-193, TF-1 cells, and
BFU-E-derived cells. cDNA was prepared from RNA using the
Superscript First Strand Synthesis System (Invitrogen) for RT-PCR.
RT-PCR was performed for 35 cycles (denaturation at 94 °C for
30 s, annealing at 57 °C for 30 s, and extension at
72 °C for 30 s). Primers for TRPM2 were 5' primer
(5'-TCGGACCCAACCACACGCTGTA-3') and 3' primer
(5'-CGTCATTCTGGTCCTGGAAGTG-3'). Control 18 S rRNA primers used in
RT-PCR were 5' primer (5'-GAAAGT CGGAGGTTCGAAGA-3') and 3' primer
(5'-ACCAACTAAGAACGGCCTG-3').
Cloning of TRPM2-L and TRPM2-S--
TRPM2 was cloned from human
bone marrow Marathon-Ready cDNA (Clontech, Palo
Alto, CA) by amplifying five adjacent cDNA fragments encoding the
complete open reading frame of TRPM2 with five PCR reactions. The
primers were chosen based on the published cDNA sequence of TRPM2
(GenBankTM accession number AB001535). For the first
fragment, the primers were chosen to amplify the region between
nucleotides 361 and 1572 (1447-nucleotide SmaI site); the
second fragment was amplified with primers to the region between
nucleotides 1350 and 2442 (1447-nucleotide SmaI and
2299-nucleotide NcoI sites); the third fragment was
amplified with primers to the region between nucleotides 2261 and 3376 (2299-nucleotide NcoI and 3358-nucleotide AccI
sites); the fourth fragment was amplified with primers to the region
between nucleotides 3351 and 4351 (3358-nucleotide AccI and
4294-nucleotide NotI sites); and the fifth fragment was
amplified with primers to the region between nucleotides 4243 and 5138 (4294-nucleotide NotI site and 4955-nucleotide stop codon).
Each of these five fragments was sequenced following T/A cloning.
Fragments 1-5 representing the published sequence of TRPM2 were cloned
into pUC18 following appropriate restriction enzyme digestion to yield
TRPM2-L (4.8 kb). The first segment was subcloned into pUC18 following
restriction digestion with HindIII and SmaI. The
second segment was ligated into pUC18 containing the first segment
using the SmaI and EcoRI sites. The third segment
was subcloned into pUC18 containing the first and second segments at
the NcoI and EcoRI sites. The fourth segment was
separately subcloned into pUC18 using the AccI and
EcoRI restriction digestion sites, and the fifth segment was
ligated into pUC18 containing the fourth segment at the NotI
site. Subsequently, the fourth and fifth segments were subcloned into
pUC18 containing the first, second, and third segments at
AccI and EcoRI. For the short version TRPM2-S
(3.0 kb), fragments 1-3 representing the isoform with the stop codon
in the third fragment were cloned into pUC18 using the same restriction
enzyme strategy described for TRPM2-L.
Expression of TRPM2-L and TRPM2-S--
For expression studies,
TRPM2-L and TRPM2-S were subcloned from pUC18 into pcDNA3.1
(Invitrogen) and into pET-42b (Novagen, Madison, WI). TRPM2-L and
TRPM2-S were also subcloned into pcDNA3.1/V5-His TOPO (Invitrogen)
following amplification by PCR. For digital video imaging studies,
TRPM2-L was subcloned into pQBI50 (QbioGene, Carlsbad, CA) and TRPM2-S
into pTracer-CMV (Invitrogen).
To demonstrate the presence of TRPM2-S in TF-1 cells and BFU-E derived
cells, RNA was prepared from these cells, and cDNA was synthesized
as described above for RT-PCR. PCR was used to amplify the third
fragment between nucleotides 2261 and 3376 using the 5' primer
(5'-TCCCTCTACAAGCGTTCCTCAG-3') and the 3' primer (5'-GGTGAGGTAGGAGTGGTAGACG-3'). The PCR products were subcloned into the T/A vector, and the insert was sequenced to confirm the presence or absence of the TAG stop codon.
Generation of Antibodies Specific to TRPM2--
Two rabbit
polyclonal antibodies were generated to TRPM2 and affinity-purified by
Bethyl Laboratories (Montgomery, TX). One of these antibodies
(anti-TRPM2-N) was generated to an epitope in the N terminus of TRPM2
(ILKELSKEEEDTDSSEEMLA, amino acids 658-677) and recognized both
TRPM2-L and TRPM2-S. A second antibody (anti-TRPM2-C) was generated to
an epitope in the C terminus of TRPM2 (KAAEEPDAEPGGRKKTEEPGDS, amino
acids 1216-1237) and was specific for TRPM2-L. Specificity of the
antibodies was confirmed using in vitro translation products
prepared with cDNAs for mTRPC2 clone 14 (19), hTRPC6 (20), TRPM2-S,
and TRPM2-L, cloned into pcDNA3 or pcDNA3.1/V5-His TOPO vector
as described previously (21). The in vitro translation
products were prepared with the TNT quick coupled
transcription/translation system (Promega, Madison, WI).
Immunoblotting of Whole Cell Lysates and Crude Membrane
Preparations--
Human 293T cells were transiently transfected using
LipofectAMINE PLUS Reagent with vector alone, TRPM2-S, TRPM2-L, or
both, in pcDNA3.1 or pcDNA3.1/V5-His TOPO. The cells were
routinely studied 48 h after transfection. For Western blotting of
whole cell lysates, the lysates were separated on 8% polyacrylamide gels, followed by transfer to polyvinylidene difluoride membranes. Following blocking, the blots were incubated with primary antibody (anti-TRPM2-N, 1:300; anti-TRPM2-C, 1:500; anti-V5, 1:2000
(Invitrogen); anti-TRPC6, 1:200 (Alomone Laboratories, Jerusalem,
Israel)), washed, and then incubated with horseradish
peroxidase-conjugated anti-rabbit or anti-mouse antibody (1:2000). ECL
was used for detection of signal. The crude membranes were also
prepared from 293T cell pellets transfected with vector alone
(pcDNA3.1), TRPM2-S, and/or TRPM2-L as described previously (21),
and Western blotting was performed as described above.
Immunoprecipitation--
To determine whether anti-TRPM2-N and
anti-TRPM2-C antibodies are able to immunoprecipitate their targets,
TRPM2-S and TRPM2-L proteins were prepared from TRPM2-S and TRPM2-L
cDNAs subcloned into pET-42b and expressed with the TNT
quick coupled transcription/translation system. In vitro
translation was performed with labeling using TranscendTM
Biotin-Lysyl-tRNA. TRPM2-S and TRPM2-L in vitro translated
proteins were incubated with anti-TRPM2-N or anti-TRPM2-C antibodies
overnight at 4 °C. Protein A-Sepharose 4 Fast Flow (Amersham
Biosciences) was then added for 30 min, followed by washing and then
boiling of beads to remove precipitated protein. Western blotting was performed with the supernatant and with pelleted proteins as described above, except that horseradish peroxidase-streptavidin was used for the
detection of positive signals.
To determine whether TRPM2-L interacts directly with TRPM2-S, 293T
cells were transfected with empty vector (pcDNA3.1), TRPM2-S in
pcDNA3.1/V5-His TOPO, TRPM2-L in pcDNA3.1, or both. The lysates were prepared from cell pellets and preabsorbed with protein
A/G-agarose. 500 µg of each supernatant was incubated with
anti-TRPM2-C (20 µg) or anti-V5 (5 µg) antibodies at 4 °C
overnight. Protein A/G-agarose was then added, the immunoprecipitates
were washed, and Western blotting was performed as described above.
Immunolocalization of TRPM2 in TF-1 Erythroleukemia Cells and in
Transfected 293T Cells--
TF-1 cells were placed in each well of
Lab-Tek Permanox Chamber Slides precoated with fibronectin. After 30 min, the cells were washed three times with phosphate-buffered saline,
fixed with methanol at
20 °C for 10 min, and permeabilized in
0.5% Triton X-100 in phosphate-buffered saline for 5 min. Incubation for 10 min in 20% goat serum preceded staining with primary antibody (anti-TRPM2-N or anti-TRPM2-C, 1:50) for 20 min at room temperature, followed by secondary antibody (goat anti-rabbit Alexa 488; Molecular Probes, Eugene, OR) for 20 min in the dark. The slides were stained with propidium iodide (PI) in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The images were acquired with a Leica
TCS SP2 Confocal Microscope.
293T cells transfected with TRPM2-L in pcDNA3.1, TRPM2-S in
pcDNA3.1/V5-His TOPO, or both were plated on polylysine-coated chamber slides 24 h after transfection and incubated for 24 h more at 37 °C. The cells were fixed, permeabilized, and incubated with primary (rabbit anti-TRPM2-C or mouse anti-V5) or secondary antibodies (fluorescein isothiocyanate donkey anti-rabbit IgG; Jackson
Laboratories, West Grove, PA; Texas Red goat anti-mouse) as described
above. The slides were stained with DAPI in Vectashield mounting medium
to visualize DNA instead of PI, which could not be distinguished from
Texas Red fluorescence. To visualize DAPI, the cells were viewed using
a Nikon Opiphot-2 microscope equipped for epifluorescence. The images
were acquired with an air-cooled CCD SenSys digital camera from
Photometrics (Tuscon, AZ) and processed using IPLab and Enhanced Photon
Reassignment software programs obtained from Scanalytics (Fairfax, VA).
Measurement of [Ca2+]i with Digital Video
Imaging--
293T cells were transfected with TRPM2-S subcloned into
pTracer-CMV, TRPM2-L subcloned into pQBI50, or both as described above. The pTracer-CMV vector contains an SV40 promoter driving expression of
a GFP gene and a CMV promoter driving expression of TRPM2-S. The pQBI50
vector contains a CMV promoter that drives expression of SuperGlo BFP
fused through a flexible linker to TRPM2-L. Successful transfection of
293T cells with TRPM2-S and TRPM2-L was verified by detection of GFP
(excitation, 478 nm; emission, 535 nm) and BFP (excitation, 380 nm;
emission 435 nm), respectively, in cells with our digital video imaging
system (21, 22). The fluorescence microscopy-coupled digital video
imaging used to measure changes in [Ca2+]i has
been described previously (22-25). To study changes in
[Ca2+]i in transfected cells, we were not able to
use Fura-2 as the detection fluorophore because its excitation and
emission wavelengths overlap with those of GFP. Instead, we used the
fluorescent indicator Fura Red (excitation, 460 and 490 nm; emission,
600-nm-long pass), a dual wavelength excitation probe. We determined
Rmin (minimum fluorescence intensity ratio
(r) of the emission following excitation at 460 nm divided
by the emission following excitation at 490 nm),
Rmax (maximum r), and the constants
Sf2/Sb2 with
Fura Red so that [Ca2+]i could be
calculated using the formula [Ca2+]i = K'D[(R
Rmin)/(Rmax
R)]
(Sf2/Sb2).
Transfected 293T cells grown on glass coverslips were loaded at 48 h with 5 µM Fura Red-AM for 30 min at 37 °C in the
presence of Pluronic F-127 to enhance loading. Transfected 293T cells
were treated with 0-10 mM H2O2 and
[Ca2+]i measured at base line and over a 20-min
interval. Statistical significance of results was analyzed with one-way analysis of variance.
Assays of Cell Viability--
293T cells transfected for 48 h with vector, TRPM2-S, TRPM2-L, or both in pQBI50 were treated with
0-10 mM H2O2 for 40 min. Cell
viability was assessed by trypan blue exclusion. Induction of apoptosis
or necrosis was also assessed in these cells following treatment with
H2O2 with the Vibrant apoptosis assay kit 2 (Molecular Probes), in which apoptotic cells are labeled with annexin V
conjugated to Alexa Fluor 488 and necrotic cells are labeled with
propidium iodide.
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RESULTS |
Cloning of TRPM2-S--
TRPM2 has been reported to be highly
expressed in lymphocytes as well as in granulocytes and other
hematopoietic cell lines (4, 17). To study TRPM2 in human hematopoietic
cells, the expression of TRPM2 in different hematopoietic lineages was
first examined. RT-PCR was performed on RNA isolated from Jurkat (T cell lymphoblast cell line), K562 (chronic myelogenous leukemia cell
line), AML-193 (acute monocytic leukemia cell line), and TF-1
(erythroleukemia cell line) cells and from BFU-E-derived primary
erythroid cells (18). The results are shown in Fig. 1. TRPM2 mRNA was highly expressed in
Jurkat, K562, AML-193, and TF-1 cells. TRPM2 was also expressed,
although at lower levels, in primary human erythroblasts at day 10 of
culture, which are largely proerythroblasts (18). TRPM2 mRNA was
detectable at low levels in 293 cells; these results differ from a
previous report in which TRPM2 mRNA was undetectable in 293 cells
(17), possibly because of differences in the RT-PCR conditions or in the 293 cells studied. No TRPM2 bands were observed when PCR was performed without the reverse transcriptase step, demonstrating that
these products did not result from contaminating DNA. The identity of
PCR bands was confirmed by sequencing.

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Fig. 1.
RT-PCR of TRPM2 in human hematopoietic
cells. RT-PCR was performed on RNA isolated from 293, Jurkat,
K562, AML-193, and TF-1 cell lines and from BFU-E-derived erythroblasts
at day 10 of culture. 18 S rRNA primers were used as a control.
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To clone TRPM2 for further study, a strategy was designed based on the
published cDNA sequence (see "Experimental Procedures"). Using
Marathon-Ready cDNA from human bone marrow as the template, five
adjacent cDNA fragments encoding the complete open reading frame of
TRPM2 were amplified by PCR. Following sequencing, the fragments were
ligated together to produce full-length TRPM2 (TRPM2-L). Sequencing of
the third fragment resulted in two alternative sequences; one was
identical to the published sequence, and the other included a TAG stop
codon at nucleotides 2984-2986. This sequence is a result of
alternative splicing of the 3' end of the intron between exons 16 and
17. As shown on Fig. 2, this isoform,
TRPM2-S, results in the deletion of the entire C terminus of
full-length TRPM2, including the four C-terminal transmembrane domains
and the putative pore region permeable to calcium. Because the TAG stop
codon immediately follows the CAG encoding glutamine (Fig. 2), no
unique amino acids are introduced in the TRPM2-S protein.

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Fig. 2.
Schema of cDNA for the two splice
variants TRPM2-L and TRPM2-S. The protein structures of TRPM2-L
and TRPM2-S are shown with the predicted transmembrane domains and the
pore region indicated. Nucleotide and amino acid sequences surrounding
the alternatively spliced stop codon between exons 16 and 17 are shown
below the TRPM2 gene, demonstrating termination of TRPM2 after the
second transmembrane domain in TRPM2-S.
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The PCR products following amplification of bone marrow cDNA with
primers for the third fragment of TRPM2 were subcloned into the T/A
vector, and 13 clones were sequenced. The sequences of seven different
clones were identical to TRPM2-S, and six were identical to TRPM2-L. To
determine whether TRPM2-S is expressed in other hematopoietic cells,
cDNA was also prepared with RNA from normal human BFU-E-derived
cells and from TF-1 cells. As described above, PCR products amplified
with primers for the third fragment were subcloned into the T/A vector,
and the clones were sequenced. Four of eight clones from normal human
BFU-E derived cells, and two of three clones from TF-1 cells were
identical to TRPM2-S; the rest were identical to TRPM2-L. These data
demonstrate the presence of TRPM2-S in three different hematopoietic
cell types.
Generation of Antibodies Specific to TRPM2--
To study the
interaction and function of TRPM2 isoforms, an affinity-purified
antibody was generated to the N terminus of TRPM2 (anti-TRPM2-N), which
recognizes both TRPM2-S and TRPM2-L. A second antibody was generated to
the C terminus of TRPM2 (anti-TRPM2-C), which recognizes only TRPM2-L.
To characterize antibody specificity, in vitro translation
was performed using cDNAs for TRPM2-S and TRPM2-L in
pcDNA3.1/V5-His TOPO and mTRPC2 clone 14 and hTRPC6 in pcDNA3
as controls. Western blotting was performed with each of these in
vitro translation products with anti-TRPM2-N, anti-TRPM2-C, anti-V5, or anti-TRPC6 antibodies. The results are shown in Fig. 3A. Anti-TRPM2-N recognized
the in vitro translation products TRPM2-S (95 kDa) and
TRPM2-L (171 kDa), both with tags that add ~6 kDa to the predicted
molecular masses. A nonspecific cross-reacting band was observed
at 105 kDa with anti-TRPM2-N antibody. Anti-V5 recognized the tagged
in vitro translation products TRPM2-S and TRPM2-L.
Anti-TRPM2-C recognized only TRPM2-L. Anti-TRPC6 was used as a control
to demonstrate the presence of the in vitro translation
product hTRPC6 (100 kDa) on the blots. The presence of TRPC2 clone 14 (132 kDa) was also confirmed as previously (Ref. 21 and results not
shown). Neither anti-TRPM2-N or anti-TRPM2-C antibodies recognized
mTRPC2 or hTRPC6.

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Fig. 3.
A, specificity of TRPM2 antibodies.
In vitro translation products were prepared using cDNAs
of mTRPC2 clone 14 (pcDNA3), hTRPC6 (pcDNA3), and TRPM2-S and
TRPM2-L in pcDNA3.1/V5-His TOPO. Equivalent amounts of each
reaction were loaded in each lane, and Western blotting was performed.
The blots were probed with anti-TRPM2-N, anti-V5, anti-TRPM2-C, and
anti-TRPC6 antibodies (AB). B, Western blot of
transfected 293T cells. The lysates were prepared from 293T cells
transfected with empty vector, TRPM2-S, and TRPM2-L. Equivalent amounts
of protein were loaded in each lane. The blots were probed with
anti-TRPM2-N, anti-V5, and anti-TRPM2-C antibodies.
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293T cells were then transfected with vector alone, TRPM2-S, or TRPM2-L
in pcDNA3.1/V5-His TOPO. The cell lysates were prepared, and
Western blotting was performed. Fig. 3B demonstrates the
ability of anti-TRPM2-N to specifically recognize TRPM2-S and TRPM2-L and the ability of anti-TRPM2-C to recognize TRPM2-L in transfected cells. These observations were confirmed with antibody directed to the
V5 tag. Fig. 3B also demonstrates the inability to detect endogenous TRPM2 protein expression in 293T cells under the identical conditions, suggesting that endogenous protein levels are low or absent.
Expression of Endogenous TRPM2--
To confirm the expression and
determine the subcellular localization of endogenous TRPM2,
immunolocalization studies were performed with TF-1 cells using
anti-TRPM2-N and anti-TRPM2-C antibodies and confocal microscopy. PI
staining was used to localize DNA. Representative results shown here
demonstrate that endogenous TRPM2 recognized by either anti-TRPM2-N
(Fig. 4A) or anti-TRPM2-C (Fig. 4B) is localized on or near the plasma membrane. In
Fig. 4C, no fluorescence was observed in control cells
stained with secondary antibody alone. Unfortunately, anti-TRPM2-N
antibodies cannot distinguish TRPM2-S from TRPM2-L; the amino acid
sequence of TRPM2-S is identical to LTRCP2-L throughout the TRPM2-S
sequence.

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Fig. 4.
Immunofluorescence of TRPM2 in TF-1 and in
transfected 293T cells. TF-1 cells fixed to glass slides were
stained with anti-TRPM2-N and secondary antibody (A),
anti-TRPM2-C and secondary antibody (B), or secondary goat
anti-rabbit Alexa 488 antibody alone (C). PI was used to
stain DNA (A-C). D, 293T cells transfected with
TRPM2-S were stained with anti-V5 as the primary antibody and Texas Red
anti-mouse IgG as the secondary antibody. DAPI was used to stain DNA.
E, 293T cells transfected with TRPM2-L were stained with
anti-TRPM2-C and fluorescein isothiocyanate anti-rabbit secondary
antibodies. 293T cells transfected with both TRPM2-S and TRPM2-L
were stained with anti-V5 and goat anti-mouse secondary antibodies
(F) and anti-TRPM2-C and donkey anti-rabbit secondary
antibodies (G). The merged images of F and
G are shown in H and demonstrate significant
overlap.
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Subcellular Localization of TRPM2-S and TRPM2-L--
To
distinguish the subcellular localization of TRPM2-S or TRPM2-L isoforms
and determine whether TRPM2-S alters the localization of TRPM2-L (10),
293T cells were transfected with TRPM2-S in pcDNA3.1/V5-His TOPO,
TRPM2-L in pcDNA3.1, or both. TRPM2-S was detected with antibody to
the V5 tag, and TRPM2-L was detected with antibody specific for the C
terminus of TRPM2. Cell staining was visualized by fluorescence
microscopy. The images at different planes through the cell were
deconvolved (Scanalytics software) to remove out-of-focus contaminating
light to generate high resolution images. In 293T cells transfected
with a single vector, TRPM2-S (Fig. 4D) or TRPM2-L (Fig.
4E) were expressed at or near the plasma membrane, as well
as throughout the cytoplasm in a nonhomogeneous pattern. When TRPM2-S
and TRPM2-L were coexpressed in the same 293T cells (Fig. 4,
F-H), the expression pattern was not different from that
observed when each was expressed alone, and the spatial distribution
showed extensive overlap. The results were identical when a range of
DNA concentrations was used to transfect 293T cells to reduce the level
of expressed protein. The results were also similar when CHO, CHO-S, or
COS-1 cells were transfected (not shown). When anti-V5, anti-TRPM2-N,
or anti-TRPM2-C antibodies were used with the appropriate secondary
antibody to stain 293T cells transfected with vector alone, no positive
fluorescence was observed (not shown). These data show that transfected
TRPM2-L and TRPM2-S are also expressed at or near the plasma membrane.
To confirm that TRPM2-S and TRPM2-L have a similar subcellular
distribution and that TRPM2-S does not alter the subcellular localization of TRPM2-L, crude membrane fractions were prepared from
293T cells transfected with vector alone (pcDNA3.1), TRPM2-S, TRPM2-L, or both as described previously (21). Western blotting was
performed with protein isolated in the crude membrane pellet or the
supernatant, and the blots were probed with anti-TRPM2-N (Fig.
5A) or anti-TRPM2-C (Fig.
5B) antibodies. A protein band of ~171 kDa was observed in
cells transfected with TRPM2-L, which localized to the membrane
fraction. A protein band of ~95 kDa was observed in cells transfected
with TRPM2-S, which also primarily localized to the membrane fraction.
When coexpressed, both TRPM2-S and TRPM2-L continued to localize in the
membrane fraction rather than in the supernatant. These data confirm
that TRPM2-S does not alter the localization of TRPM2-L and that
TRPM2-S and TRPM2-L have a similar subcellular localization. They also
suggest that the nonhomogeneous cytoplasmic staining observed with
immunofluorescent microscopy in 293T cells transfected with TRPM2-S or
TRPM2-L (Fig. 4, D-H) represents intracellular membrane
structures in which TRPM2 is produced or transported.

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Fig. 5.
Membrane localization of TRPM2-S and
TRPM2-L. Western blots were performed on crude membrane pellets or
the supernatants of 293T cells transfected with pcDNA3.1 vector
alone (V), TRPM2-S, TRPM2-L, or both. The blots were probed
with anti-TRPM2-N (A) or anti-TRPM2-C antibodies
(B).
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Direct Protein Interaction between TRPM2 Isoforms--
To study
the interaction of TRPM2 isoforms with each other, the ability of
anti-TRPM2 antibodies to immunoprecipitate their targets was first
examined. TRPM2-S and TRPM2-L cDNAs in pET-42b were expressed by
in vitro translation and labeled using Biotin-Lysyl-tRNA. Each of these in vitro translation products was incubated
with anti-TRPM2-C or anti-TRPM2-N antibodies and bound protein
precipitated with protein A-Sepharose. Western blotting of pelleted
fractions or the supernatants demonstrates that anti-TRPM2-C
immunoprecipitates TRPM2-L but not TRPM2-S (Fig.
6A). The results also
demonstrate that anti-TRPM2-N immunoprecipitates both TRPM2-S and
TRPM2-L but that TRPM2-L is not immunoprecipitated as efficiently as
TRPM2-S by this antibody. The higher molecular masses of proteins shown here compared with that in reticulocyte lysates or 293T cells transfected with TRPM2-S/TRPM2-L in pcDNA3.1 or pcDNA3.1/V5-His TOPO are results of linkage of GST to TRPM2-S/-L in the pET-42b vector.

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Fig. 6.
Interaction of TRPM2-S with TRPM2-L.
A, to demonstrate the ability of anti-TRPM antibodies to
immunoprecipitate, in vitro translated proteins were
prepared using empty vector or cDNAs of TRPM2-S or TRPM2-L in
pET-42b and labeling with TranscendTM Biotin-Lysyl-tRNA.
The proteins were immunoprecipitated with anti-TRPM2-C (C)
or anti-TRPM2-N (N) antibodies, and Western blotting was
performed with precipitated proteins (P) or supernatant
before the first wash (S). The proteins were detected by
horseradish peroxidase-streptavidin followed by ECL. B, 293T
cells were transfected with vector alone (pcDNA3.1), TRPM2-S in
pcDNA3.1/V5-His TOPO, TRPM2-L in pcDNA3.1, or both. The cell
lysates were immunoprecipitated with anti-TRPM2-C or anti-V5
antibodies. Western blotting was performed with anti-TRPM2-C antibody,
and the blots were then stripped and reprobed with anti-V5
antibody.
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To determine whether TRPM2-S directly interacts with TRPM2-L, 293T
cells were transfected with empty vector, TRPM2-S in
pcDNA3.1/V5-His TOPO, TRPM2-L in pcDNA3.1, or both. Each cell
lysate was immunoprecipitated with anti-TRPM2-C to precipitate TRPM2-L
or anti-V5 to precipitate TRPM2-S. The blots were probed with
anti-TRPM2-C or anti-V5 antibodies. The results are shown on Fig.
6B. Anti-TRPM2-C antibody immunoprecipitated TRPM2-S
when TRPM2-L was coexpressed, and anti-V5 antibody immunoprecipitated TRPM2-L in the presence of TRPM2-S, demonstrating the direct
interaction of TRPM2-S with TRPM2-L.
TRPM2-S Inhibits Calcium Influx through
TRPM2-L--
H2O2 has previously been
demonstrated to evoke calcium influx through TRPM2 and to increase
[Ca2+]i (3, 4). To confirm these results, 293T
cells were transfected with empty vector or TRPM2-L in pQBI50.
Successful transfection of empty vector or TRPM2-L was confirmed by
detection of BFP, fused through a flexible linker to TRPM2-L, in single cells with our digital video imaging system.
[Ca2+]i was measured in these cells by detection
of Fura Red fluorescence at base line and at intervals over 20 min
following exposure to 0.1, 1, or 10 mM
H2O2. These experiments confirmed a significant
and dose-dependent increase in
[Ca2+]i in TRPM2-L-transfected cells in response
to H2O2. The increase in
[Ca2+]i in TRPM2-L-transfected cells was
significantly greater than that observed in cells transfected with
vector alone at all concentrations of H2O2 at
exposure times of 5 min or greater (p
0.05). These
results are shown in Fig.
7A.

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Fig. 7.
Stimulation of Ca2+ influx
through TRPM2 splice variants by H2O2.
293T cells were transfected with vector alone (pQBI50), TRPM2-S (in
pTracer-CMV), TRPM2-L (in pQBI50), or both. 48 h later,
successfully transfected single cells were identified by GFP or BFP
fluorescence. [Ca2+]i was measured in Fura
Red-loaded, transfected cells at base line and at intervals over 20 min
after treatment with H2O2. A,
[Ca2+]i was measured in 293T cells transfected
with vector alone (V) or TRPM2-L (L) and treated
with 0.1, 1.0, or 10 mM H2O2. Mean
[Ca2+]i + S.E. is shown. Four to thirteen cells
were studied at each H2O2 concentration.
B, the change in [Ca2+]i is shown in
cells transfected with vector alone, TRPM2-S, TRPM2-L, or both
following treatment with 1.0 mM
H2O2 for 20 min. Mean change in
[Ca2+]i + S.E. is shown for 13 (V), 16 (TRPM2-S), 20 (TRPM2-L), or 28 (TRPM2-S+L) transfected cells studied. The
asterisk indicates a significant difference compared with
vector alone, TRPM2-S-transfected, and TRPM2-S+L- transfected cells
(p 0.01).
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To determine whether TRPM2-S can modulate
H2O2-induced calcium influx through TRPM2-L,
293T cells were transfected with vector alone, TRPM2-S in pTracer-CMV,
TRPM2-L in pQBI50, or both. Successfully transfected individual 293T
cells were identified for study by detection of GFP (TRPM2-S), BFP
(empty vector, TRPM2-L), or both with our digital video imaging system.
[Ca2+]i was measured in Fura Red-loaded,
transfected cells before and at intervals for 20 min after exposure to
1.0 mM H2O2. The mean peak increase
in [Ca2+]i above base line for the four groups of
transfected cells is shown in Fig. 7B. The increase in
[Ca2+]i following treatment with 1.0 mM H2O2 in 293T cells transfected
with TRPM2-S was not statistically different from vector alone
transfected cells. As noted above, the peak increase in
[Ca2+]i in TRPM2-L-transfected cells was
significantly greater than that seen in cells transfected with vector
alone or TRPM2-S (p
0.01). In cells transfected with
both TRPM2-L and TRPM2-S, the peak increase in
[Ca2+]i was significantly less than that observed
in cells transfected with TRPM2-L alone (p < 0.001),
demonstrating the ability of TRPM2-S to inhibit calcium influx through
TRPM2-L.
TRPM2-S Suppresses Susceptibility to Cell Death Induced through
TRPM2 by H2O2--
TRPM2-expressing cells are
susceptible to cell death induced by exposure to
H2O2 (3). Here, the ability of TRPM2-S to
modulate cell death induced by H2O2 was
examined. 293T cells were transfected with vector alone, TRPM2-S,
TRPM2-L, or combinations in pQBI50. Empty vector was transfected with
TRPM2-S or TRPM2-L to maintain the equivalent amount of DNA used in
cotransfection of both TRPM2-S and TRPM2-L. At 48 h, the cells
were treated with 0, 0.1, 1.0, or 10 mM
H2O2 for 40 min. The cell viability was then
assessed by trypan blue exclusion. The results are shown on Fig.
8A. Treatment of
TRPM2-L-transfected cells with H2O2 resulted in
a significant and dose-dependent decrease in cell viability
compared with untransfected cells, cells transfected with vector alone,
or cells transfected with TRPM2-S (p
0.01). In 293T
cells expressing both TRPM2-S and TRPM2-L, the cell viability was
significantly enhanced compared with TRPM2-L expressing cells
(p
0.001).

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Fig. 8.
A, viability of cells transfected with
TRPM2 splice variants and treated with H2O2.
293T cells were transfected with vector alone, TRPM2-S, TRPM2-L, or
combinations in pQBI50. Each group of transfected cells was treated
with 0, 0.1, 1.0, or 10 mM H2O2 for
40 min, and cell viability assessed by trypan blue exclusion. The
asterisk indicates a significant difference from the other
groups of transfected cells (p 0.01). Mean trypan
blue exclusion + S.E. for three experiments is shown. B,
Western blot of 293T cells transfected with TRPM2 splice variants. 293T
cells were transfected with equivalent amounts of vector alone
(pQBI50), TRPM2-S, TRPM2-L, or both. The cell lysates were prepared,
and 100 µg of each was loaded in each lane. The blots were probed
with anti-TRPM2-N or anti-TRPM2-C and secondary antibody followed by
ECL.
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To confirm that this suppression of
H2O2-induced cell death did not result from
decreased expression of TRPM2-L in cotransfected cells, Western
blotting was performed on cells transfected with vector alone, TRPM2-S,
TRPM2-L, or both in pQBI50 under the same conditions as above for
48 h. The results, shown in Fig. 8B, demonstrate nearly
equivalent expression of TRPM2-S and TRPM2-L in cells that were
transfected with both cDNAs simultaneously when compared with cells
transfected with each cDNA alone. The higher molecular masses of
proteins shown here compared with that in reticulocyte lysates or 293T
cells transfected with TRPM2-S/TRPM2-L in pcDNA3.1 or
pcDNA3.1/V5-His TOPO is a result of linkage to BFP.
To assess the ability of TRPM2-S to inhibit apoptosis or necrosis in
response to oxidative stress, 293T cells transfected for 48 h with
vector alone (pQBI50), TRPM2-S, TRPM2-L, or both were treated with 0 or
10 mM H2O2 for 40 min. Apoptosis
was assessed by labeling of cells with Alexa Fluor 488 annexin V
conjugates; annexin V binds to phosphatidylserine on the surface of
early apoptotic cells. Necrosis was assessed by labeling with propidium iodide, which binds to nucleic acids in necrotic cells but does not
penetrate live cell membranes or early apoptotic cells. Representative results are shown in Fig. 9. Treatment of
293T cells transfected with vector alone (Fig. 9A) or with
TRPM2-S (Fig. 9B) with 0 or 10 mM
H2O2 resulted in barely detectable apoptosis
(Alexa 488 annexin) or necrosis (PI). In contrast, 293T cells
transfected with TRPM2-L demonstrated little apoptosis or necrosis with
vehicle (0 mM H2O2) but
70%
apoptotic cells when treated with 10 mM
H2O2 (Fig. 9C). When 293T cells were
transfected with both TRPM2-S and TRPM2-L, the number of apoptotic
cells was greatly reduced (<10%) compared with cells transfected with
TRPM2-L alone (Fig. 9D). Necrosis was not a prominent
finding in any of these H2O2-treated cells
after 40 min. Three experiments were performed with similar results.

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Fig. 9.
Apoptosis in 293T cells transfected with
TRPM2 splice variants. 293T cells were transfected with vector
alone (pQBI50, A), TRPM2-S (B), TRPM2-L
(C), or TRPM2-S and TRPM2-L in pQBI50 (D). The
cells were treated with 0 or 10 mM
H2O2 for 40 min and then incubated with
propidium iodide and Alexa Fluor 488/annexin V. Representative white
light images of cells (DIC), fluorescence of PI,
fluorescence of Alexa 488 (Annexin), or the merged image of propidium
iodide and Alexa 488 staining are shown for each group of transfected
cells.
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DISCUSSION |
In this report, a new isoform of TRPM2, TRPM2-S, was cloned from
human bone marrow and consists of only the N terminus and the first two
transmembrane domains. Here, we demonstrated that expression of TRPM2-S
inhibits calcium influx, enhances cell viability, and reduces apoptosis
and cell death, which occur following exposure of full-length
TRPM2-expressing cells to H2O2. Because
H2O2 causes an increase in intracellular
calcium that precedes cell death in numerous cell types including
cardiac and smooth muscle cells, macrophages, and neurons (1), TRPM2-S
may have a generally important role in the determination of cell fate
following exposure to oxidative stress.
The first major finding of this report is the identification of a short
isoform of TRPM2 expressed physiologically in human hematopoietic
cells. This is the third TRPM family member for which a short isoform
has been identified and is similar to the short isoforms of MLSN
(TRPM1) (10) and MTR1 (TRPM5) (12) in that all have a deletion of the C
terminus including transmembrane domains and the putative
calcium-permeable pore region. N-terminal fragments of
Drosophila and mammalian TRPCs have previously been shown to
bind to and suppress the activity of full-length TRPC proteins
(26-28). These N-terminal domains of TRPC were found to participate in
heteromultimer complex formation. The physiological short form of MLSN,
MLSN-S, which has no transmembrane domains, interacts with and
suppresses the activity of full-length MLSN (MLSN-L) (10). MLSN-L
localizes near or in the plasma membrane, whereas MLSN-S is uniformly
distributed in the cytoplasm. Published data suggest that the
suppression of activity by MLSN-S results from direct interaction of
MLSN-S and MLSN-L, inhibiting translocation of MLSN-L to the plasma
membrane (10). Here we identified a short isoform of TRPM2 that
interacts with and suppresses the activity of full-length TRPM2, but
the mechanism does not appear to be the same as reported for MLSN-S.
Although TRPM2-S directly interacts with TRPM2-L, our experiments
demonstrate that TRPM2-S and TRPM2-L have a similar subcellular
localization and that TRPM2-S does not alter the localization of
TRPM2-L. Unlike MLSN-S, TRPM2-S retains two transmembrane domains that
localize it to the cell membrane.
The second major finding of this report is that TRPM2-S is able to
inhibit calcium influx induced by H2O2 through
full-length TRPM2. TRPM2 has been shown to be regulated by at least
three different mechanisms. 1) TRPM2 has a Nudix box in its C terminus that has homology to NUDT9, an ADP-ribose pyrophosphatase degrading ADP-ribose (ADPR). Although it can function as an ADPR pyrophosphatase, it has a much lower level of activity than NUDT9 (11). The Nudix box
may also serve as an ADPR-binding site, and ADPR has been shown to
directly gait TRPM2 opening (11, 17). Because in oxidative stress ADPR
production is increased (29), this is a mechanism through which
H2O2 may regulate TRPM2-L. 2) NAD has also been
shown to directly activate TRPM2 (3, 17), although one group of
investigators was unable to show that NAD stimulates current through
TRPM2 (4). 3) H2O2, but not ADPR or NAD, was able to stimulate calcium influx through an TRPM2 mutant with a small
deletion in the C terminus (4). These data suggest that
H2O2 and oxidative stress can mediate calcium
influx through a third mechanism independently of ADPR or NAD. This
pathway will need to be defined before the mechanism through which
TRPM2-S inhibits H2O2-mediated calcium influx
through TRPM2-L can be identified. Because TRPCs have been proposed to
function as homo- or heterotetramers (30) and TRPM2-S and TRPM2-L are
shown here to directly interact, TRPM2-S may participate in
heterotetramer formation, altering the tertiary structure of the
TRPM2-L homotetramer and inhibiting calcium permeability.
Alternatively, because it is missing the C terminus, TRPM2-S could also
function to impair localization of TRPM2-L to signaling complexes (6)
or act as a dominant negative blocking an unknown aspect of TRPM2-L
regulation critical for calcium channel activation.
The third and most important finding of this report is that TRPM2-S
inhibits the decreased cell viability and increased susceptibility to
cell death that result from activation of TRPM2-L by oxidative stress.
Our observation that exposure of TRPM2-L-expressing cells to
H2O2 reduces cell viability and enhances
apoptosis is consistent with previous reports (3, 17). The significance
of our work is the identification of a physiological splice variant of
TRPM2, which suppresses cell death in response to oxidative stress. The genomic sequence of TRPM2 spans 32 exons (16), raising the possibility that multiple splice variants with different capabilities may exist. In
fact, two other splice variants of TRPM2 have been identified in
granulocytes and undifferentiated HL-60 cells, which have small deletions in their N terminus (amino acids 538-557) or C terminus (amino acids 1292-1325) (4). H2O2 does not
induce calcium influx in cells transfected with the N-terminal deletion
but is effective in stimulating calcium influx through the C-terminal
deletion. No functional studies were performed to examine isoform
interactions or the impact of expression of these variants on cell
viability. As suggested by our work with TRPM2-S, expression or
activation of different isoforms of TRPM2 may be a mechanism through
which cells can control their reaction to damaging oxidants. In this event, regulation of splicing of TRPM has important implications for
controlling the onset of apoptosis or cell death in response to
oxidative stress and needs to be explored. Reduction of TRPM2 levels
with antisense oligonucleotides also suppressed
[Ca2+]i oscillations and cell death induced by
tumor necrosis factor
in RIN-5F cells and the monocyte cell line
U937 (3). This suggests that the level of TRPM2 expression, as well as
the ratio of isoforms expressed (TRPM2-S versus TRPM2-L),
may be important in regulating the proliferative or apoptotic response
to stimulation with or withdrawal of certain hematopoietic growth
factors. This possibility is being pursued.
In summary, we have identified a physiologic splice variant of TRPM2
that is expressed in hematopoietic cells and interacts directly with
full-length TRPM2. Using 293T cells cotransfected with TRPM2-S and
TRPM2-L, we demonstrated the ability of TRPM2-S to inhibit
H2O2-induced calcium influx through TRPM2-L and
to suppress the susceptibility to cell death induced through TRPM2-L by
H2O2. Our studies suggest that TRPM2-S may have
an important role in many tissues because it can modulate calcium
influx and cellular responses to oxidative stress.