Receptor-activated Ca2+ Influx via Human Trp3 Stably
Expressed in Human Embryonic Kidney (HEK)293 Cells
EVIDENCE FOR A NON-CAPACITATIVE Ca2+ ENTRY*
Xi
Zhu
§,
Meisheng
Jiang, and
Lutz
Birnbaumer¶
From the
Department of Pharmacology and
Neurobiotechnology Center, Ohio State University, Columbus, Ohio
43210 and Departments of ¶ Anesthesiology and
Biological Chemistry, School of Medicine, and the
Molecular Biology and Brain Research Institutes, UCLA,
Los Angeles, California 90095
 |
ABSTRACT |
Ca2+ release from its internal
stores as a result of activation of phospholipase C is accompanied by
Ca2+ influx from the extracellular space. Ca2+
influx channels may be formed of proteins homologous to
Drosophila Trp. At least six non-allelic Trp
genes are present in the mouse genome. Full-length human, bovine,
mouse, and rat cDNAs for Trp1, 3, 4, 6 have been
cloned. Expression of these genes in various mammalian cells has
provided evidence that Trp proteins form plasma membrane
Ca2+-permeant channels that can be activated by an agonist
that activates phospholipase C, by inositol 1,4,5-trisphosphate, and/or
store depletion. We have stably expressed human Trp3 (hTrp3) in human embryonic kidney (HEK)293 cells. Measurement of intracellular Ca2+ concentrations in Fura2-loaded cells showed that cell
lines expressing hTrp3 have significantly higher basal and
agonist-stimulated influxes of Ca2+, Mn2+,
Ba2+, and Sr2+ than control cells. The increase
in Ca2+ entry attributable to the expression of hTrp3
obtained upon store depletion by thapsigargin was much lower than that
obtained by stimulation with agonists acting via a
Gq-coupled receptor. Addition of agonists to
thapsigargin-treated Trp3 cells resulted in a further increase in the
entry of divalent cations. The increased cation entry in Trp3 cells was
blocked by high concentrations of SKF 96365, verapamil,
La3+, Ni2+, and Gd3+. The
Trp3-mediated Ca2+ influx activated by agonists was
inhibited by a phospholipase C inhibitor, U73122. We propose that
expression of hTrp3 in these cells forms a non-selective cation channel
that opens after the activation of phospholipase C but not after store
depletion. In addition, a subpopulation of the expressed hTrp3 may form
heteromultimeric channels with endogenous proteins that are sensitive
to store depletion.
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INTRODUCTION |
The activities of a large number of enzymes are regulated through
changes of intracellular Ca2+ concentration
([Ca2+]i).1
Under resting conditions, cells keep
[Ca2+]i at approximately 100 nM. A rise of the cytosolic Ca2+ triggers a
cascade of Ca2+-sensitive events that are both immediate,
such as secretion, contraction, and mobilization of energy resources
(e.g. glycogenolysis), and long term, such as changes in the
transcription of many genes. Some of the Ca2+-responsive
transcription processes are known to cause proliferation and programmed
cell death (1-3). In both nonexcitable and excitable cells,
Ca2+ signaling pathways can be activated by a ligand
binding to cell surface receptors that activate phospholipase C (PLC).
These include receptors that activate heterotrimeric G proteins and
receptors signaling through activation of protein tyrosine kinases. The activation of PLC leads to the production of inositol
1,4,5-trisphosphate (IP3), which binds to IP3
receptors, a class of intracellular ligand-operated Ca2+
release channels. The opening of IP3 receptors allows
Ca2+ to exit from its internal storage pools, causing a
rapid increase in [Ca2+]i. The
increased cytosolic Ca2+ level is reduced quickly by
Ca2+ pumps located on both the endoplasmic reticulum and
the plasma membrane, causing
[Ca2+]i to decrease. The
Ca2+ signal is prolonged, however, by the opening of a set
of plasma membrane Ca2+-permeant channels that allow
Ca2+ to enter cells from the extracellular space, where the
concentration of Ca2+ is in the millimolar range. In many,
and possibly all cells, the entering Ca2+ is taken up
rapidly by a storage compartment from which it is re-released in the
continued presence of the triggering extracellular signal. This may,
although not necessarily, be accompanied by periodic oscillations of
the [Ca2+]i and allows for
regulation of cytosolic as well as membrane-associated functions of the
affected cells (for reviews, see Refs. 1 and 4).
Putney (5, 6) coined the term capacitative Ca2+
entry (CCE) for Ca2+ entry that is activated upon
stimulation of cells with agonists that promote Ca2+
release from internal stores. After the discovery that CCE can be
stimulated by mere store depletion, as occurs after inhibition of
sarcoplasmic endoplasmic reticulum Ca2+-activated ATPases
with thapsigargin (TG) (7, 8), channels that mediate this type of CCE
have been referred to as store-operated channels. In most cells,
Ca2+ entry after stimulation by agonists can be explained
by activation of store-operated channels, but neither the mechanism of
how the store depletion signal is transmitted to the plasma membrane
nor the molecular nature of the plasma membrane channels mediating Ca2+ has been clearly identified. Moreover, although
store-operated plasma membrane Ca2+-permeant channel
activities have been found in many cell types (9-13), channels
regulated by other mechanisms, such as a G protein, IP3,
inositol 1,3,4,5-tetrakisphosphate (IP4), Ca2+,
or ATP, seem to coexist with store-operated channels (14-17). The
molecular basis of this diversity and the relation of store depletion-insensitive Ca2+-permeant channels to store
depletion-sensitive Ca2+-permeant channels are unclear.
However, it is likely that multiple Ca2+ influx channels
are involved in Ca2+ entry initiated by the activation of
PLC-linked receptors.
In Drosophila eyes, the light-induced current is carried by
channels formed from at least two related photoreceptor-specific proteins, Trp and Trpl (Trp-like). Trp was identified by molecular cloning as a protein missing from the transient receptor potential (trp) mutant (18). Trpl was identified biochemically as a
calmodulin-binding protein and was cloned by standard techniques
revealing a structure that shares a high degree of homology with Trp
(19). Because the phototransduction pathway in insects resembles the
PLC/IP3 signaling pathway in mammalian cells, it was
speculated that mammalian homologs of Trp may exist and form
Ca2+ influx channels. In support of this, expression of
Trpl in Sf9 cells led to development of an
agonist-stimulated non-selective Ca2+-permeable ion
conductance, and that of Trp led to the development of a TG-stimulated
ion conductance (20-22). However, neither Trp-formed channels nor
Trpl-formed channels displayed the ion permeation properties of
endogenous insect CCE channels and, while forming channels, Trpl was
not activated by TG treatment. More recently, work from the
laboratories of Minke (23) and Montell (24) showed that Trp and Trpl
can form heteromultimeric ion channels with properties that differ from
those of their parental molecules, suggesting that native
voltage-independent Ca2+ influx channels in insects are
likely to be formed of more than one type of subunit, i.e.
to be heteromultimers.
Several mammalian sequences homologous to the Drosophila
Trps2 have been identified
from the data base of expressed sequence tags and by reverse
transcriptase-polymerase chain reaction using degenerate
oligonucleotide primers based on the Drosophila sequences (25-30). In the mouse we found the existence of at least six
non-allelic Trp genes that can be divided into four major
types based on primary amino acid sequence similarity (26, 31).
Full-length cDNAs for Trp1, 3, 4, and 6 from human, murine, rat and
bovine sources have been reported (25-27, 29-32). Functional
expression of human Trp1, Trp3, bovine Trp4, or mouse Trp6 in COS,
Chinese hamster ovary, or human embryonic kidney (HEK)293 cells results
in enhancement of either agonist-stimulated Ca2+ entry or
of IP3- or TG-stimulated inward currents that are at least
in part carried by Ca2+ (26, 29, 31, 32). More importantly,
agonist-stimulated Ca2+ influx in murine L cells was
blocked by transfection of murine Trp cDNA fragments in
the antisense direction (26, 31), showing that mammalian Trp proteins
are involved in agonist-stimulated Ca2+ influx.
For the present work we developed stable HEK293 cell lines expressing
human Trp3 (hTrp3) and studied their Ca2+ influx
properties. We report that the expression of hTrp3 caused an increase
of [Ca2+]i under basal and
agonist-stimulated conditions. The influx pathway formed by hTrp3
permeates Ca2+, Sr2+, and Ba2+
equally well, whereas the pathways intrinsic to HEK293 cells seem to be
more Ca2+ selective. The influx due to hTrp3 can be blocked
by SKF 96365, a known CCE blocker, or by verapamil, a nonspecific
blocker for L-type voltage-sensitive Ca2+ channels.
Although treatment with TG caused a small increase of Ca2+
influx over the basal in cell lines expressing hTrp3, a large portion
of the influx pathway due to hTrp3 is not sensitive to store depletion
and seems to depend primarily on the activation of PLC.
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EXPERIMENTAL PROCEDURES |
Materials--
SKF 96365 hydrochloric acid was purchased from
Calbiochem. TG, U73122, (±) verapamil were from Research Biochemicals
International. Carbachol, 3-amino-9-ethyl-carbozole tablets,
deoxycholic acid, phenylmethanesulfonyl fluoride, soybean trypsin
inhibitor, leupeptin, and Nonidet P-40 were purchased from Sigma.
35S-Express protein labeling mix (10 Ci/liter) and
N-[3H]methylscopolamine (82 Ci/mmol) were
purchased from NEN Life Science Products.
Peptide-N-glycosidase F and endoglycosidase H were from
Boehringer Mannheim. Protein A-Sepharose (4 Fast Flow) was from
Pharmacia Biotech. Fura2 acetoxymethyl ester (Fura2/AM) and Pluronic
F-127 were from Molecular Probes (Eugene, OR). Monoclonal antibody
12CA5 was from Babco (Berkeley, CA). All tissue culture reagents were
purchased from Life Technologies, Inc. unless indicated otherwise.
cDNAs and Expression Vectors--
The wild type hTrp3 was
subcloned into the mammalian expression vector pcDNA3 (26). The
insert, located in between the EcoRI and the XbaI
site of the polylinker of pcDNA3, contains nucleotide
15 (A of
the first ATG is nucleotide 1) to 3285 of hTrp3 cDNA which includes a poly(A) tail (n = 30) and is flanked
by a 36-nucleotide sequence from the Marathon cDNA adaptor
(CLONTECH) on each side. To introduce an epitope
for hemagglutinin (HA) at the C terminus of hTrp3, oligonucleotide A,
5
-CCCAGCATGCTGTACCCGTACGATGTTCCTGATTACGCGAGATGTGAATGATGCAGCA-3
, which contains a sequence (underlined) encoding the HA epitope (YPYDVPDYA) in between codons for Leu-845 and Arg-846 at the C terminus
of hTrp3 cDNA, was synthesized. A partial
hTrp3 sequence was amplified by polymerase chain reaction
using hTrp3 in pcDNA3 as a template and oligonucleotide
A and Sp6 as primers. The polymerase chain reaction product was
subcloned back into the original plasmid by ligation of an
EcoRI/SphI fragment generated from
hTrp3/pcDNA3, the SphI/XbaI
fragment of the polymerase chain reaction product, and the
EcoRI/XbaI fragment of pcDNA3. The presence
of the HA epitope sequence in the hTrp3-HA/pcDNA3
plasmid was confirmed by sequencing. The rat V1aR/pcDNA3
expression vector was a gift from Dr. Mariel Birnbaumer.
Cell Lines and Cell Culture Conditions--
HEK293 cells were
cultured in Dulbecco's modified Eagle's medium containing 4.5 mg/ml
glucose, 10% heat-inactivated fetal bovine serum, 50 units/ml
penicillin, and 50 mg/ml streptomycin. The
hTrp3-HA/pcDNA3 (100 ng) was transfected into the HEK
cells, plated at 2.8 × 106 cells/10-cm tissue culture
dish 20 h before transfection, by the calcium phosphate/glycerol
shock method (33). After 24 h, cells were harvested, diluted in
medium supplemented with 400 µg/ml G418, and transferred to wells of
96-well plates at different dilutions. G418-resistant transformants
were expanded into 12-well plates. To identify the clones expressing
hTrp3-HA, cells were seeded in 96-well plates precoated with
poly-D-lysine (100 µg/ml). Immunocytochemical staining
was performed as described by Vannier et al. (34) using a
monoclonal HA antibody (12CA5) as the primary antibody, anti-mouse IgG
conjugated with peroxidase (Amersham) as the secondary antibody, and
3-amino-9-ethyl-carbozole as the colorant. Positive cells were stained
red and were visualized through a light microscope. Eleven cell lines
expressing the HA epitope were further expanded and analyzed for the
expression of HA-tagged hTrp3 by immunoprecipitation with 12CA5 as
described below. All cell lines synthesized an immunoprecipitated
protein of approximately 100 kDa when analyzed by SDS-polyacrylamide
gel electrophoresis, which corresponds to the predicted size of hTrp3. Two cell lines, HEKTrp3-9 (T3-9) and HEKTrp3-65 (T3-65) were used for
further analysis. Control cells were transfected with
V1aR/pcDNA3 under the same conditions, two
G418-resistant clones were randomly selected and designated as
control-1 (C1) and control-2 (C2). The stable cell lines were diluted
twice weekly and maintained in medium supplemented with 400 µg/ml
G418.
Immunoprecipitation--
Cells (2 × 106) were
plated in 6-cm tissue culture dishes at least 16 h before the
experiments. Cells were washed once with Hanks' balanced salt solution
and then incubated in 1 ml of methionine/cysteine-free Dulbecco's
modified Eagle's medium (ICN) containing 5% fetal bovine serum at
37 °C for 1 h. The medium was replaced by the same medium containing 50 µCi/ml 35S-express protein labeling mix
containing [35S]Met and [35S]Cys for a
desired time. When needed, the incorporated radioactivity was chased by
replacing the labeling medium with 4 ml of regular Dulbecco's modified
Eagle's medium and continuing incubation at 37 °C for the desired
time period. At the end of the incubation, cells were rinsed twice with
Dulbecco's phosphate-buffered saline solution without Ca2+
and Mg2+, scraped off from the culture dish in 1 ml of
ice-cold Dulbecco's phosphate-buffered saline containing 1 mM EDTA and 0.1 mM phenylmethanesulfonyl fluoride, and pelleted by centrifugation at 5,000 rpm in a
microcentrifuge at 4 °C for 5 min. Cells were then homogenized in
0.5 ml of RIPA buffer (150 mM NaCl, 5 mM EDTA,
1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM
Tris-HCl, pH 8.0) containing 0.1 mM phenylmethanesulfonyl fluoride, 1 µg/ml soybean trypsin inhibitor, and 0.5 µg/ml
leupeptin. Immunoprecipitation was performed using the anti-HA
monoclonal antibody (12CA5) as described by Innamorati et
al. (35). For glycosidase treatment, cell lysate was incubated
with peptide-N-glycosidase F (2 units/ml) or endoglycosidase
H (10 milliunits/ml) at room temperature for 1 h before antibody
(12CA5) was added. The immunoprecipitated proteins were eluted in 80 µl of 2 × Laemmli buffer (1 × = 62.5 mM
Tris-HCl, 1% SDS, 10% glycerol, 10%
-mercaptoethanol, pH 6.8) and
separated by SDS-polyacrylamide gel electrophoresis in 9% acrylamide
gels at 40 mA for 2 h. The gels were stained with Coomassie Blue
to visualize the molecular markers, destained, and then dried for
autoradiography.
Measurement of [Ca2+]i--
Changes of
[Ca2+]i in individual cells were
monitored after loading cells with Fura2 by fluorescence videoimaging
microscopy using an Attofluor Digital Imaging and Photometry attachment
of a Carl Zeiss Axiovert inverted microscope as described before (26).
Changes of [Ca2+]i in cell
populations were measured using an Aminco-Bowman Series 2 luminescence
spectrofluorometer (SLM Instruments, Inc.). Briefly, cells grown to
confluence in 15-cm dishes were trypsinized and collected in a 50-ml
capped polypropylene tube. After a centrifugation for 5 min at 400 × g and removal of the supernatant, the cell pellets were
resuspended at room temperature in an extracellular solution (ECS)
composed of 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2,
10 mM glucose, 0.1% bovine serum albumin, 15 mM Hepes, pH 7.4. Cells were incubated in ECS supplemented
with 5 µM Fura2/AM and 0.05% Pluronic F-127 at 37 °C
for 40 min. Cells were then washed once and resuspended in ECS at
2 × 106 cells/ml. Aliquots of 2 ml were kept in the
dark at room temperature until use. Before each measurement, cells were
washed twice in ECS or twice in ECS to which no CaCl2 was
added if a Ca2+-free/Ca2+ readdition protocol
was used. 2 ml of cell suspension was transferred to a quartz cuvette
and maintained at 32 °C under continuous stirring. Changes in
intracellular Fura2 fluorescence intensity were measured by alternating
excitation at 340 and 380 nm at 3-s intervals and detecting emission at
510 nm. Autofluorescence of the cells at 340 and 380 nm was determined
from unloaded cells of an equivalent cell density and subtracted from
values obtained for the Fura2-loaded cells. Drugs were added by a small
aliquot (<20 µl) of concentrated stocks (dissolved either in water
or dimethyl sulfoxyide) to achieve the final concentrations. At the end
of each recording, 0.1% Triton X-100 was added to the cells to
determine the maximal fluorescence ratio (Rmax).
This was followed by an addition of 20 mM EGTA to determine
the minimal fluorescence ratio (Rmin).
[Ca2+]i was calculated according
to Grynkiewicz et al. (36).
In most experiments, Ca2+ entry under the basal or
stimulated conditions was measured by a
Ca2+-free/Ca2+ readdition protocol, in which
cells were incubated in a nominally Ca2+-free ECS
stimulated or not by the addition of an agonist or a store depletion
drug. The stimulation normally caused a transient [Ca2+]i increase due to the
release of Ca2+ from intracellular Ca2+ stores.
After allowing the first [Ca2+]i
peak to decrease to a steady-state level (normally 3-7 min),
CaCl2 was added to give a final concentration of 1.8 mM. Typically, this led to a second
[Ca2+]i increase. This
extracellular Ca2+-dependent
[Ca2+]i increase is assumed to be
caused by Ca2+ influx.
Measurements of Influxes for Sr2+, Ba2+,
and Mn2+--
A Ca2+ free/cation addition
protocol, analogous to the Ca2+-free/Ca2+
readdition protocol in which Sr2+ or Ba2+ was
added during the cation readdition phase, was used to study the entry
of Sr2+ and Ba2+ into cells. The extent of
influx of these divalent cations is expressed in the figures as changes
in the ratio of 340 to 380 nm fluorescence without the estimation of
their intracellular concentrations.
In a similar manner, MnCl2 was added to a final
concentration of 50 µM to cells incubated in a nominally
Ca2+-free ECS under either unstimulated or stimulated
conditions. Fluorescence quenching was studied using the Fura2
isosbestic excitation wavelength at 360 nm and recording emitted
fluorescence at 510 nm. At the end of each recording, 0.1% Triton
X-100 was added to cells to determine the maximal quenching of Fura2 by Mn2+. Results of fluorescence quenching are expressed (in
percent) as the amount of fluorescence decreased from the initial value (before the addition of Mn2+) divided by the maximal loss
after the addition of Triton X-100.
 |
RESULTS |
Activity of HA Epitope-tagged hTrp3--
We showed previously (26)
that transient expression of hTrp3 in COS-M6 cells results in a 2-fold
increase of Ca2+ influx in response to the activation of a
coexpressed M5 muscarinic receptor. To study the function and
biochemical properties of hTrp3 in more detail, we decided to develop
stable cell lines expressing hTrp3 at high levels. To aid the
identification of cell lines expressing hTrp3, we added an HA epitope
at the C terminus of hTrp3 (hTrp3-HA). The resulting cDNA construct
was first transfected to COS-M6 cells, and its function on
Ca2+ influx was compared with the wild type
hTrp3, which was transfected in parallel in the same
experiment. Ca2+ influx induced by 20 µM
carbachol (CCh) was identical in cells transfected with the wild type
hTrp3 or hTrp3-HA (Fig.
1). We transfected
hTrp3-HA/pcDNA3 into HEK293 cells and selected 11 cell
lines that expressed the HA-tagged hTrp3. Two cell lines, HEKTrp3-9
(T3-9) and HEKTrp3-65 (T3-65), expressed relatively high levels of
hTrp3 and were used in studies presented in this report. Two control
cell lines (C1 and C2) were obtained by transfecting cDNA for the
rat V1aR into the HEK293 cells. Thus the hTrp3 cells and the control
cells underwent the same type of treatment and selection processes and
were kept under the same culturing conditions.

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Fig. 1.
Introduction of an HA epitope at the C
terminus of hTrp3 does not alter its ability to enhance
Ca2+ influx in response to stimulation of a
Gq-coupled receptor. COS-M6 cells were transiently
cotransfected with cDNAs encoding wild type hTrp3 (a),
hTrp3-HA (b), or murine luteinizing hormone receptor
(c, control) plus the rat M5 muscarinic receptor as
described previously (26). Changes of
[Ca2+]i in individual cells loaded
with Fura2 were measured by videoimaging microscopy 40 h
post-transfection as described in Zhu et al. (26). CCh at 20 µM was added at 1 min and was present throughout the
experiment as indicated by the horizontal bar. A solution
containing 0.5 mM EGTA was replaced by a solution containing 1.8 mM CaCl2 at 4 min. Data are
averages of [Ca2+]i pooled from
cells that responded to CCh from six experiments. The numbers of the
cells are 93 (a), 121 (b), and 154 (c).
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Glycosylation of hTrp3--
Immunoprecipitation of HA-tagged hTrp3
from metabolically labeled T3-9 and T3-65 cells showed multiple bands
at around 100 kDa (bands 1, 2, 3, 4) and a haze of radioactivity above
these bands (Fig. 2A).
Treatment with endoglycosidase H selectively eliminated only one of the
middle bands (band 2) and increased the intensity of the lower band
(band 1), suggesting that band 2 is a high mannose-containing immature
form of the glycosylated hTrp3. Treatment with
peptide-N-glycosidase F removed bands 2-4 and the haze
while greatly enhancing the intensity of band 1. Thus, bands 2, 3, 4 and the haze of radioactivity seen on the SDS-polyacrylamide gel
electrophoresis represent hTrp3 at different stages of glycosylation
during remodeling of the protein-attached sugar moieties, whereas band
1 represents the non-glycosylated hTrp3. In COS cells transiently
transfected with hTrp3-HA, the predominant protein was the
high mannose-containing immature glycosylated form (band 2), as shown
by its sensitivity to the endoglycosidase H treatment (see Fig. 7 in
Ref. 31). This is not surprising since transiently transfected COS
cells are known to synthesize a large quantity of the exogenous
proteins of which only a fraction is processed to mature cell surface
form (37).

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Fig. 2.
Immunoprecipitation of hTrp3-HA from two
stable HEK293 cell lines (T3-9 and T3-65) by an anti-HA monoclonal
antibody, 12CA5. Cells were metabolically labeled with
[35S]Met/Cys for 2 h or as indicated.
Immunoprecipitation was carried out as described under "Experimental
Procedures." Panel A, glycosylation analysis of hTrp3.
Cell lysate was either not treated (N) or treated with
endoglycosidase H (H) or peptide N-glycosidase F
(F) for 60 min at room temperature before the addition of
anti-HA antibody. Panel B, time course of
[35S]Met/Cys incorporation into different forms of hTrp3
in T3-9 cells. Panel C, a pulse-chase experiment showing the
maturation process of hTrp3 in T3-9 cells.
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The maturation process of hTrp3 can be followed in a pulse-chase
experiment, as shown in Fig. 2B. After 15 min of labeling with a mixture of [35S]Met and [35S]Cys,
the high mannose glycosylated hTrp3 was the major form. At 30 min, the
more mature forms of glycosylated hTrp3 started to appear. At 60 and
120 min, the intensities of all of the bands increased proportionally
compared with that at 30 min. The haze of radioactivity became more
evident. When cells were labeled for 15 min and then the labeled
35S was chased with culture medium containing unlabeled
amino acids, the intensities of both the non-glycosylated and the high
mannose-containing form of hTrp3 decreased as the chase time increased
(Fig. 2C). Bands 3 and 4 and the haze remained after 3 h of chase. The proportions of bands 3 and 4 and the haze remained
constant for up to 8 h (not shown).
Basal and Stimulated Ca2+ Influx in hTrp3-expressing
HEK Cells--
Ca2+ influx activities in the T3-9 and
T3-65 cells under basal or stimulated conditions were compared with
that of control cells by studying extracellular
Ca2+-dependent
[Ca2+]i changes in Fura2-loaded
cells suspended at a density of 2 × 106 cells/ml. For
the most part, we show results obtained from T3-9 and C1 cells. Similar
results were obtained with T3-65 cells. C1 and C2 cells did not differ
in their way of handling [Ca2+]i.
Fig. 3 shows traces of
[Ca2+]i changes during the course
of typical experiments in which Trp3 (upper traces) or
control (lower traces) cells were either not stimulated
(panel A) or stimulated with 200 µM CCh (panel B) or 0.2 µM TG (panel C).
Ca2+ influx was studied using the
Ca2+-free/Ca2+ readdition protocol described
under "Experimental Procedures." CCh was used to activate the PLC
pathway via an endogenous muscarinic receptor found in the HEK293
cells. Fig. 4A shows the
abundance of the muscarinic receptor sites in each cell line determined according to Liao et al. (38). Similar results were obtained by stimulating cells with 100 µM ATP, which activates an
endogenous purinergic receptor. To study Ca2+ influx
induced by store depletion, we used 0.2 µM TG to block the endoplasmic reticulum Ca2+-ATPase, which passively
depletes the internal store without increasing the production of
IP3 (4). Fig. 4 summarizes results obtained for two Trp3
cells (T3-9 and T3-65) and two control cells (C1 and C2).

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Fig. 3.
Calcium influx in stable HEK293 cell line
expressing hTrp3 (T3-9) or rat V1aR (control, C1).
Cells grown to confluence were trypsinized, loaded with Fura2, and
maintained in suspension. Changes in
[Ca2+]i were measured in cell
population at a density of 2 × 106 cells/ml using a
SLM-Aminco-Bowman spectrofluorometer. Cells were incubated in a
nominally Ca2+-free solution either untreated (panel
A) or treated with 200 µM CCh (panel B)
or 0.2 µM TG (panel C) as indicated. The
activities of Ca2+ influx were studied by readdition of 1.8 mM Ca2+ to the solution.
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Fig. 4.
Panel A, abundance of endogenous
muscarinic receptor (mAchR) in control and Trp3 HEK293
cells. The number of the receptors/cell for each cell line was
determined by Scatchard analysis of the binding of
N-[3H]methylscopolamine
([3H]NMS) in increasing concentrations to
intact cells as described in Liao et al. (38). Data are
averages ± range of results obtained from two experiments.
Panel B, basal levels of
[Ca2+]i in control and Trp3 cells
maintained in a physiological ECS containing either no Ca2+
(open bars) or 1.8 mM Ca2+
(filled bars). Cell lines were developed, and
[Ca2+]i in cell populations was
measured in Fura2-loaded cells as described under "Experimental
Procedures." For [Ca2+]i in
Ca2+-free solutions, cells were washed twice and then
resuspended in a nominally Ca2+-free ECS. For
[Ca2+]i in 1.8 mM
Ca2+, cells were washed twice and then resuspended in the
same ECS except that 1.8 mM Ca2+ was included.
Data are averages ± S.E. of results from the number of
experiments indicated in parentheses. Panel C,
Ca2+ readdition elicited
[Ca2+]i increases in control and
Trp3 cells at basal or stimulated by agonist or TG. Cells were
incubated in a nominally Ca2+-free solution unstimulated
(open bars) or stimulated with 200 µM CCh
(filled bars) or 0.2 µM TG (shaded
bars). Then Ca2+ was added to the ECS to a final
concentration of 1.8 mM. Data are maximal
[Ca2+]i reached within 1 min of
Ca2+ addition minus the
[Ca2+]i before the
Ca2+ addition from each experiment; averages ± S.E.
are from the number of experiments indicated in parentheses.
Filled star, because of relatively low level of
Ca2+ mobilization in T3-65 and C2 by CCh alone (not shown),
ATP (100 µM) was included to release the internal
Ca2+ maximally before readdition of extracellular
Ca2+.
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Although in a nominally Ca2+-free buffer, basal
[Ca2+]i in Trp3 cells is not
significantly different from that of control cells; when the cells are
maintained in a normal physiological solution containing 1.8 mM Ca2+, the resting
[Ca2+]i in Trp3 cells is 40-70%
higher than that in control cells (Fig. 4B). Addition of 1.8 mM Ca2+ to the extracellular medium of cells
incubated in the Ca2+-free solution causes a 75-130
nM increase of [Ca2+]i
in the Trp3 cells (Figs. 3A and 4C). In control
cells, the increase was less than 20 nM, of which a
fraction could represent titration of Fura2 leakage or a small
proportion of leaking cells in the whole population. Therefore, under
non-stimulated conditions, control HEK293 cells exhibit very low
Ca2+ influx, whereas Ca2+ influx channels in
the Trp3 cells appear to have a higher spontaneous activity.
In control cells maintained in a nominally Ca2+-free
solution, CCh caused a rapid and transient increase of
[Ca2+]i because of the release
from internal Ca2+ stores. Readdition of 1.8 mM
Ca2+ to the medium causes
[Ca2+]i to increase about 120 nM. In TG-treated cells, readdition of Ca2+
causes a larger increase in
[Ca2+]i (up to 240 nM). In the Trp3 cells, the
[Ca2+]i following Ca2+
readdition after CCh or TG treatment is higher than under basal conditions, increasing by about 230 and 270 nM for CCh and
TG, respectively (Fig. 4C).
A low cytosolic Ca2+ level is maintained by
Ca2+ pumps located on plasma membrane and endoplasmic
reticulum, as well as other Ca2+-buffering systems inside
cells, which actively extrude Ca2+ out of the cell or into
its intracellular stores. Opening of the Ca2+ influx
channels results in a rapid rise of
[Ca2+]i, which in turn causes an
increase in extrusion of Ca2+ mediated by the
Ca2+ pumps. Therefore, at any given time,
[Ca2+]i is a result of the dynamic
interplay of Ca2+ pumps, intracellular Ca2+
channels (e.g. IP3 receptors), and plasma
membrane Ca2+ influx channels. Upon readdition of
Ca2+ to the extracellular medium, three influx activities
could contribute positively to the increase of [Ca2+
]i in CCh- or TG-stimulated Trp3 cells. The
first is the basal activity mediated by Trp3. The second is influx
intrinsic to the HEK cells stimulated by CCh or TG. The third is a
Trp3-mediated influx stimulated by the same drugs. Because a
significant fraction of Ca2+ entering from external space
is removed by the Ca2+ pumps, the net increase of
[Ca2+]i will not be the sum of
influx arising from the three activities. Therefore, from the
experiments shown in Figs. 3 and 4, it is difficult to conclude whether
stimulation by CCh or TG increases a Trp3-mediated Ca2+
influx pathway or if the stimulated increase in
[Ca2+]i observed in the Trp3 cells
results merely from the opening of Ca2+ influx channels
endogenous to the HEK cells. More difficult is to see whether store
depletion causes more Ca2+ influx in Trp3 cells than in
control cells because the increase in
[Ca2+]i in the two Trp3 cell lines
is not significantly higher than that in C2 cells (Fig. 4C).
To test whether any stimulated Ca2+ influx is caused by the
expression of hTrp3, we selectively blocked the endogenous
Ca2+ influx with 10 µM Gd3+. As
shown in Fig. 5, A,
B, and E), in the presence of 10 µM
Gd3+, the endogenous Ca2+ influx activated by
CCh and TG decreased >85% in control cells. In contrast, addition of
10 µM Gd3+ to the Trp3 cells reduced <10%
of the basal Ca2+ influx (Fig. 5E), about 15%
of Ca2+ influx stimulated by CCh (Fig. 5, C and
E), and 55-60% stimulated by TG (Fig. 5, D and
E). Assuming that the Trp3-mediated influx pathway is
insensitive to 10 µM Gd3+, we conclude that
for Trp3 cells treated with CCh, a significant proportion of
Ca2+ influx is mediated by Trp3, whereas in cells treated
with TG, most of the Ca2+ influx is carried by the
endogenous CCE channels.

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Fig. 5.
Effect of 10 µM
Gd3+ on Ca2+ influx in control (panels
A and B) and Trp3 cells (panels C and
D). Cells were treated with either 200 µM CCh (panels A and C) or 0.2 µM TG (panels B and D) in a
nominally Ca2+-free solution with or without 10 µM GdCl3 added 2 min after the application of
the drugs. CaCl2 was added as indicated to a final concentration of 1.8 mM. Panel E, summary of
Gd3+ effect on basal and stimulated Ca2+ influx
in Trp3 and control cells. Cells were incubated in a nominally Ca2+-free solution unstimulated (open bars) or
stimulated with 200 µM CCh (filled bars) or
0.2 µM TG (shaded bars). GdCl3 was
added to a final concentration of 10 µM to cells 2 min
before the addition of 1.8 mM Ca2+ to the
external solution. Data are maximal
[Ca2+]i reached within 1 min of
Ca2+ addition minus the
[Ca2+]i before Ca2+
addition from each experiment, averages ± S.E. from the number of
experiments indicated in parentheses. Filled
star, for T3-65 and C2 cells, ATP (100 µM) was
included in CCh to maximally release the internal Ca2+
before readdition of extracellular Ca2+.
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Although in the presence of 10 µM Gd3+, the
maximal increase in [Ca2+]i in
Trp3 cells in response to the addition of 1.8 mM Ca2+ under basal and TG-stimulated conditions is very
similar, the rate of [Ca2+]i
increase in TG-treated cells appears to be faster than in the
non-stimulated cells (Fig.
6A). In fact, in both cell lines expressing hTrp3, maximal
[Ca2+]i was reached within 40 s after the addition of external Ca2+, and then
[Ca2+]i gradually decreased. At 1 min after the addition of Ca2+ the increase in
[Ca2+]i in TG-stimulated cells was
equal to that in non-stimulated cells. Then
[Ca2+]i in the TG-stimulated cells
continued to drop, whereas that in non-stimulated cells stayed
constant. Fig. 6B shows TG-stimulated Ca2+
influx resistant to 10 µM Gd3+ in control and
Trp3 cells. Date are from subtraction of
[Ca2+]i changes of TG-stimulated
cells from that of non-stimulated cells. Control cells have very low
CCE activity when 10 µM Gd3+ is present,
whereas Trp3 cells have a small and transient CCE activity that lasts
for less than 1 min. Therefore, although very small, there is an
increase of store-operated Ca2+ influx due to expression of
hTrp3 in the HEK cells. Furthermore, TG also appears to block the basal
Ca2+ influx seen in Trp3 cells. This could be due to the
closing of the constitutively active hTrp3 channel or the enhancement
of intracellular Ca2+ removal. Neither function has been
described for TG.

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Fig. 6.
Gd3+-resistant CCE in Trp3 cells.
Panel A, comparison of Ca2+ influx under basal
(open symbols) and TG-stimulated conditions (filled
symbols) in Trp3 (T3-9 at upper left and T3-65 at
upper right) and control cells (C1 at lower left
and C2 at lower right). Cells were incubated in a nominally
Ca2+-free solution unstimulated or stimulated with 0.2 µM TG. GdCl3 was added to a final
concentration of 10 µM to cells 2 min before the addition
of 1.8 mM Ca2+ to the external solution. Data
are from the same experiments shown in Fig. 5E and
averages ± S.E. of [Ca2+]i
obtained at 3-s intervals minus the
[Ca2+]i before Ca2+
addition. Panel B, Gd3+-resistant CCE for Trp3
(circles) and control (squares) was obtained by
subtracting the averages of
[Ca2+]i increase in TG-stimulated
cells (filled symbols in panel A) from that in
unstimulated cells (open symbols in panel A).
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Divalent Cation Selectivity of Influx Channels in Control and the
Trp3 HEK Cells--
We compared the influx of Ba2+ and
Sr2+ through the Trp3-mediated pathway in Trp3 cells and
the endogenous pathway in control cells. Increasing concentrations of
both Sr2+ and Ba2+ have been shown to produce a
shift in Fura2 excitation wavelength spectrum similar to that produced
by Ca2+, but with somewhat higher dissociation constants
(39, 40). Therefore, increases in the fluorescence ratio of Fura2
obtained at excitation wavelengths of 340 and 380 nm reflect increases of intracellular concentrations of Ba2+ or
Sr2+. Control or Trp3 cells were incubated in nominally
Ca2+-free solutions either unstimulated or stimulated by
CCh before 1.8 mM Sr2+ or Ba2+ was
added into the ECS substituting for Ca2+. Fig.
7, A-D, shows that the rates
of Ba2+ and Sr2+ influx into Trp3 cells are
much higher than into control cells. Because Ba2+ is a poor
substrate for Ca2+ pumps responsible for extrusion from the
cytosol (39), a declining phase of intracellular [Ba2+]
is normally not seen, as is the case with Ca2+ and
Sr2+ (Fig. 7, C and D). In
CCh-stimulated control cells, the initial rate of fluorescence ratio
increase with Ca2+ (2.0 units/min) is at least two times
faster than the rates with Sr2+ (0.6 unit/min) and
Ba2+ (0.6 unit/min). In contrast, the rates of fluorescence
ratio increase in Trp3 cells are very similar regardless of which
divalent cation was added (5.5 for Ca2+, 6.8 for
Sr2+, and 6.2 for Ba2+, in units/min).
Therefore, in HEK293 cells, the endogenous influx pathway activated by
CCh seems to be more selective for Ca2+ than
Ba2+ and Sr2+, whereas the Trp3-mediated influx
is not selective for these cations.

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Fig. 7.
Influx of Sr2+, Ba2+,
and Mn2+ into Trp3 (T3-9) and the control (C1)
cells under basal (left) or stimulated (right)
conditions. Left, at the time point indicated,
Sr2+, Ba2+, or Mn2+ was added to
cells incubated in a nominally Ca2+-free solution to a
final concentration of 1.8 mM, 1.8 mM, or 50 µM, respectively. Right, 200 µM
CCh was added to cells incubated in a nominally Ca2+-free
solution 3 min before the addition of the divalent cations.
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Mn2+ can enter cells through certain types of
Ca2+ influx channels and quenches Fura2 fluorescence (41,
42). The addition of 50 µM MnCl2 to cells
suspended in a nominally Ca2+-free solution causes a slow
but constant decrease of fluorescence, indicative of Mn2+
entry into the cells under basal conditions (Fig. 7E). The
rate of Mn2+ entry into Trp3 cells was faster than into
control cells. In CCh-stimulated cells, Mn2+ entry was
increased in both the control and the Trp3 cells (Fig. 7F).
Effect of Ca2+ Channel Blockers--
Although not
specific, SKF 96365 has been shown to block agonist-stimulated
Ca2+ influx in many cells (43). Fig.
8A shows that at 25 µM, SKF 96365 inhibits completely the basal
Ca2+ influx of Trp3 cells. In CCh-treated cells, the drug
inhibits Ca2+ influx in both Trp3 and control cells.
However, SKF 96365 seems to inhibit the Trp3-mediated influx more
effectively than the endogenous influx. The residual Ca2+
influx left in the Trp3 cells is comparable to that seen in control cells. The IC50 for SKF 96365 was about 5 µM
as estimated from measuring the CCh-stimulated Ba2+ influx
in the Trp3 cells (Fig. 8B). Verapamil, a blocker of L-type voltage-gated Ca2+ channels, not known to affect CCE or
agonist-stimulated Ca2+ entry in general, also prevents
divalent cation entry via the Trp3-mediated pathway with an
IC50 of about 4 µM (Fig. 8C). Both the basal and the stimulated cation influx in Trp3 cells can be blocked
by high concentrations of Ni2+ (6 mM),
La3+ (150 µM), and Gd3+ (200 µM).

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Fig. 8.
Ca2+ influx mediated by hTrp3 is
blocked by SKF 96365 and verapamil. Panel A, basal influx of
Ca2+ in the Trp3 cells (T3-9) is blocked completely by 25 µM SKF 96365, added 3 min before reintroduction of 1.8 mM Ca2+. Panel B, effect of 25 µM SKF 96365 on CCh-stimulated Ca2+ influx in
the Trp3 cells. SKF was added 1 min before the reintroduction of 1.8 mM Ca2+. Panel C, effect of 25 µM SKF 96365 on CCh-stimulated Ca2+ influx in
control cells (C1). The experiment was performed as in
panel B. Panel D, dose-response effect of SKF
96365 on Ba2+ influx stimulated by CCh in the Trp3 cells.
Cells were treated with 200 µM CCh in a nominally
Ca2+-free solution for 5 min. SKF 96365 was added to final
concentrations as indicated 1 min before the addition of 1.8 mM BaCl2. Panel E, dose-response
effect of ± verapamil on Ba2+ influx stimulated by
CCh in the Trp3 cells. The experiment was performed as in panel
D.
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PLC-dependent and Store Depletion-insensitive Cation
Influx in Trp3 Cells--
To explore further the activation mechanism
of Trp3-mediated Ca2+ influx, we tested whether depleting
internal stores with TG would prevent agonist-stimulated
[Ca2+]i increase. In control cells
maintained in a nominally Ca2+-free solution, the addition
of 200 µM CCh after incubating cells with 200 nM TG for 6 min caused no significant
[Ca2+]i increase (not shown). In
the Trp3 cells, a small rise in
[Ca2+]i (27 ± 6 nM net increase at the peak, n = 4) was
induced by CCh after cells had been incubated with TG for 6 min.
However, when the cells were treated with TG in a medium containing 1.8 mM Ca2+ and the endogenous influx pathway was
blocked by 10 µM Gd3+, the addition of CCh
caused a transient increase of
[Ca2+]i of 105 ± 8 nM (n = 11) in Trp3 cells (Fig.
9A). A similar increase of
[Ca2+]i was not seen in control
cells. In the experiment shown in Fig. 9B, the endogenous
Mn2+ influx stimulated by either CCh or TG was blocked by
10 µM Gd3+. Addition of CCh to Trp3 cells
pretreated with TG caused an increase in the rate of Mn2+
entry, which was not seen in control cells (right panel).
The rate of Mn2+ entry under these conditions (TG followed
by CCh) was not significantly different from the rate of entry observed
in Trp3 cells treated only with CCh (left panel). In similar
experiments, Ba2+ or Sr2+ was added to the
TG-treated cells. Stimulation by CCh in the presence of extracellular
Ba2+ or Sr2+ caused additional influx of these
cations in Trp3 cells (Fig. 9, C and D) but not
in control cells. These results suggest that in HEK cells expressing
hTrp3, not only does receptor stimulation activate the influx of
divalent cations better than store depletion, but signals from store
depletion do not prevent or occlude the Trp3-mediated influx activated
via receptor stimulation. Most of the influx activity that resulted
from overexpression of hTrp3 alone in the HEK293 cells is thus
activated by a mechanism other than store depletion.

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Fig. 9.
Influx of divalent cations through the
hTrp3-mediated pathway can be activated in cells previously depleted of
internal Ca2+ stores. Fura2-loaded HEK293 cells
expressing hTrp3 (T3-9) or control cells (C1) were treated with 0.2 µM TG either in a solution containing 1.8 mM
Ca2+ (panel A) or in a nominally
Ca2+-free solution (panels B-D). Panel
A, in the presence of 1.8 mM extracellular
Ca2+, the endogenous Ca2+ influx was blocked by
the addition of 10 µM Gd3+. Stimulation by
200 µM CCh caused a transient increase of
[Ca2+]i only in the Trp3 cells.
Panel B, Mn2+ entry in the presence of 10 µM Gd3+ recorded by Fura2 fluorescence
quenching using the Fura2 isosbestic excitation wavelength at 360 nm
and emission at 510 nm. Panel B, left, 50 µM MnCl2 was added as indicated 3 min after
the cells had been incubated with 200 µM CCh in a
nominally Ca2+-free solution. Under these conditions, CCh
only increased the rate of Mn2+ entry over the basal (not
shown) rate in the Trp3 cells but not the control cells. Panel
B, right, 50 µM MnCl2 was
added as indicated 3 min after cells had been incubated with 0.2 µM TG. 200 µM CCh was added 4 min later as
indicated. An increase of Mn2+ entry was seen in the Trp3
cells. In panels C and D, a similar protocol was
used. Ba2+ (panel C) or Sr2+
(panel D) was added to TG-treated cells to a final
concentration of 1.8 mM. CCh (200 µM) was
added 4 min later. The increase of Fura2 fluorescence ratio in response
to CCh seen in Trp3 cells was caused by the entry of Ba2+
or Sr2+.
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The signaling cascade initiated by an agonist includes the activation
of its receptor, of a G protein, of PLC, and thus the production of
IP3 and the release of Ca2+ from its internal
stores. Although store depletion does not appear to be the cause for
agonist-activated Ca2+ influx through the Trp3-mediated
pathway, activation of PLC seems to be necessary. When 15 µM U73122, an inhibitor of PLC (44), was used, not only
Ca2+ mobilization (not shown) but also Ca2+ and
Ba2+ influx stimulated by CCh was blocked (Fig.
10). Basal Ca2+ influx in
the Trp3 cells was little affected by the treatment of U73122.
Therefore, either the production of IP3 or the active form
of the PLC is required for the activation of the agonist-stimulated Trp3-mediated influx pathway.

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Fig. 10.
CCh-activated influx of Ca2+
(upper panel) and Ba2+ (lower
panel) via hTrp3 is prevented by a phospholipase C inhibitor,
U73122. HEKTrp3-9 cells were used. U73122 was added to the ECS to
a final concentration of 15 µM 3 min before the addition
of 0.2 µM TG. Experiments were performed as described in
Fig. 9, A and C. Note that the store
depletion-insensitive influx activated by CCh as seen in Fig. 9,
A and C, is blocked by the PLC inhibitor.
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DISCUSSION |
Calcium mobilization from internal stores and subsequent
Ca2+ entry from extracellular space are the two major
components of Ca2+ signaling following activation of PLC by
cell surface receptors. It has long been established that
Ca2+ is released from its internal stores via
IP3 receptors in response to the increase of
IP3 production (1). Only recently has evidence accumulated
showing that Ca2+ influx can occur via plasma membrane
channels formed of Trp homologs (26, 29, 32). The two
Drosophila photoreceptor Trp proteins have different
functional features. DTrp was found to form a
Ca2+ influx channel activated either by IP3 or
by store depletion with TG, when expressed in insect Sf9
cells (22), Xenopus oocytes (23, 28), or 293T cells (24). On
the other hand, DTrpl forms a non-selective cation channel
that is activated by stimulation of Gq-coupled receptors
but is not sensitive to store depletion (20, 21, 24). In addition,
DTrpl also displayed significant basal activity (20). By
comparing the amino acid sequences of the mammalian Trp proteins with
that of Drosophila Trp or Trpl, it is difficult to predict
whether any of the mammalian Trps would functionally resemble
DTrp or DTrpl. A recent report by Philipp et al. (29) showed that bovine Trp4 transiently expressed in HEK293 cells forms a channel that is relatively more selective for
Ca2+ than for monovalent cations and is activated to the
same extent by either IP3 or TG, suggesting that Trp4 may
functionally resemble the DTrp. Zitt et al. (32)
also reported that hTrp1 expressed in Chinese hamster ovary cells can
be activated by store depletion treatment, although the channel formed
in this case is non-selective. Thus, hTrp1 seems to resemble partly
both DTrp and DTrpl. Experiments to be reported
elsewhere3 indicate that the
stable expression of hTrp3 in the HEK293 cells gives rise to a novel
Ca2+-permeant cation influx current that is spontaneously
active, non-selective, and can be further stimulated by activation of Gq-coupled receptors. In the present experiments, the
majority of the increased Ca2+ influx due to hTrp3 is
neither activated by store depletion alone nor occluded by store
depletion with TG. Thus, hTrp3 functionally resembles
Drosophila Trpl.
Ca2+ influx mediated by channels insensitive to store
depletion have been shown to coexist with the store depletion-activated pathway in many systems. Orrenius and colleagues (42, 46, 47) reported
that in cells such as hepatocytes, T lymphocytes, adrenal glomerulosa
cells, fibroblasts, platelets, and anterior pituitary cells,
Ca2+ influx activated by emptying the agonist-sensitive
stores could not completely mimic the influx activated by receptor
agonists. More importantly, the store-insensitive cation influx in
hepatocytes was less selective for Ca2+ than influx
activated by TG. Clementi et al. (48) also reported that
stimulation of PC12 cells with carbachol activated two Ca2+
influx responses, of which one was store-dependent and the
other was directly dependent on receptor activation. Work by Montero et al. (49) showed that in differentiated HL60 cells,
N-formyl-leucyl-phenylalanine activated an additional
Ca2+ influx after the internal Ca2+ store had
been completely emptied by TG. In electrophysiological studies,
multiple types of Ca2+-conducting currents bearing distinct
characteristics are often observed by investigators using carefully
designed protocols. In mast cells, Ca2+ enters through both
a Ca2+ release-activated current (ICRAC) and a
50-picosiemen channel that is activated more directly by receptor/G
protein coupling (16, 50). In A431 epithelial cells, up to four
different types of Ca2+-permeant channels could be involved
in Ca2+ influx following the activation of G protein (shown
by the action of GTP
S) or perfusion of IP3 (14). In
addition, IP3 was found to activate plasma membrane
channels in B lymphocytes (51) and substance P receptor-transfected
Chinese hamster ovary cells (52). Moreover, IP3 may
activate the same channel that is sensitive to store depletion. In
inside-out patches of vascular endothelial cells, IP3 was
shown to modulate the activity of a Ca2+ influx channel
which is indistinguishable from that activated by treatment with
2
,5
-di(tert-butyl)1,4-benzohydroquinone, i.e. by store depletion (17). Thus, under physiological conditions, it is
likely that after receptor activation, Ca2+ enters through
multiple Ca2+-permeant channels. The relative contribution
of each influx pathway is likely to differ depending on the cell type
and probably the type and the degree of the stimulation. An estimate
has been made for rat mast cells in which, under physiological
conditions, the amount of Ca2+ conducted by
ICRAC is three times of that carried by the 50-picosiemen channel (16, 53).
The activation mechanism of agonist-stimulated Ca2+ influx
via hTrp3 is unclear. Controversy exists with respect to the mechanism of activation of the Drosophila Trpl expressed in the
Sf9 cells. Dong et al. (54) reported that
DTrpl is activated by intracellular perfusion with
IP3, whereas Obukhov et al. (55) found that in excised patches, DTrpl is activated by the active forms of
the
subunits of the G protein G11 and Gq,
but not by IP3. The fact that agonist-stimulated
store-insensitive Ca2+ influx via hTrp3 can be prevented by
an inhibitor of PLC suggests that the channel formed by hTrp3 is
activated by a factor downstream of PLC activation, and upstream of
store depletion. Since our experiments were carried out under
conditions in which store depletion occurred either before or upon the
addition of the agonist, we cannot rule out the possibility that
Ca2+ mobilization is also a necessary step for
agonist-induced Ca2+ influx through Trp3. If that is the
case, stimulation of hTrp3 would require both store depletion and the
generation of this other factor downstream of PLC activation. The
likely candidates for this factor are IP3 and its
derivatives, for instance IP4. According to a so-called
conformational coupling model (56), it is also possible that the
channel is activated through direct interaction between the channel and
the activated IP3 receptor. Less likely, although not
impossible, is that the channels could be stimulated by the activated
PLC itself. A more recent study by Zitt et al. (45) showed
that hTrp3 may be activated by Ca2+. However, treatment
with TG also induces an increase in
[Ca2+]i. This increase does not
seem to activate Ca2+ influx mediated by Trp3 as well as
that stimulated by receptor activation, suggesting that factors more
than just intracellular Ca2+ are involved in activating
Trp3. The 50-picosiemen channel found in mast cells is not activated by
IP3 but instead requires the activation of receptor/G
protein system (16). Thus, whether hTrp3 expressed in the HEK293 cells
forms a channel that resembles any of the Ca2+ influx
channels found in native tissues or isolated primary cells remains to
be elucidated.
Because of some homology between the last four putative transmembrane
segments of Trp and those of the subdomains of voltage-gated Ca2+ and Na+ channels, it has been proposed
that a Trp-based channel may be a tetramer (19). Based on our finding
that the mammalian genome contains at least six Trp genes,
we speculated that a channel assembled by Trps can be either
homotetrameric or heterotetrameric and that this could be a mechanism
to create functionally diverse Ca2+-permeant channels,
including store depletion-activated, store depletion-insensitive, and
channels activated by both store depletion and independently by
agonists (31). In the stable HEK293 cell lines, a homotetrameric hTrp3
may have become the predominant influx channel formed because of
overexpression of this protein. However, it may not represent any of
the native Ca2+ influx channels present in this or other
cell types. Because the cDNA for hTrp3 was isolated from HEK293
cells (26), we believe that there is endogenous hTrp3 protein in these
cells. However, we do not know whether channels formed by hTrp3 alone
are present in the native HEK293 cells because a component of
Ca2+ influx resembling that expressed in the Trp3 cell
lines is either missing or too small to be detected in the control
cells. One possibility is that the endogenous hTrp3 in the HEK293 cells
plays a very minor role in Ca2+ influx in response to
agonist stimulation. The other possibility is that hTrp3 and other
endogenous Trp proteins form heterotetrameric channels that are
responsible for agonist and store depletion-activated Ca2+
entry in these cells. Evidence for the formation of a heteromultimeric Trp-based Ca2+ influx channel was recently obtained by
Gillo et al. (23) who observed that coexpression of
Drosophila Trp and Trpl in Xenopus oocytes leads
to the appearance of a channel with an ion selectivity and
La3+ sensitivity different from those seen in oocytes
expressing either protein alone. Interestingly, the new channel is
activated to the same extent either by IP3 or by TG, even
though one of its components, DTrpl, is not sensitive to
store depletion. More recently, Montell and colleagues (24)
demonstrated that the N termini of DTrp and DTrpl
interact with each other both in vitro and in vivo. Coexpression of the two proteins in 293T cells gave rise to
a store depletion-sensitive cation influx channel that had features
from both DTrp and DTrpl. Interestingly, cells
expressing both DTrp and DTrpl did not have an
increased basal inward current as found in cells expressing
DTrpl alone. The authors proposed that DTrpl in
Drosophila photoreceptors does not form homomultimers by
itself, and its spontaneous activity is prevented by forming heteromultimeric channels with DTrp or other Trp-related
proteins. A similar conclusion may be drawn for hTrp3 since it behaves
very similarly to DTrpl when expressed alone. Because
Drosophila Trp proteins can form store-operated
heteromultimeric channels even if only one subunit is capable of
detecting the signal from store depletion, it is possible that the
exogenously expressed hTrp3 in the stably transfected cell lines also
forms heteromultimers in combination with the endogenous Trp proteins,
of which some can be activated by store depletion. This would explain
the small but significant Gd3+-resistant TG-stimulated
Ca2+ influx in Trp3 cells (Fig. 6C). The smaller
TG-stimulated increase of Ca2+ influx than the
CCh-stimulated increase found in transiently transfected COS cells (26)
can also be explained this way. On the other hand, the contribution of
hTrp3-containing heteromultimeric channels to TG-stimulated
Ca2+ influx could be more significant than that being
revealed by the protocol shown in Fig. 6 if these channels are more
sensitive to Gd3+ than the homomultimeric channel formed by
hTrp3. This is even more so when the new channels formed cannot be
distinguished from the native Ca2+ influx channels present
in the HEK293 cells by Ca2+ channel blockers. Therefore,
although our results show that overexpression of hTrp3 in HEK293 cells
gives rise to a cation entry pathway insensitive to regulation by a
store-operated manner, we cannot at this point rule out the possibility
that Trp3 is involved in forming native store-operated channels in
HEK293 and other cells. Other Trp-unrelated proteins may also
participate in the formation of the Ca2+ influx channels.
It is yet to be firmly established that Trps are the true or the only
subunits of store- or agonist-stimulated Ca2+ entry
channels, as this will require purification of the channel complexes
followed by reconstitution into either vesicles or planar lipid
bilayers and analysis of channel activity. Further, even if they were
channel subunits, other auxiliary proteins are expected to be required
for the formation the native channel(s).
In conclusion, Ca2+ influx following the activation of the
PLC/IP3 signaling pathway is an important phenomenon.
Channels with different properties have been described in various cell
types (for review, see Refs. 3 and 53). These include the
store-operated channels that differ both in conductance and ion
selectivity. These also include channels that do not appear to be
store-operated. The presence of six Trp homologs in mammals and the
possibility that they form heteromultimeric channels provide a
plausible explanation for the tissue- and cell-specific behavior of
Ca2+ influx pathways (and channels) found in different
systems.
 |
ACKNOWLEDGEMENTS |
We thank Drs. G. Innamorati, H. Sadeghi, B. Vannier, N. Qing, M. Birnbaumer, and E. Stefani for technical advice.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants GM54235 (to X. Z.) and HL45198 (to L. B.).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: Dept. of Pharmacology,
Ohio State University, 206 Rightmire Hall, 1060 Carmack Rd., Columbus,
OH 43210.
1
The abbreviations used are:
[Ca2+]i, intracellular
Ca2+ concentration; PLC, phospholipase C; IP3,
inositol 1,4,5-trisphosphate; CCE, capacitative Ca2+ entry;
TG, thapsigargin; IP4, inositol
1,3,4,5-tetrakisphosphate; HEK, human embryonic kidney; HA,
hemagglutinin; V1aR: vasopressin receptor type 1a; C1 and C2, control-1
and control-2 cells, respectively; ECS, extracellular solution; CCh,
carbachol; GTP
S, guanosine 5
-3-O-(thio)triphosphate.
2
The nomenclature of mammalian Trps is used
according to Zhu et al. (26). Thus, the human sequence that
appeared in Wes et al. (27) and Zitt et al. (32)
is hTrp1; the partial murine sequence published by Petersen et
al. (28) and the full-length bovine and rat sequences reported by
Philipp et al. (29) and Funayama et al. (30),
respectively, are Trp4.
3
R. S. Hurst, X. Zhu, G. Boulay, E. Stefani, and
L. Birnbaumer, submitted for publication.
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