From the Laboratoire de Physiologie Cellulaire, INSERM EMI 0228, Bâtiment SN3, USTL, 59655 Villeneuve d'Ascq, France
Received for publication, November 27, 2002, and in revised form, February 5, 2003
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ABSTRACT |
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Ca2+ influx via store-operated
channels (SOCs) following stimulation of the plasma membrane receptors
is the key event controlling numerous processes in nonexcitable cells.
The human transient receptor potential vanilloid type 6 channel, originally termed Ca2+ transporter type 1 (CaT1)
protein, is one of the promising candidates for the role of endogenous
SOC, although investigations of its functions have generated
considerable controversy. In order to assess the role of CaT1 in
generating endogenous store-operated Ca2+ current
(ISOC) in the lymph node carcinoma of
the prostate (LNCaP) human prostate cancer epithelial cell line, we
manipulated its endogenous levels by means of antisense hybrid
depletion or pharmacological up-regulation (antiandrogen treatment)
combined with functional evaluation of ISOC.
Antisense hybrid depletion of CaT1 decreased ISOC in LNCaP cells by ~50%, whereas
enhancement of CaT1 levels by 60% in response to Casodex treatment
potentiated ISOC by 30%. The functional
characteristics of ISOC in LNCaP cells were
similar in many respects to those reported for heterologously expressed CaT1, although 2-aminoethoxydiphenyl borate sensitivity and lack of
constitutive current highlighted notable departures. Our results suggest that CaT1 is definitely involved in
ISOC, but it may constitute only a part of the
endogenous SOC, which in general may be a heteromultimeric channel
composed of homologous CaT1 and other transient receptor potential subunits.
In a variety of nonexcitable cells, Ca2+ influx is
signaled by the depletion of intracellular Ca2+ stores, a
process initially termed "capacitative calcium entry" or, later,
"store-operated calcium entry" (1). This Ca2+ influx is
mediated via plasma membrane store-operated Ca2+-permeable
channels (SOCs).1 Besides
playing a major role in replenishing internal Ca2+ stores
in the endoplasmic reticulum (ER) and maintaining ER filling status,
these channels are also involved in regulating of numerous important
physiological processes, ranging from cell growth and proliferation to
apoptosis and cell death.
The best studied SOCs in terms of function and biophysical properties
are Ca2+ release-activated Ca2+ channels
(CRACs), which were originally described in T-lymphocytes and mast
cells (2, 3). This specific type of SOC is highly selective for
Ca2+ under physiological conditions and has a tiny
single-channel conductance, estimated by fluctuation analysis at 24 femtosiemens in 100 mM extracellular
Ca2+ (4).
However, recent electrophysiological studies have clearly established
the existence of a number of subtypes of endogenous store-operated
Ca2+ currents in various cells, differentiated by their
selectivity, unitary conductance, and pharmacology (for a review, see
Ref. 5). Despite considerable efforts in SOC studies, the mechanisms linking store depletion to SOC activation are still unknown, and several fundamentally different hypotheses have been proposed (reviewed
in Refs. 6-9): direct coupling of SOCs to store proteins (10,
11), diffusible messengers (12), or exocytotic insertion of SOCs
preformed in vesicles (13). This variety in the biophysical properties
and activation mechanisms of SOCs reflects the continuing lack of
precise knowledge about the molecular identity of these channels.
The widely investigated family of mammalian homologues of the
Drosophila transient receptor potential (TRP) and TRP-like
(TRP-L) channels is considered the most likely source of candidate
proteins for the role of SOCs (reviewed in Refs. 5 and 14-16). It was recently reported that the epithelial Ca2+ channel (ECaC)
Ca2+ transporter type 1 (CaT1), a member of the vanilloid
subfamily of TRP cationic channels, transient receptor potential
vanilloid type 6 (TRPV6) according to the latest nomenclature (16),
exhibited the unique biophysical properties of CRACs, which led to the
conclusion that CaT1 comprised all or part of the endogenous CRAC pore
(17). However, since then, although new reports (18-21) have confirmed the general resemblance between CaT1- and CRAC-transported currents, they also identified a number of major differences challenging the
hypothesis that CaT1 per se was sufficient to form the
endogenous CRAC. Interestingly, Schindl et al. (19) recently
demonstrated that the regulatory and pharmacological properties of
heterologously expressed CaT1 depend on the cell type used for
expression and on expression levels, suggesting that it may interact
with endogenous TRP channels to produce the resultant heterotetrameric
store-dependent channel.
Beyond its academic interest, the relationship of CaT1 protein to CRAC
acquires great potential practical importance as it becomes
overexpressed in prostate cancers (22, 23). Overexpression of this
protein may represent a novel marker for prostate cancer progression as
well as a target for therapeutic strategies. However, functional
evidence linking CaT1 to endogenous store-operated Ca2+
entry (SOCE) in prostate cancer cells is still missing. In our recent
work (24), we described SOC-mediated current in LNCaP prostate cancer
epithelial cells for the first time. In this study, we used the patch
clamp technique combined with interventions that produce controlled
alterations in the endogenous CaT1 (antisense depletion and
pharmacological up-regulation) to further characterize this current and
show the involvement of CaT1 in generating it. This strategy differs
fundamentally from most of the previous ones, which were based on
overexpression of foreign CaT1 in various cell types, since we
manipulated only the endogenous levels of this protein, thus revealing
its physiological significance in situ. Only recently, a
similar strategy, aimed at reducing the number of endogenous channels
that presumably include CaT1 as an essential subunit by providing an
excess of exogenous CaT1 with a mutated, function-incompatible pore
region, was used to demonstrate the involvement of this protein in
endogenous SOCE in Jurkat T-lymphocytes (21). Although our data are
generally consistent with the functional role of CaT1 in store-operated Ca2+ entry, they also suggest that this particular TRP
channel may represent only a part of the store-operated channel in
prostate cancer epithelial cells.
Cell Cultures--
LNCaP cells from the American Type Culture
Collection were cultured in RPMI 1740 medium (BioWhittaker, Fontenay
sous Bois, France) supplemented with 5 mM
L-glutamine (Sigma) and 10% fetal bovine serum (Seromed,
Poly-Labo, Strasbourg, France). The culture medium also contained
50,000 IU/liter penicillin and 50 mg/liter streptomycin. Cells were
routinely grown in 50-ml flasks (Nunc, Poly-labo) and kept at 37 °C
in a humidified incubator in an air/CO2 (95/5%)
atmosphere. For electrophysiological experiments, the cells were
subcultured in Petri dishes (Nunc) coated with polyornithine (5 mg/liter; Sigma) and used after 3-6 days.
Electrophysiology and Solutions--
Macroscopic currents in
LNCaP cells were recorded in the whole-cell configuration of the patch
clamp technique, using a computer-controlled EPC-9 amplifier (HEKA
Electronics). Patch pipettes were made from borosilicate glass
capillaries (WPI) on a PIP-5 (HEKA Electronics) puller. The
resistance of the pipettes filled with the basic pipette solution (see
below) varied from 4 to 6 megaohms. Series resistance compensation was
used to improve voltage clamp performance during whole-cell current recordings.
The composition of the regular bath (extracellular) solution was 120 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 2 mM MgCl2, 5 mM
glucose, 10 mM HEPES, pH 7.3 (adjusted with Na(OH)). The
high Ca2+, Na+-free extracellular solution used
for store-operated Ca2+ current recordings contained 120 mM tetraethyl-ammonium-Cl, 10 mM
CaCl2, 5 mM glucose, 10 mM HEPES,
pH 7.3 (adjusted with tetraethyl-ammonium(OH)). The basic
Cs+-based, Ca2+-free pipette (intracellular)
solution included 120 mM CsCl, 1 mM
MgCl2, 10 mM HEPES, 10 mM BAPTA, pH
7.3 (adjusted with Cs(OH)). Necessary supplements (IP3,
thapsigargin) were added directly to the respective solutions, from
appropriately prepared stock solutions. All chemicals for
electrophysiological recordings were from Sigma except for
thapsigargin, which was purchased from Calbiochem.
During electrophysiological experiments, cells were maintained in the
regular extracellular solution. External solutions were changed using a
multibarrel puffing micropipette with common outflow positioned in
close proximity to the cell under investigation. During the experiment,
the cell was continuously superfused with the solution via puffing
pipette to reduce possible artifacts related to the change from static
to moving solution and vice versa. Complete external
solution exchange was achieved in less than 1 s.
Analysis of membrane currents was performed off-line.
Usually 3-5 current traces that directly preceded administering
store-depleting intervention were averaged to derive mean base-line
current, which was then subtracted from the currents during
administration of the intervention. The resultant current was
considered as the store-operated current
(ISOC).
Analysis of CaT1 Expression (RT-PCR)--
Total RNA was isolated
from LNCaP cells using the guanidium thiocyanate/phenol/chloroform
extraction procedure (25). After a DNase I (Life Technologies)
treatment to eliminate genomic DNA, 2.5 µg of total RNA was reverse
transcribed into cDNA at 42 °C using random hexamer primers
(PerkinElmer Life Sciences) and murine leukemia virus reverse
transcriptase (PerkinElmer Life Sciences) in a 20-µl final volume,
followed by PCR as described below. In order to control the
amplification of genomic DNA, PCR was also carried out on the
nonreverse transcribed RNA, where the reverse transcriptase was omitted
in the reverse transcription (RT) mix of each sample. The PCR primers
used to amplify the RT-generated CaT1, Bcl-2, and
In order to study the effects of the CaT1 antisense treatments on the
rate of CaT1 and Bcl-2 mRNA synthesis in LNCaP cells, a
semiquantitative multiplex PCR was performed using primers amplifying either CaT1 and Antisense Assays--
The LNCaP cells were treated for up to 5 days with either 0.5 µM phosphorothioate antisense
oligodeoxynucleotides (ODNs) (Eurogentec) targeted to the coding region
of the CaT1 protein and 2.5 µM cytofectin (GS 3815 to
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at a 2:1
molar ratio, unsized) (Eurogentec) or sense ODNs by adding them
directly to the culture medium. The 18-mer ODNs used in these studies
had the following sequences: 5'-GGGCAGTGACAAACCCAT-3' for antisense ODN
and 5'-ATGGGTTTGTCACTGCCC-3' for sense ODN.
Data Analysis and Statistics--
Each experiment was repeated
several times. The data were analyzed using PulseFit (HEKA Electronics)
and Origin 6.0 (Microcal, Northampton, MA) software. Results were
expressed as means ± S.E. where appropriate. Student's
t test was used for statistical comparison of the
differences, and p < 0.05 was considered significant.
Endogenous Whole-cell Store-operated Membrane Currents in LNCaP
Cells--
Transmembrane store-operated Ca2+ current
(ISOC,Ca) can be activated by interventions that
lead to the enhanced emptying of ER intracelluar
Ca2+ stores (26). In this study, two major approaches were
used to activate this current in LNCaP prostate cancer epithelial
cells: (i) cell dialysis via patch pipette with IP3 (100 µM or 25 µM) or (ii) cell dialysis/exposure
to the sarcoendoplasmic reticulum calcium ATPase pump inhibitor
thapsigargin (TG; 100 nM). Both interventions facilitated
the emptying of ER stores, in the first case, by opening
IP3-sensitive channels in the ER and, in the second case,
by inhibiting Ca2+ reuptake into the ER. In addition, in
all experiments, the patch pipette contained high concentrations of the
Ca2+ chelator BAPTA (10 mM), which has been
shown in numerous studies (e.g. Ref. 27) to be sufficient on
its own to deplete the ER stores, due to rapid capture of the
Ca2+ that leaks passively from the ER into the cytosol. In
keeping with this principle, the LNCaP cells responded to the infusion of 10 mM BAPTA-containing intracellular solution via the
patch pipette by generating an inwardly rectifying membrane current (Fig. 1A). With 10 mM Ca2+ in the bath, this current developed to
a maximal density of 1.3 ± 0.3 pA/pF (at
Exposure of the cell to 100 nM TG after full development of
the Ca2+-dependent inward current in response
to BAPTA did not produce any further enhancement of the current
amplitude (data not shown), suggesting that sarcoendoplasmic reticulum
calcium ATPase pump inhibition did not cause any additional ER
Ca2+ store depletion beyond that produced by high cytosolic
BAPTA alone and that the observed Ca2+ current is
transferred via activated SOCs (i.e. is indeed
ISOC,Ca). Inclusion of 100 nM TG in
the patch pipette together with 10 mM BAPTA slightly
accelerated the ISOC,Ca development time course
(to about 60 s) but did not alter its maximal amplitude compared
with BAPTA alone (see, for example, Fig. 3). We therefore used dialysis
with 10 mM BAPTA as the only intervention to activate
ISOC,Ca in subsequent experiments unless
otherwise specified. It should be noted that our experimental
conditions did not favor the activation of Mg2+-sensitive
cationic channels (known as MIC or MagNuM), easily confused with SOCs
under certain conditions (28-30), so we were absolutely confident that
the only current we were dealing with was transferred through the SOCs.
Application of a prolonged hyperpolarizing pulse to
We also examined the sensitivity of ISOC,Ca in
LNCaP cells to the polyvalent cations La3+ and
Ni2+, which had been shown to block native CRAC and
expressed CaT1 currents (3, 17). Fig. 4, E and F,
presents ISOC,Ca recordings from two
representative cells exposed to La3+ and Ni2+,
respectively. Inspection of these recordings shows that
La3+ appears to be a much more potent blocker than
Ni2+. A 10 µM concentration of
La3+ reduced the current virtually to its base-line level,
indicating complete inhibition, whereas Ni2+, even at 0.5 mM, was able to block 40% at most, and as much as 3 mM Ni2+ was required for complete current
inhibition (Fig. 1F). This high sensitivity of
ISOC,Ca in LNCaP cells to La3+ is
characteristic of CaT1-mediated current (17).
Next, we investigated whether inclusion of IP3 in the patch
pipette had any impact on the properties of
ISOC,Ca in LNCaP cells. Fig.
2A shows that, with 100 µM IP3 in the BAPTA-containing pipette
solution, the inward Ca2+ current developed considerably
faster and to about a 2-fold higher maximal amplitude of nearly 3 pA/pF
(n = 7) compared with BAPTA alone (see Fig.
1A) and then declined quite rapidly. This increase in
ISOC,Ca amplitude and the dramatic change in its
development kinetics and rundown are not surprising, considering that
IP3 produces active store depletion by opening IP3-sensitive Ca2+-permeable channels in the
ER, which also leads to facilitated Ca2+-dependent inactivation of SOCs
(e.g. Refs. 26 and 32). Nevertheless, despite the dramatic
change in kinetics, ISOC,Ca evoked by the
combined action of BAPTA plus IP3 still exhibited an
I-V relationship (n = 7) (Fig.
2B) nearly identical to that observed with BAPTA alone (see
Fig. 1B), suggesting that both interventions recruit the
same store-operated channels. This conclusion is further validated by
the fact that the IP3-induced
ISOC,Ca showed similar sensitivity to the
polyvalent cations La3+ and Ni2+
(n = 4) (Fig. 2, C and D).
Pharmacology of the Endogenous ISOC in LNCaP
Cells--
Pharmacological modulation of endogenous
ISOC,Ca in LNCaP cells was studied on currents
activated by intracellular BAPTA (10 mM) plus TG (100 nM) to ensure reliable store depletion and to ensure that
the action of the compounds would less likely be distorted by the
rundown process seen when IP3 was used in the pipette.
The following organic compounds were used: 2-APB, an IP3
receptor antagonist and inhibitor of "classic"
ICRAC (18, 33); SK&F 96365, an SOC- and
TRP-mediated Ca2+ entry blocker (34, 35); econazole, an
antimycotic drug; and ruthenium red, a polycationic dye. The last two
have been shown to be quite potent inhibitors of ECaC1/CaT2-mediated
current (33). Among these drugs, 2-APB appeared to be the most
effective ISOC,Ca inhibitor in LNCaP cells. At
its effective concentration (100 µM according to previous
studies), it caused almost 100% current inhibition, which was
completely reversible upon drug withdrawal (Fig.
3A). 50 µM 2-APB
also caused 100% current inhibition. Ruthenium red at the 0.3 µM concentration required to block ECaC1/CaT2 current completely (36) produced 15% ISOC,Ca inhibition
at most in our experiments, and as much as 9 µM was required to increase the blockade to 50% (Fig. 3B). It is
noteworthy that ECaC2/CaT1 has been shown to have about 100-fold lower
affinity for ruthenium red (IC50 = 9 ± 1 µM) compared with ECaC1/CaT2 (IC50 = 121 ± 13 nM) (37). With the other two drugs, SK&F 96365 (10 µM) inhibited ISOC,Ca by 22%, but econazole (10 µM) apparently had no effect (Fig. 3,
A and C).
CaT1 Expression in LNCaP Cells--
To date, the human TRPV6
channel, originally termed CaT1, is considered the most likely
molecular candidate for the role of native SOCs (17, 19, 21). Peng
et al. (22) already reported high expression levels of CaT1
transcripts in LNCaP cells, although no functional evidence has been
yet presented for the role of the corresponding channel-forming protein
in the endogenous SOCE in these cells. To determine whether or not CaT1
is directly involved in generating Ca2+ current in response
to the depletion of intracellular Ca2+ stores in LNCaP
cells, we altered the levels of its transcripts by either suppressing
them via antisense ODN technology or enhancing them with a specific
pharmacological treatment (the antiandrogen, Casodex, generic name
bicalutamid), while simultaneously monitoring the changes in
store-operated Ca2+ current. Prior to carrying out this
type of manipulation, we initially tested CaT1 mRNA expression in
LNCaP cells by RT-PCR, using specific primers that only amplify CaT1
mRNA (Fig. 4A). As shown
in Fig. 4B, the CaT1-specific primers generated a PCR product of the expected size in LNCaP cells, suggesting that CaT1 protein was expressed in these cells.
The CaT1 antisense ODN was designed in the ATG region of the CaT1
mRNA sequence to inhibit endogenous CaT1 expression (Fig. 4A). The exact mechanism, by which the antisense ODNs are
able to block protein translation, is not fully understood. One
hypothesis implies activation of the RNase H enzyme (38-40), which
cleaves RNA-DNA complexes. The formation of an
antisense-mRNA hybrid activates this enzyme, resulting in the
cleavage of both the antisense and mRNA strands and leading to the
down-regulation of the specific mRNA. Thus, we tested the
efficiency of the CaT1 antisense ODNs treatment in reducing the CaT1
mRNA content in LNCaP cells by RT-PCR. Consistent with the above
mentioned hypothesis, Fig. 4C shows a significant reduction
in the amount of CaT1 PCR product after 72-h treatment with 0.5 µM antisense ODNs compared with the control
(i.e. LNCaP cells treated under the same conditions with
sense ODNs). At the same time, the antisense treatment did not affect
the mRNA content of the CaT1-unrelated antiapoptotic oncoprotein
Bcl-2 (Fig. 4, C and D), suggesting that the
action of CaT1 antisense ODNs is highly selective.
Antisense Hybrid Depletion of CaT1 Suppressed Store-operated
Ca2+ Current in LNCaP Cells--
Maximal effects of
antisense ODNs treatment on cell responses to Ca2+
store-depleting interventions were observed after a 72-h incubation, with almost no change over the next 2 days. Immediately after the
whole-cell configuration was established, using 10 mM BAPTA plus 100 µM IP3 pipette solution, both CaT1
sense- and antisense-treated LNCaP cells showed nearly identical
base-line current with linear I-V and small amplitude. At
Fig. 5A compares averaged time
courses of the development of inward ISOC,Ca in
CaT1 sense- and antisense-treated LNCaP cells in response to dialysis
with BAPTA plus IP3 intracellular solution. Progression of
the dialysis was accompanied by a relatively rapid increase in
ISOC,Ca density in both cell types (Fig.
5A), due to depletion of intracellular Ca2+
stores associated under the combined action of BAPTA plus
IP3. However, the maximal density of
ISOC,Ca in CaT1-depleted cells (1.4 ± 0.36 pA/pF) was about half that in the control (2.8 ± 0.34 pA/pF)
(Fig. 5, A and D). Inspection of the time courses
of Fig. 5A also indicated significantly slower current
development in CaT1-deficient cells in response to BAPTA plus
IP3, with virtually no rundown after it reached maximal
amplitude. This observation was further validated by quantification of
the first derivatives of the time courses, which showed that CaT1 depletion slowed the current development rate from 0.18 ± 0.03 pA/pF/s to 0.08 ± 0.02 pA/pF/s and the current rundown from
0.04 ± 0.01 pA/pF/s to 0.01 ± 0.006 pA/pF/s. Reducing the
IP3 concentration in the pipette to 25 µM
resulted in a considerably longer delay in
ISOC,Ca activation in both sense- and
antisense-treated LNCaP cells (Fig. 5B), which is
qualitatively consistent with previous observations in RBL-1 cells
(26). This treatment also had much less effect on the maximal current
amplitudes (Fig. 5D), as well as development and rundown
kinetics (Fig. 5B), than 100 µM
IP3. Moreover, the increase in the delay period was much
more pronounced for CaT1-depleted cells (Fig. 5B).
Collectively these results prove not only that CaT1 is directly
involved in transferring a substantial portion of
ISOC in LNCaP cells but also that it determines
the store sensitivity and Ca2+-dependent
inactivation properties of endogenous SOCs.
Ca2+-carried ISOC activated in
control and CaT1-depleted LNCaP cells in response to dialysis with the
Ca2+ chelator BAPTA (10 mM) plus TG (100 nM) was characterized by almost 2-fold lower maximal
current densities and noticeably slower development time courses (Fig.
5, C and D), compared with currents activated by
BAPTA plus IP3. These differences are generally consistent with previous observations showing that the magnitude of
ISOC as well as the temporal parameters of its
development depend on the tools used to deplete intracellular calcium
stores (26). Still, like BAPTA plus IP3-activated
currents, the ISOC,Ca evoked in response to
BAPTA plus TG dialysis was also nearly 50% lower in CaT1-depleted
cells than control (Fig. 5, C and D), suggesting that the decrease in current amplitude following CaT1 depletion was
associated with a change in the number of underlying channels or their
intrinsic properties, irrespective of the means used to activate them.
To determine whether CaT1 depletion altered the pharmacological
sensitivity of the residual store-dependent current, we
also compared the action of 2-APB (100 µM), SK&F 96365 (10 µM), ruthenium red (9 µM), and
econazole (10 µM) on ISOC,Ca in
CaT1 sense- and antisense-treated LNCaP cells. A summary of these
results is presented in Fig. 5E. The most notable
differences were noted in the action of SK&F 96365 and ruthenium red.
The first agent blocked the residual current to a greater extent in antisense-treated cells, whereas the second was more effective with
respect to the original ISOC,Ca in sense-treated
cells. These results suggest that most of the current that "goes
away" upon CaT1 depletion is ruthenium red-sensitive but insensitive
to SK&F 96365.
Pharmacological Up-regulation of CaT1 Levels Potentiates
Store-operated Ca2+ Current in LNCaP Cells--
It has
been reported that the level of CaT1 transcripts in LNCaP cells is
negatively controlled by androgen receptors (22). Therefore, to further
demonstrate the correlation between CaT1 transcript expression and the
magnitude of ISOC,Ca, we compared this current
in regular LNCaP cells and LNCaP cells treated for 72 h with
Casodex (1 µM), an androgen receptor antagonist (22).
Fig. 6A shows that, during
Casodex treatment, the level of CaT1 mRNA in LNCaP cells gradually
increased, exceeding the base line by almost 60% after 72 h. This
enhancement was accompanied by an ~30% up-regulation of
ISOC,Ca evoked by BAPTA plus IP3 or
BAPTA plus TG infusion (Fig. 6, B and C),
providing additional support for the hypothesis that endogenous CaT1 is
involved in generating this current.
The major objective of this study was to characterize endogenous
store-operated Ca2+ current in LNCaP prostate cancer
epithelial cells and test the hypothesis that the channel-forming CaT1
protein contributed to generation of this current. Although there is
substantial agreement between the biophysical properties of
CaT1-mediated current in a heterologous expression system and
endogenous ICRAC (17, 18), there were no
previous data demonstrating a direct correlation between endogenous
expression of CaT1 protein and the function of endogenous SOCE in the
same cells. We used an approach consisting of manipulating endogenous
CaT1 levels by means of antisense hybrid depletion or pharmacological
up-regulation, combined with functional evaluation of
ISOC,Ca, to obtain this data for LNCaP prostate
cancer epithelial cells, which have been shown, in recent independent
studies, to possess robust capacitative Ca2+ entry (24) and
express quite high levels of CaT1-specific mRNA (22). This approach
shows that CaT1 is definitely involved in SOCE in LNCaP cells, but it
may constitute only a part of the endogenous store-operated channel.
Hybrid Depletion Experiments--
Functional scrutiny of CaT1
protein heterologously expressed in CHO-K1 cells led to the
initial conclusion that it comprised all or part of the endogenous CRAC
pore (17). Recently, however, this conclusion was seriously challenged,
since a direct step-by-step comparison of the properties of
heterologously expressed CaT1 in HEK 293 cells with the "classic"
endogenous CRAC in RBL-2H3 cells under identical conditions revealed
substantial differences (18, 20). Although there may be a number of
reasons for this inconsistency, many of which are undoubtedly related
to the channel expression system and cell-specific modulation of its
function (19), CaT1 represents one of the best suited candidates for the role of SOC discovered to date. This is especially true, since "classic" CRAC represents only one phenotypic manifestation of SOCs, which are, in general, characterized by a whole range of cell-specific properties.
We used a different approach to assess the role of CaT1 in
store-operated Ca2+ entry in LNCaP prostate cancer cells,
which are known to have an up-regulated gene for this protein (22), by
developing antisense oligonucleotides to inhibit expression of
endogenous CaT1 protein selectively without affecting any of the other
components in endogenous SOC. These nucleotides certainly reduced the
CaT1 mRNA content in LNCaP cells to a significant extent,
suggesting that protein levels are likely to be reduced as well.
Knockout of CaT1 mRNA was accompanied by a decrease of ~50% in
ISOC density.
The slower development of residual store-dependent current,
which accompanied a general ISOC reduction in
CaT1-deficient cells, highlights the role of CaT1 not only in
transferring this current but also in establishing the coupling
efficacy of endogenous SOCs to the filling status of the stores and
Ca2+-dependent inactivation. Moreover,
inhibition of androgen receptors, conditions that favor overexpression
of CaT1 in LNCaP cells (22), potentiated ISOC.
Together with the fact that CaT1 depletion had the opposite effect on
the current, this suggests a direct correlation between endogenous CaT1
levels and the size of the store-operated current. These findings are
consistent with the hypothesis that CaT1 either represents a
functionally significant subunit in a homo- or heteromeric assembly of
endogenous SOC or is an important regulator of this channel.
Since CaT1 per se is a channel-forming protein rather than a
channel regulator (17, 18), the results of the hybrid depletion experiments led us to conclude that it either forms a monomeric SOC
channel in LNCaP prostate cancer cells or is an integral part of the
heteromeric SOC channel, which loses and/or alters its function when
CaT1 is removed.
How Similar Is Endogenous SOC in LNCaP Cells to CaT1?--
Our
functional studies of endogenous SOCs in LNCaP cells favor the second
possibility. Indeed, the properties of the endogenous ISOC described in this study are quite similar,
in many respects, to the ones reported for heterologously expressed
CaT1 in CHO-K1 cells (see Ref. 17). First, the endogenous SOCs in LNCaP
cells and CaT1 channels have the same unique activation mechanism
(i.e. dependence on the ER and cytoplasmic Ca2+
levels). Second, macroscopic current properties such as sharp inward
rectification, Ca2+-dependent inactivation,
high Ca2+ selectivity under normal conditions, the order of
relative conductance for divalent cations Ca2 > Sr2+
Despite these similarities, there are also notable differences,
primarily relating to the lack of the significant constitutively active
current characteristic of heterologously expressed CaT1 and the lack of
slight potentiation by 2-APB of constitutively active CaT1 currents
observed in both RBL and HEK 293 cells (17, 19). On the contrary, our
results show strong inhibition of endogenous SOC similar to that of the
store-dependent CaT1 currents of RBL-cells by 2-APB (19).
It is important to note that most of the distinctive properties of the
original ISOC in LNCaP cells compared with
CaT1-induced current were also maintained in the residual
ISOC following CaT1 depletion. This is
apparently due to the fact that CaT1 may only be a part of the
endogenous SOC in LNCaP cells, whereas, for CaT1-specific properties to
predominate, CaT1 must form a monotetrameric channel as a result of its
strong overexpression (19). However, the stronger inhibition of control versus residual ISOC by ruthenium red
is consistent with the CaT1 nature of the portion of the current
eliminated by antisense, since CaT1 is known to be quite sensitive to
this drug (37).
What Are the Other Possible Candidates?--
Thus, on the basis of
our functional data, it seems unlikely that CaT1 is the only
Ca2+ transport protein involved in endogenous
ISOC in LNCaP cells. Recent studies have shown
that, in addition to CaT1, these cells also express high levels of
mRNA for two other members of the rapidly growing family of
structurally related Ca2+ transport proteins, CaT2 (also
termed ECaC1 or TRPV5 (see Ref. 16) and CaT-L (also known as htrp8A and
htrp8B), which has subsequently been shown to be encoded by the same
gene as CaT1, but for consistency with original literature we keep the
initial designation (22, 23)). Although CaT2 and CaT-L channels are
Ca2+-selective, as well as showing inward rectification and
Ca2+-dependent feedback inhibition, their
functional properties generally seem to be even more dissimilar to
those of endogenous SOCs in LNCaP cells. Heterologously expressed CaT-L
protein, for instance, induces a constitutive Ca2+ current
(23), which, as has already been pointed out, is not detectable in
LNCaP cells. In addition, such specific organic inhibitors of
ECaC1/CaT2-mediated current as econazole and ruthenium red (36) had
little or no effect on endogenous ISOC in LNCaP cells. On the other hand, 2-APB, an IP3 receptor antagonist
and "classic" endogenous CRAC currents inhibitor (30), which has no
effect on ECaC1/CaT2 current at 100 µM (36), totally
blocked endogenous ISOC in LNCaP cells.
Collectively, these data support the hypothesis that none of the
endogenously expressed Ca2+ transport proteins, CaT1, CaT2,
or CaT-L alone can be responsible for the endogenous
ISOC in LNCaP cells.
It should be noted that, in addition to the
Ca2+-transporting proteins mentioned above, all members of
the vanilloid family of TRP channels, TRPV (see Ref. 16), LNCaP cells
are also likely to express other TRP channels as well. Indeed, it has
been shown that the gene for Trp-p8 in the melastatine family, TRPM
(16), is up-regulated in prostate cancer cells (41). Although the functional properties of Trp-p8 in prostate cells have not yet been
elucidated, in general, this fact suggests that various TRP channel
proteins, many of which have indeed been implicated in store-operated
Ca2+ entry, may take part in the formation of functional
store-operated channels in prostate cancer cells. As was suggested, SOC
may have either a hetero- or homotetrameric TRP-based structure, which may account for its varying cell-specific properties (15, 42). Moreover, depending on the endogenous expression of other
store-dependent TRP channels in the particular cell type,
an artificially produced excess or deficiency of CaT1 may impact the
natural stoichiometry of SOCs, yielding channels with diverse
biophysical properties (19). Our data suggest that the most likely
endogenous SOC in LNCaP cells is a heterotetramer composed of several
TRP channel subunits, including endogenous CaT1, which, however, does
not play a dominant role in defining the main properties of the
resultant channel.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin cDNAs
were designed on the basis of established GenBankTM
sequences. Primers were synthesized by Invitrogen. The
primers for human CaT1 cDNA were as follows:
5'-TGAGGATTGCAAGGTGCACCAT-3' (nucleotides 201-222,
GenBankTM accession number AF304463) and
5'-CCCCAAAGTAGATGAGGTTGCA-3' (nucleotides 468-490). The expected DNA
length of the PCR product generated by these primers was 290 bp. The
sense and antisense primers for Bcl-2 were as follows: 5'-
GATTGTGGCCTTCTTTGAGTTCGGT-3' (nucleotides 469-493,
GenBankTM accession number M14745) and
5'-CTACTGCTTTAGTGAACCT-3' (nucleotides 784-803). The expected DNA
length of the PCR product generated by these primers was 335 bp.
Amplification of the
-actin cDNA was used to control the
integrity of the cDNA and also as an internal control to quantify
the expression of a given gene in duplex RT-PCR. The following
oligonucleotide primers were used to amplify
-actin cDNA (227 bp): 5'-CAGAGCAAGAGAGGCATCCT-3' and 5'-ACGTACATGGCTGGGGTGTTGAA-3'. In order to confirm the identity of the amplified products,
restriction analysis was carried out on each PCR product using specific
restriction enzymes. PCR was performed on the RT-generated cDNA
using a GeneAmp PCR System 2400 thermal cycler (PerkinElmer Life
Sciences). To detect CaT1 cDNAs, PCR was performed by adding 2 µl
of the RT template to the following mixture (final concentrations): 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl2, 200 µM each dNTP, 1 µM sense and antisense primers, and 1 unit of AmpliTaq
Gold (PerkinElmer Life Sciences) in a final volume of 25 µl. DNA
amplification conditions included an initial 10-min denaturation step
at 95 °C (which also activates the Gold variant of Taq
polymerase) and 40 cycles of 30 s at 95 °C, 30 s at
58 °C, 30 s at 72 °C, and finally 7 min at 72 °C. The
identity of each PCR-amplified product was confirmed by restriction
analysis using specific restriction enzymes.
-actin (internal standard) or Bcl-2 and
-actin mRNAs. Control experiments using LNCaP cell cDNA
showed that the amount of each amplimer obtained in a multiplex PCR was
independent of the presence of other primers (cross-correlation
analysis), excluding the possibility of strong interference between
primers. The number of cycles and the final cDNA concentrations
were then adjusted to be in the exponential phase of the amplification
of each product. Finally, the amount of each PCR product in multiplex reaction increased linearly with the amount of starting cDNA (5-50 ng of RNA equivalents), ensuring that changes in the ratio of PCR
product to control gene product truly reflects a change in mRNA
abundance of the gene relative to the control gene. The duplex PCR was
then performed using the cDNA (18 ng of RNA equivalent to
the RT reaction) of CaT1 sense- and antisense-treated LNCaP cells. The
conditions were as follows: 10 min at 95 °C and then 32 cycles of
30-s extension at 72 °C each and a final 7-min extension step. Half
of the PCR samples were analyzed by electrophoresis in a 2% agarose
gel and stained with ethidium bromide (0.5 µg/ml) and viewed by Gel
Doc 1000 (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
100 mV,
n = 9) in about 90 s following establishment of
the whole-cell configuration (Fig. 1A) and then stayed
relatively constant or declined slightly over a period of up to 5 min.
Current amplitude was measured at the beginning of the
hyperpolarization phase to
100 mV (see pulse protocol in Fig.
1A). The I-V relationship of the current showed
strong inward rectification and an apparent reversal potential at
+43 ± 1.1 mV (n = 9) (Fig. 1B). The
current was dependent on extracellular Ca2+ concentration
and disappeared completely in nominally Ca2+-free
extracellular solution, suggesting that it is carried solely by
Ca2+ ions.
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Fig. 1.
Whole-cell Ca2+-carried
ISOC in LNCaP cells. A,
the time course of the development of ISOC,Ca at
100 mV in representative LNCaP cell in response to the dialysis with
10 mM BAPTA-containing pipette solution; time 0 corresponds
to the establishment of the whole-cell configuration; the
inset shows superimposed base-line current just after the
rupture of the membrane (1) and fully developed
ISOC,Ca (2) together with the voltage
protocol used to elicit the currents (n = 9);
ISOC,Ca amplitude was measured at
100
mV at the beginning of the hyperpolarization. B,
I-V relationship of a fully developed
ISOC,Ca derived from the traces presented in
A (n = 9). C, trace of the fully
developed ISOC,Ca (continuous
line) in response to the depicted pulse protocol with
superimposed fit of the current decay phase with double exponential
function (open symbols); parameters of the fit
are presented under "Results" (n = 4).
D, time course of the changes of ISOC
at
100 mV in the representative LNCaP cell sequentially exposed to
Ca2+-, Ba2+-, Sr2+- and
Mn2+-containing (10 mM) extracellular solutions
(marked by horizontal bars); time 0 corresponds
to the establishment of the whole-cell configuration (n = 5). E, superimposed traces from representative cell of the
fully developed ISOC,Ca under control conditions
and in the presence of 10 µM La3+
(n = 3); the pulse protocol used to elicit the currents
is shown at the top. F, same as in E
but for another cell sequentially exposed to 0.5 and 3 mM
Ni2+ (n = 3).
100 mV revealed
time-dependent inactivation of the inward
ISOC,Ca, which could be described by the sum of
two exponential functions (
fast = 9.1 ± 1.8 ms and
slow = 55.7 ± 7.8 ms, n = 4) and a steady-state level (Fig. 1C). Substituting 10 mM Ba2+ for 10 mM Ca2+
reduced current amplitude by about 35%. Further equimolar replacement of Ba2+ with Sr2+ produced almost no change in
current, whereas using Mn2+ instead of Sr2+
caused a strong current decrease. Quantification of Ca2+-,
Sr2+-, Ba2+- and Mn2+-carried
ISOC (Fig. 1D) in five cells yielded
the sequence of the relative channel conductance: Ca2+
(1) > Sr2+ (0.69)
Ba2+ (0.65)
Mn2+ (0.23), very similar to the analog sequences
found for native SOCs in other preparations (3, 4, 31) as well as that of heterologously expressed CaT1 protein, a likely molecular candidate for the role of native SOCs (17).
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Fig. 2.
Intracellular IP3 changes the
time course of Ca2+-carried
ISOC in LNCaP cells. A, the time course of
the development of ISOC,Ca at 100 mV in a
representative LNCaP cell in response to the dialysis with 10 mM BAPTA plus 100 µM
IP3-containing pipette solution; time 0 corresponds to the
establishment of the whole-cell configuration (n = 7).
B, I-V relationship of IP3-evoked
ISOC,Ca acquired at the maximum of the time
course of A (n = 7). C,
superimposed traces from a representative cell of the maximal
IP3-evoked ISOC,Ca (control)
following application of 10 µM La3+; the
pulse protocol is shown at the top (n = 4).
D, same as in C, but for another cell
sequentially exposed to 0.5 and 3 mM Ni2+
(n = 4).
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Fig. 3.
Pharmacology of
ISOC,Ca in LNCaP cells. A,
the effects of 100 µM 2-APB (n = 5) and
10 µM SK&F 96365 (n = 5) on the
whole-cell ISOC,Ca (measured at 100 mV)
elicited in the representative regular LNCaP cell by the dialysis with
10 mM BAPTA plus 100 nM TG-supplemented pipette
solution; the periods of drug application are marked by
horizontal bars. B and C,
same as in A, but for the effects of 0.3 and 9 µM of ruthenium red (RR) (n = 5) (B) and 10 µM econazole (n = 3) (C) in two other LNCaP cells.
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Fig. 4.
Antisense treatment reduces CaT1 mRNA in
LNCaP cells. A, CaT1 mRNA nucleotide sequences
showing the CaT1 regions targeted by CaT1-specific forward and backward
primers used for RT-PCR as well as by CaT1 antisense oligonucleotide
used for hybrid depletion. B, RT-PCR analysis of the CaT1
mRNA (290 bp) expression in LNCaP cells with the use of
CaT1-specific primers. C, RT-PCR analysis demonstrating the
reduction of the CaT1 mRNA (290 bp) in LNCaP cells treated for
72 h with antisense oligonucleotide compared with the cells
treated for the same period of time with sense oligonucleotide.
D, RT-PCR analysis demonstrating that treatment of LNCaP
cells with CaT1 antisense or sense oligonucleotides does not affect the
mRNA content for CaT1-unrelated Bcl-2 protein (335 bp); in each
case, a duplex PCR was performed with -actin (227 bp) used as an
internal standard of the total mRNA of each sample. H2O
was used as a negative control. PCR-amplified products were analyzed by
a 2% agarose gel electrophoresis and visualized by EtBr staining.
M, molecular weight marker. Each experiment was repeated
three times.
100 mV, the current was 1.0 ± 0.1 pA/pF and 0.9 ± 0.1 pA/pF, respectively. This base-line current was considered to reflect a
passive leak and was, therefore, subtracted from the currents achieved
at later in cell dialysis.
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Fig. 5.
CaT1 depletion reduces
ISOC in LNCaP cells. A and
B, averaged time courses of the development of
ISOC,Ca (at 100 mV) in LNCaP cells treated for
72 h with CaT1 sense (filled circles) and
antisense (open circles) oligonucleotides
(mean ± S.E., n = 12 for each cell type) in
response to the dialysis with 100 µM (A) or 25 µM (B) IP3-supplemented
BAPTA-containing pipette solution. C, same as in
A and B, but in response to the dialysis with
BAPTA plus TG (100 nM). D, quantification of the
differences in the maximal ISOC,Ca density in
CaT1 sense-treated (black column) and
antisense-treated LNCaP cells in response to IP3 (100 and
25 µM) and TG (100 nM). E,
comparison of the effects of 2-APB (100 µM), SK&F 96365 (10 µM), ruthenium red (9 µM), and
econazole (10 µM) on ISOC,Ca in
CaT1 sense- and antisense-treated LNCaP cells (mean ± S.E.,
n = 4-6).
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Fig. 6.
Antiandrogen treatment enhances CaT1
expression and ISOC in LNCaP cells.
A, RT-PCR analysis with the use of CaT1-specific primers
demonstrating gradual enhancement of the CaT1 mRNA (290 bp) with
time of LNCaP cell treatment with specific androgen receptor
antagonist, Casodex (1 µM). This experiment was repeated
three times. B, time courses of the development of
ISOC,Ca (at 100 mV) in representative control
(filled circles) and Casodex-treated (72 h,
open circles) LNCaP cells in response to the
dialysis with 100 µM IP3-supplemented
BAPTA-containing pipette solution. C, quantification of the
differences in the maximal ISOC,Ca density in
the control (black column) (n = 8) and Casodex-treated (72 h) (n = 9) LNCaP cells in
response to IP3 (100 µM) and TG (100 nM).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ba2+
Mn2+, and
sensitivity to blockade by Ni2+ and La3+ are
also nearly identical.
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FOOTNOTES |
---|
* This work was supported by grants from INSERM, La Ligue Nationale Contre le Cancer, and Association pour la Recherche sur le Cancer (France) and International Association-99-01248.
Both authors contributed equally to this work.
§ Supported by INSERM and the Ministry of Science of France. Present address: Bogomoletz Institute of Physiology, Bogomoletz Str., 4, 01024 Kiev-24, Ukraine.
¶ To whom all correspondence should be addressed: Laboratoire de Physiologie Cellulaire, INSERM EMI 0228, Bâtiment SN3, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France. Tel.: 33-3-20-33-60-18; Fax: 33-3-20-43-40-66; E-mail: phycel@univ-lille1.fr.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M212106200
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ABBREVIATIONS |
---|
The abbreviations used are:
SOC, store-operated
channel;
ER, endoplasmic reticulum;
CRAC, calcium release-activated
calcium channel;
SOCE, store-operated calcium entry;
LNCaP, lymph node
carcinoma of the prostate;
TRP, transient receptor potential;
TRP-L, TRP-like;
CaT, Ca2+ transporter type 1;
ECaC, epithelial calcium channel;
ODNs, oligodeoxynucleotides;
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid;
TG, thapsigargin;
IP3, inositol 1,4,5-trisphosphate;
2-APB, 2-aminoethoxydiphenyl borate;
SK&F 96365, 1-[-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl]-1H-imidazole
hydrochloride;
TRPV6, transient receptor potential vanilloid
type 6;
RT, reverse transcription;
pF, picofarads.
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