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INTRODUCTION |
In a wide variety of nonexcitable and in many excitable
cells, activation of G-protein-coupled receptors initiates a linear sequence of events leading to depletion of intracellular calcium storage compartments (the endoplasmic reticulum,
ER)1 via the production of
inositol 1,4,5-trisphosphate (IP3) by phospholipase C, and
the subsequent induction of calcium influx from the extracellular space
(1).
Depletion of the ER appears to be a prerequisite for the activation of
calcium influx, because several experimental maneuvers that induce
depletion of the ER, such as the introduction of IP3 into
the cell or blockade of the microsomal calcium ATPase with thapsigargin
(TG) and other selective blockers, are equally effective activators of
calcium influx (1).
The activation of calcium influx after store depletion has been termed
store-operated calcium entry (SOCE) and appears to be a well preserved
mechanism from insects to humans (2). The finding that the transient
receptor potential protein (TRP) from the Drosophila
photoreceptor encodes a calcium-permeable channel activated after
depletion of intracellular calcium stores, provided the first evidence
for the identification of the molecular entity responsible for SOCE
(3). An intense search in mammalian tissues led to the isolation of
several mammalian TRP homologues, some of which are activated after
depletion of the ER (4).
The diverse TRP superfamily of channels has been divided in three
subfamilies (TRPC, TRPV, and TRPM) based on structural motifs (5).
Members from the TRP superfamily are found in a wide range of
organisms, from yeast to humans (6). One of the most prominent features
in this superfamily is the wealth of regulatory mechanisms responsible
for channel activation, including changes in osmolarity (TRPV4),
ligands such as vanilloid (TRPV1), cold and menthol (TRPM8), store
depletion and diacylglycerol (TRPC subfamily) (for review see Ref.
6).
Although the search for new TRPC homologues in a wide variety of
organisms has proven very fruitful, the identification of the mechanism
communicating the depleted state of the ER to the plasmalemmal channel
remains elusive. Several models have been proposed to explain the
communication between the ER and store operated channels (SOC), which
can be divided into two main classes: one involving a physical coupling
between the ER and the plasmalemmal channel and another suggesting the
presence of a diffusible messenger responsible for communicating the
depleted state of the ER to the plasma membrane (7).
Recent evidence favoring the physical coupling model shows direct and
functional interactions between the IP3 receptor
(IP3R) and human TRP3 channel (8). Furthermore, a common
binding site for calmodulin (CaM) and IP3R has been
identified at the carboxyl termini of TRP channels (9). Interestingly,
we have previously shown that the carboxyl-terminal domain of the
Drosophila TRP channel confers the thapsigargin sensitivity
to TRPL, a channel that is not originally activated after depletion of
the ER (10). We have shown also that CaM inhibits SOCE in vascular
endothelium (11).
The modulation of channel activity by CaM appears to be a common
feature found in several TRP members. In the Drosophila
photoreceptor, CaM mediates termination of the light response in
vivo via modulation of TRP and TRPL channels (12). CaT1, a TRP
member recently identified, is modulated by a competitive interaction
between CaM and protein kinase C (13). A CaM binding site has been
identified in human TRP1 and TRP3 and mouse TRP4-TRP7 (9).
In the present study, we have characterized the role of CaM and
IP3R in the communication between the ER and endogenous
TRP1 channels from Chinese hamster ovary (CHO) cells using rapid
confocal microscopy and electrophysiology. The results presented here
show: 1) a consistent delay of several hundreds of milliseconds between the first detectable release of calcium from the ER and the activation of TRP1 channels; 2) CaM antagonists significantly reduced the delay
period; 3) introduction of CaM into the cell via the patch pipette
significantly increased the delay period; 4) single channel measurements revealed a competitive interaction between CaM and IP3 in the modulation of TRP1 single channel activity; 5) a
peptide from the IP3R restored channel activity in excised
inside-out patches; and 6) introduction of dsRNA from a segment of the
TRP1 sequence resulted in very potent and specific reduction of TRP1 protein and SOCE.
All of these results show that a competitive interaction between CaM
and IP3R is sufficient to explain the activation of TRP1 channels and SOCE, after depletion of the ER in CHO cells. Our results
show also that CaM has a dominant effect on TRP1 channel activity,
which may play a role in preventing calcium influx under resting conditions.
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MATERIALS AND METHODS |
Reagents and Solutions--
All salts used were analytical grade
purchased from Sigma. The PBS solution contained (mM): 140 NaCl, 2 MgCl2, 2 CaCl2, HEPES, and was adjusted
to pH 7.2 with NaOH. The NMDG-Cl solution contained (mM):
140 N-methyl-D-glucamine chloride, 2 MgCl2, 2 CaCl2, 10 HEPES, and was adjusted to
pH 7.2 with NaOH. The solution NA contained (mM): 140 Na-aspartate, 2 MgCl2, 10 HEPES, pH 7.2, with NaOH. In all
whole cell experiments, the pipette (NA) solution contained in addition
2 mM ATP and 1 mM GTP from Sigma. Where
indicated, the calcium in PBS and NA solutions was adjusted to either 1 or 100 nM with EGTA as previously described (14). Bovine
brain calmodulin, the S-100 calcium-binding protein from bovine brain, and the calmodulin antagonists W7 and trifluoperazine dimaleate (TFP)
were purchased from Calbiochem (San Diego, CA).
Cell Culture--
CHO cells were purchased from American
Type Culture Collection (ATCC) and maintained in culture using
Dulbecco's modified Eagle's medium (Sigma) supplemented with
1% antibiotics, 2 mM glutamine, and 10% bovine fetal
serum (Invitrogen, Gaithersburg, MD). Cells were grown on
plastic Petri dishes and maintained with 5% CO2 in a
humidity controlled incubator (NUAIRE, Plymouth, MN).
Cloning of Partial cDNA Encoding TRP1--
Degenerated
oligonucleotide primers from TRP-conserved regions were
5'-TGGGGCC(ATC)(TC)TGCAGAT(AC)TC(AT)CTGGGA-3' and
5'-TG(CT)AG(AC)(TG)CGTTT(CA)AAGGT(GA)AG-3' synthesized by Life
Technologies (Karlsruhe, Germany). The first primer was designed
according to Okada et al. (15); the second primer was
designed based on the highly TRP conserved amino acid sequence EWKFAR.
mRNA from CHO cultures was isolated following standard procedures
and cDNA was synthesize in vitro. A PCR was performed
for 19 cycles and the products were analyzed in a polyacrylamide gel.
Over five independent PCR reactions from different cultures produced
similar results, a single product of 450 bp. The PCR product was cloned
in the pCRII vector (Invitrogen, Carlsbad, CA). After sequencing the
fragment, the nucleotide sequence was introduced in the FASTA program
(National Center for Biotechnology Information) to identify homologues.
The search indicated that the cloned sequence was over 93.7% identical
to rat TRP1 (accession number AF061266). The partial clone from
CHO-TRP1 has been submitted to GenBankTM (accession number
AF448492).
Stable Expression of the Human Bradykinin Type 2 Receptor in CHO
Cells--
The full-length cDNA encoding the human bradykinin type
2 receptor (Bk2R) was cloned into the EcoRI site of the
pcDNA3 vector (Invitrogen) using standard ligation techniques. 50%
confluent CHO cells were transfected with 5 µM of the
construct mixed with LipofectAMINE Plus (Life Technologies).
Transfected CHO cells were maintained in G418 (Life Technologies) for 1 month, and antibiotic-resistant clones were isolated. The
G418-resistant cells were maintained in culture and used in the studies described.
Double-stranded RNA Interference--
The entire 450-bp TRP1 DNA
fragment isolated by PCR was cloned in pBluescript (Stratagene, La
Jolla, CA) and linearized with BamHI to produce single
stranded RNA using the MEGAscript kit (AMBION, Austin, TX) with the T3
polymerase. In a separate experiment the vector with insert was
linearized with EcoRV to produce the complementary RNA
strand with the T7 polymerase. Equal amounts of both single stranded
RNA products were mixed and heated at 60 °C and cooled down slowly
for 30 min until room temperature was reached. The product obtained was
compared by nondenaturating electrophoresis with both single stranded
RNA products to confirm the formation of the double stranded RNA
(dsRNA). 5 µg of dsRNA were mixed with LipofectAMINE Plus (Life
Technologies) and the mixture was placed on 60-mm Petri dishes
containing 50% confluent CHO cells. 48 and 72 h later the same
cells were transfected again with 5 µg of dsRNA each time. Calcium
measurements were performed 24-48 h after the third transfection.
Control cells were exposed to LipofectAMINE alone using the same
protocol. For Bk2R RNAi experiments a fragment from nucleotides 420 to
762 was isolated by PCR and cloned in pBluescript. The dsRNA was
prepared as described above for TRP1. CHO cells were transfected three
times with the dsRNA using a similar procedure to the one described above.
Western Blotting Analysis--
Total membrane extracts from
control cells and RNAi-treated cells were resolved by SDS-PAGE and
transferred to nitrocellulose paper as previously reported (16). To
ensure equal amounts of protein loading, the amount of total protein
was assessed using the MicroBCA protein assay kit (Pierce) and albumin
as control. 10 µg of protein was loaded for every experimental
condition. Antibody anti-TRPC1 (ALAMONE Labs. number ACC-010) was
diluted 1:1000 in buffer A containing (in mM): 50 Tris, 150 NaCl, pH 7.4, and 2% bovine serum albumin (Sigma). After 30 min
incubation at 37 °C, the nitrocellulose sheets were washed 5 times
for 15 min each time with buffer A and incubated for 30 min with
peroxidase-labeled mouse anti-rabbit antibody diluted 1:5000 (Sigma).
After a second washing period, bound antibody was visualized with the
Supersignal Ultra chemiluminescence detection kit (Pierce). The human
type 2 bradykinin receptor was detected with antibody AS 346-2 (a
generous gift from Dr. Müller-Esterl, Johannes Gutenberg
University at Mainz) as previously described (16).
Free Calcium Determinations--
The free calcium reported in
the different solutions was adjusted by the addition of EGTA purchased
from Sigma. To determine the amount of EGTA needed to obtain the free
calcium for each experimental condition, we used the program "Bound
and Determined" (BAD 4.35) as previously described (14). Free calcium
concentrations reported in the text were corroborated using FURA-2
tetrapotassium salt (Molecular Probes, Eugene OR) and an Aminco-Bowman
Series II spectrophotometer.
Synthetic Peptides from IP3R--
The peptide
IP3R-P1 (EYLSIEYSEEEVWLTWTD) was purchased from ResGen
(Carlsbad, CA). This peptide has been previously reported to bind
several TRP channels and compete with CaM for a common binding site
(9). The scrambled peptide IP3R-P2 (TWLSTDEVELYIEWEYSE) was
used as control. This peptide contains the same amino acids as
IP3R-P1 but in random order. Peptides were introduced into the cell via the patch pipette or perfused on isolated patches (single
channel experiments) via a Picospritzer II (General Valve, Fairfield, NJ).
Measurements of Free Cytosolic Calcium in Cell
Populations--
Free calcium in CHO cells was monitored as previously
described (16). Briefly, cells were incubated in physiological solution containing (mM): 120 NaCl, 1.2 KH2PO4, 1.2 Mg2SO4,
4.75 KCl, 10 glucose, 20 HEPES, 0.05% bovine serum albumin, and 5 µM fura-2-AM (Molecular Probes). Following a 30-min
incubation at room temperature (22 °C), cells were washed twice with
physiological solution and treated for 2 min with 0.25% trypsin. Cells
were later dispersed with a plastic pipette and subjected to
centrifugation, and resuspended in PBS. Fluorescence was measured using
an Aminco-Bowman Series II spectrophotometer. Excitation wavelength
alternated between 340 and 380 nm, and fluorescence intensity was
monitored at an emission wavelength of 510 nm. Calibration of the
fura-2 associated with the cells was accomplished using Triton lysis in
the presence of a saturating concentration of calcium followed by
addition of EGTA (pH 8.5). Free calcium was calculated as previously
described (16).
Combined Confocal Calcium and Electrophysiology
Measurements--
CHO cells were loaded with 5 µM
Fluo-4-AM using the loading procedure described above for FURA-2-AM.
Cells were placed on glass coverslips and allowed to attach to the
glass surface. Combined patch clamp and confocal microscopy
measurements were performed using an EPC9 amplifier (Heka Instruments,
Bonn, Germany) and a real-time confocal system (Noran Instruments,
Oxon, England). The EPC9 amplifier was synchronized with the confocal
acquisition unit via a TTL pulse. The microscope objective used was
Nikon 60X Plan-Apo 1.40 oil immersed. The confocal acquisition window was set to 300 × 300 pixels to improve acquisition speed. At this resolution the system acquired one image every 10 ms. Five images were
averaged and the result was saved, producing one averaged image every
50 ms. All images were stored on the hard disk of an
Indigo2 (Silicon Graphics, Mountain View, CA) for off-line
analysis. CHO cells showed diameters in the range of 15-20 µm, the
focal point was moved to approximately the middle of the cell, using a
focal resolution of 10 µm. Conventional whole cell experiments and
perforated patch mode were performed on Fluo-4-AM-loaded CHO cells. The
voltage protocol consisted of a ramp from 
100 to +100 mV delivered
continuously every 50 ms for the duration of the experiment. Patch
pipette resistance ranged from 6 to 10 M
when filled with the NA
solution. Cell capacitance was 10-12 pF. To compare among different
cells, the amount of current from each cell was corrected by the cell
capacitance and reported throughout the text as pA/pF. TG and Bk were
applied to the cell via a Picospritzer II (General Valve). Drug
applications were synchronized with electrophysiology and confocal
acquisition via a TTL pulse delivered by a DG535 Digital Delay/Pulse
Generator (Stanford Research Systems, Sunnyvale, CA). Whole cell data
was recorded and stored on a PC computer hard disk for off-line
analysis using Igor Pro 4 software (Wavemetrics, Lake Oswego, OR).
Fluorescence intensity was measured with procedures written in-house
with Igor Pro.
Single Channel Experiments--
For cell-attached and inside-out
experiments, the pipette contained the PBS solution with the calcium
reported in each condition, as indicated in the figure legends. Data
was stored on FM tape (VETTER PCM recorder) for off-line analysis.
Single channel recordings were digitized at 10 kHz using a Digidata
1200 (Axon Instruments, Union City, CA) and filtered at 5 kHz during
analysis using pClamp software (Axon Instruments). An in-house designed
multibarreled microperfusion system was used to apply the different
concentrations of CaM and IP3.
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RESULTS |
Simultaneous Measurements of Confocal Calcium and Whole Cell
Currents--
Fluo-4-loaded single CHO cells stably transfected with
the human Bk2R responded to Bk with elevation of intracellular calcium in a dose-dependent manner, showing a maximum response to
75 nM Bk (data not shown). Over 90% of the cells studied
showed this response. These results are consistent with the affinity of
this receptor for Bk measured in different expression systems (16). Nontransfected CHO cells did not respond to Bk at the concentrations tested in this study (up to 1 µM).
Combined whole cell patch clamp and rapid confocal intracellular
calcium measurements with Fluo-4-loaded single CHO cells uncovered a
consistent delay between the first detectable increment of
intracellular calcium and the activation of calcium influx current.
Approximately 85% of the cells studied (43 of 50) responded to a
saturating Bk concentration (100 nM) with a rapid elevation in intracellular calcium observed within the first 50 ms after Bk
application, followed 900 ± 100 ms later by an increment in inward current (Fig. 1). In these
experiments the extracellular solution was PBS and the intracellular
solution was the NA solution with 100 nM free calcium
("Materials and Methods"). Under these conditions the inward
current activated by Bk showed a reversal potential of +45 ± 5 mV
(n = 40), only a current carried primarily by calcium
ions would show such a positive reversal potential given the solutions
used. Replacing the bath solution for the NMDG-Cl solution with 2 mM calcium ("Materials and Methods") did not result in
a significant change of the reversal potential (+48 ± 4 mV,
n = 17), further supporting the calcium selectivity of the Bk-activated inward current.
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Fig. 1.
Simultaneous measurements of rapid confocal
calcium and electrophysiology. A typical calcium
mobilization experiment with a single cell, measuring simultaneous
Fluo-4 fluorescence and whole cell currents in response to 100 nM bradykinin. Each panel shows single cell fluorescence
and whole cell currents obtained at time 5 s, and 0.2, 0.4, 0.6, 0.8, and 1 s after bradykinin application. The horizontal
(red) arrow points to the zero current level. The
voltage protocol was changed from 0 mV (holding potential) to 100 mV
followed by a ramp to +100 mV with 50 ms duration. The pipette (NA)
solution contained 100 nM free calcium adjusted with EGTA
as indicated under "Materials and Methods," the bath solution was
PBS with 2 mM calcium. The scale to the
left shows 10 µm. Scale to the left middle of
the figure illustrates the fluorescence intensity in a pseudo-color
scale from 0 to 10 arbitrary fluorescent units. Scale at the left
bottom shows 25 ms and 4 pA/pF.
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The delay between the first detectable increment of intracellular
calcium and the activation of calcium influx current was slightly
longer in cells studied in the perforated patch mode using nystatin in
the patch pipette (800 ± 50 ms, n = 35). The differences between the whole cell and the perforated patch mode were
not statistically significant, probably because of the fact that whole
cell experiments were completed within the first minute after
disrupting the plasma membrane. Under these conditions, the dilution of
intracellular components was not favorable.
The initial fluorescence increment (IFI) obtained after Bk stimulation
was localized to well defined spots in the cells (Fig. 1). Because no
inward current was measured in this initial time periods, the IFI most
likely reflects the release of calcium from intracellular storage
compartments (ER). By the time the inward current was activated, the
elevation in fluorescence intensity was observed throughout the
cytosol, part of which most likely reflects the influx of calcium from
the extracellular space.
CaM Increases the Delay Period between Initial Release from ER and
Activation of SOC--
Fig.
2A illustrates representative
experiments using combined rapid confocal microscopy and whole cell
current measurements. Under control conditions, after Bk stimulation, a
typical delay of 900 ± 100 ms between IFI and activation of
inward current was observed. Introduction of two CaM selective
antagonists, W7 and TFP, in the patch pipette significantly reduced the
delay period between IFI and inward current activation, when compared
with control experiments (Fig. 2). Under these conditions, the delay period observed was 200 ± 100 ms with 5 µM TFP
(n = 16) and 150 ± 50 ms with 500 nM
W7 (n = 22). This reduction represents 1/4 of the control value. Increasing TFP to 20 µM or W7 to 5 µM did not result in further reductions of the delay
period (data not shown) therefore, the remaining studies were carried
out with these CaM antagonists concentrations.
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Fig. 2.
CaM modulates the delay period between
depletion of the ER and activation of SOCE. A,
representative examples of simultaneous Fluo-4 fluorescence and whole
cell currents in response to 100 nM bradykinin under
control conditions, and with 500 nM W7 or 10 µM CaM in the patch pipette. The numbers on
each panel are in seconds. Time 0 was considered the
acquisition time at which the IFI was detected. All experiments were
aligned to this time point. Fluorescence intensity was color and height
coded based on a scale in arbitrary fluorescent units (0 to 10). Scale
for whole cell currents shows 50 ms and 6 pA/pF. Horizontal
arrows (in red) point to the 0 current level. The
pipette (NA) solution contained 100 nM free calcium
adjusted with EGTA and the bath solution PBS with 2 mM
calcium. B, mean ± S.D. of the inward current
amplitude (pA/pF) stimulated by 100 nM Bk. The zero time
point represents the point at which the first detectable increment in
cell fluorescence (IFI) was observed. Data shows the inward current
amplitude in control cells (filled circle), cells exposed to
500 nM W7 (open triangle), 5 µM
TFP (filled triangle), and 10 µM CaM
(open square) in the pipette solution. The solid
lines show the fit to a single exponential function from which the
time constants illustrated in C were obtained. C,
time constants (time to maximum current) and delay times (time between
first detectable fluorescence increment (IFI) and activation of inward
current) for cells under control conditions and exposed to W7, TFP, or
CaM in the patch pipette. Symbols correspond to the
conditions shown in B. Each data point represents mean ± S.D.
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Introduction of CaM in the patch pipette resulted in a significant
increment of the delay period between IFI and the activation of inward
current. In experiments where 10 µM CaM was introduced in
the patch pipette, the delay period increased to 10 ± 2.1 s, a value around 10 times greater than control (n = 18, Fig. 2B). The CaM effects were observed with 100 nM free intracellular calcium (in the patch pipette)
adjusted with EGTA ("Materials and Methods"). Buffering calcium in
the patch pipette to 1 nM prevented the effects of CaM on
the delay period between IFI and activation of inward current (data not
shown). With 1 nM free intracellular calcium the delay
period observed in the presence of 10 µM CaM was 950 ± 150 ms (n = 7), a value not significantly different
from control (p > 0.1). These results suggest that the
CaM-calcium complex modulates the delay period between the initial
release of calcium from the ER and the activation of SOC. CaM blockade
reduces significantly the delay period, and SOCE initiates faster.
CaM and the CaM antagonists affected also the time to reach the maximum
current. In control conditions, the whole cell current density reached
its peak with a time constant of 15 ± 4.1 s, to a value of
6.12 ± 0.13 pA/pF at 100 mV (n = 25, Fig.
2C). With 10 µM CaM in the pipette, the
maximum current density was reached with a time constant of 25 ± 5.2 s to a value of 3.65 ± 0.34 pA/pF (n = 18, Fig. 2C). The CaM antagonists reduced the time to reach the maximum current density to 6.2 ± 1.8 s with TFP
(n = 16, Fig. 2C) and 6.8 ± 1.5 s
with W7 (n = 22, Fig. 2C). The maximum
current density obtained with CaM was about 60% from control (Fig.
2B). Both CaM antagonists did not alter significantly the
maximum current density compared with control (6.16 ± 0.43 and
6.12 ± 0.32 pA/pF for W7 and TFP, respectively, Fig.
2B). These results resemble the effects of CaM on the
TG-induced calcium influx current from vascular endothelial cells
(11). To determine whether the effects observed with CaM were
the result of calcium buffering by this calcium-binding protein, we
performed similar experiments to those indicated above but replaced CaM
with 20 µM S-100 protein from bovine brain, a
calcium-binding protein with some structural similarity to CaM.
With 20 µM S-100 in the patch pipette, the delay period
between IFI and activation of inward current was 850 ± 120 ms
(n = 8), a value not significantly different from the
value obtained in the absence of S-100 (910 ± 140 ms,
n = 6). These results strongly suggest that the effects
observed with CaM are not the result of buffering the calcium in the cell.
Because any maneuver that alters the rate of release of calcium from
the ER may affect also the time of activation of SOC (17), we designed
experiments to explore the effect of incubating cells with W7 and TFP
on the rate of calcium release from the ER. To explore this we took two
avenues: first we explored the increments in intracellular calcium in
CHO cells incubated for 5 min with the CaM antagonist and compared the
time course of calcium increments with control cells (not exposed to
CaM antagonists). To ensure that the CaM antagonists were permeating
adequately into the cells, we performed additional experiments in which
the CaM antagonists were introduced in the cell via the patch pipette, as we have done in previous experiments. With both experimental procedures the extracellular (PBS) solution contained less than 1 nM free calcium, to minimize the effects of calcium influx. Under these experimental conditions, fluorescence increments would reflect primarily the release of calcium from the ER.
Fig. 3A illustrates
representative experiments of the increment in single cell fluorescence
after application of 100 nM Bk in a cell under control
conditions and a cell incubated for 5 min with W7. Fig. 3B
shows the time course of the increments in fluorescence in response to
Bk in control cells (CON, n = 16) and
W7-treated cells (n = 14). Fig. 3C shows
representative examples in an expanded time scale to illustrate the
time course of the rise in average cell fluorescence in a control cell
(CON) and a cell exposed to TFP or W7 for 5 min. The time
course of the rapid increment in intracellular calcium was well fitted
by a sigmoidal function, providing half-activation constants of
340 ± 20 ms for the control condition, 325 ± 25 ms with W7,
and 320 ± 20 ms with TFP. Similar time constants were obtained in
cells in which the CaM antagonists were introduced via the patch
pipette (data not shown). Under these conditions, half-activation
constants were 350 ± 22 ms for control cells (n = 8), 318 ± 21 ms for W7 (n = 12), and 321 ± 25 ms for TFP (n = 14).
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Fig. 3.
CaM does not affect the rate of release of
calcium from the ER. A, representative experiment
illustrating the rapid increment in intracellular fluorescence of
Fluo-4-loaded cells induced by 100 nM Bk in control
conditions and in a cell incubated for 5 min with 500 nM W7
measured by confocal microscopy. B, mean ± S.D. from
several independent experiments like those in A. The data
points for TFP were not included because they overlapped with control
and W7. C, time scale magnification to illustrate better the
rapid increment in fluorescence in response to Bk. Solid
lines represent the fit to a sigmoidal function from which the
half-time activations were obtained. The time constants obtained were
340 ± 20 ms for the control condition, 325 ± 25 ms with W7,
and 320 ± 20 ms with TFP. The bath solution was PBS with 1 nM free calcium adjusted by the addition of EGTA
("Materials and Methods").
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As indicated by these results, neither of the CaM antagonists altered
significantly the time course of average cell fluorescence increments
in response to Bk. Because these experiments were performed in low
extracellular calcium (<1 nM), the increment in
fluorescence reflects essentially the release of calcium from ER.
Therefore, these results strongly suggest that the effects of CaM and
the CaM antagonists on the inward current are not the result of
altering the time course of calcium release from ER, but rather because of the modulation of the communication between release of ER and SOC activation.
Identification of the Channels Activated after Depletion of the
ER--
In the next series of experiments we explored the ionic
selectivity of the Bk-activated inward current. With the PBS
extracellular solution containing 2 mM calcium, the
current-voltage relationship presented a reversal potential of 40 ± 5 mV (n = 22). A similar inward current was
activated also with 200 nM of the calcium ATPase inhibitor,
TG. Fig. 4A illustrates a
representative experiment with the TG-induced inward current. The
current reversal potential obtained from the current-voltage
relationship was 46 ± 6 mV (n = 15), a value not
significantly different from the reversal potential obtained for the
Bk-activated current.
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Fig. 4.
Identification of the single channel
conductance mediating SOCE. A, representative whole cell
currents obtained under control conditions (before the addition of TG),
and after the addition of 200 nM TG with 2 mM
extracellular calcium (TG + Ca2+) or 1 nM calcium (TG Ca2+) in the PBS
solution. B, single channel conductance induced by 100 nM Bk or 200 nM TG in the cell-attached mode.
The holding potential was 100 mV. The pipette PBS solution contained
1 nM free calcium. C, inside-out patches
containing a channel activated by 200 nM TG in the
cell-attached mode with 1 nM (TG Ca2+) or 2 mM (TG + Ca2+)
calcium in the patch (extracellular) pipette solution. D,
current-voltage relationships (mean ± S.D.) for channels
activated by TG or Bk with 2 mM or 1 nM
extracellular calcium, as indicated by symbol legends.
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Reducing the extracellular calcium to less than 1 nM
("Materials and Methods") resulted in a shift of the reversal
potential to 1 ± 4 and 0 ± 3 mV for the Bk
(n = 12) and the TG (n = 9)-induced currents, and a linear current-voltage relationship (Fig.
4A). These results indicate that, in the absence of
extracellular calcium, the TG-Bk-induced current permeates primarily
monovalent cations. The switch in selectivity from divalent to
monovalent cations upon removal of extracellular calcium has been
previously observed for other depletion-activated channels (18,
19).
Application of 100 nM Bk in the cell-attached mode produced
the activation of single channel currents in 22 of 35 attempts; the
remaining patches did not show channel activity upon agonist stimulation. 200 nM TG induced similar single channel
currents in 15 of 22 attempts. Fig. 4B illustrates
representative experiments showing the channel activity induced by Bk
or TG. In the majority of the experiments, both agonists induced the
activity of 2-4 channels, suggesting some form of channel clustering.
Channel activity was induced in the cell-attached mode and then
explored at different voltages in the inside-out configuration. For
these experiments we used either 2 or 1 nM extracellular
free calcium ("Materials and Methods"). Examples of channel
activity in the inside-out configuration are illustrated in Fig.
4C. With 2 mM calcium, the channel reversal
potential was 55 ± 5 and 0 ± 3 mV with 1 nM
calcium (Fig. 4D). The current-voltage relationships for the
TG and the Bk-induced channel activity were indistinguishable (Fig.
4D). The single channel conductance measured from the slope obtained between 100 and +100 mV in low extracellular calcium (1 nM) was 10 and 2.5 pS with 2 mM extracellular
calcium (Fig. 4D).
Excising the patches of membrane from previously stimulated cells to
form the inside-out configuration resulted in rapid run-down of channel
activity as illustrated in Fig. 5.
Channel activity in the inside-out mode could be recovered by the
application of 10 µM IP3 (Fig.
5A). The effect of IP3 was inhibited when
the patch of membrane was simultaneously exposed to 1 µM
CaM (Fig. 5B). Fig. 5C illustrates the number of
channels multiplied by the open probability
(NPo) measured over time for the representative experiment shown in Fig. 5A, and Fig. 5D shows
the NPo plot for the experiment in
B.
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Fig. 5.
IP3 recovers channel activity in
excised patches. A, typical channel activity induced in the
cell-attached (on-cell) mode with 100 nM Bk. Patch was
later excised to the inside-out configuration and channel activity was
run-down rapidly. Channel activity was recovered by the addition of 10 µM IP3 to the intracellular face of the
channel. B, similar experiment to that illustrated in
A, except that in the inside-out mode, the channel was
exposed simultaneously to 10 µM IP3 and 1 µM CaM. Scale shows 2 pA and 2 s. Downward
deflections represent inward current carried by the influx of calcium.
C, open probability multiplied by the number of channels
(NPo) measured over time for the experiment
illustrated in A. D, NPo measured
overtime for the experiment illustrated in B. Pipette
(extracellular) solution was PBS with 2 mM calcium and bath
(intracellular) solution was NA solution with 100 nM free
calcium adjusted by the addition of EGTA as indicated under
"Materials and Methods." The holding potential was 100 mV.
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We characterized further the antagonistic effects of CaM and
IP3 on single channel activity. Fig.
6A shows the recovery of channel activity with three different IP3 concentrations.
As illustrated in the figure, the maximum activity was obtained with 10 µM IP3. However, even with 100 µM IP3 the effect of CaM was evident (Fig. 6A). Fig. 6B shows the Po
plot for the experiment illustrated in Fig. 6A. Fig.
6C shows the pooled data from 15 independent single channel
experiments in which three different concentrations of CaM and
IP3 were explored. As indicated in the figure, the inhibitory effect of CaM prevailed, even at 100 µM
IP3, which was the concentration that induced the maximum
increment in channel Po. With 100 µM IP3, the Po
decreased from 0.8 ± 0.1 with 0.1 CaM to 0.4 ± 0.09 with 1 µM CaM, to 0.2 ± 0.05 with 10 µM CaM (Fig. 6C). The dose-response curves were well fitted by a
Hill equation, providing Hill coefficients of 1.8 for 0.1 CaM, 3 for 1 µM CaM, and 2.8 for 10 µM CaM. These
results indicate that at least two CaM may be required for the
inhibition of channel activity.
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Fig. 6.
Antagonistic effects of IP3 and
CaM in the modulation of channel activity. A,
representative experiment of channel activity in the inside-out
configuration under control conditions (CON), and with 1, 10, and 100 µM IP3 perfused to the
intracellular face of the channel. With 100 µM
IP3 the patch was exposed also to 1 and 10 µM
CaM. Downward deflections represent inward current carried by the
influx of calcium B, changes in the channel
Po over time induced by the procedures
illustrated in A. C, changes in channel
Po (mean ± S.D.) induced by three
concentrations of CaM (0.1, 1, and 10 µM) at three
IP3 concentrations (1, 10, and 100 µM). The
solid lines indicate the fit to a Hill equation with Hill
coefficients of 1.8 for 0.1 CaM, 3 for 1 µM CaM, and 2.8 for 10 µM CaM. Pipette (extracellular) solution was PBS
with 2 mM calcium and bath (intracellular) solution was NA
solution with 100 nM free calcium adjusted by the addition
of EGTA as indicated under "Materials and Methods." The holding
potential was 100 mV.
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These results indicate that the inhibitory effect of CaM on channel
activity prevailed even at saturating concentrations of IP3, suggesting a dominant effect of CaM on channel
activity. The consequences of this finding in the modulation of SOCE
will be discussed later in this article.
The effect of IP3 on channel activity can have at least two
possible explanations. One is that IP3 may be interacting
directly with the channel. The second possibility is that
IP3 may activate the IP3R, which in turn might
modulate channel activity at the plasma membrane. For the second
hypothesis to be true, the excised patches of membrane must retain
fragments of the ER with functional IP3Rs.
Modulation of TRP channel activity by peptides from the type 3 IP3R has been previously shown in excised patches from
cells expressing hTRPC3 (20). Furthermore, IP3R and CaM
share a common binding site in several TRPC channels (21).
To explore if the effects of IP3 observed in the present
study were mediated by IP3R, a series of experiments were
designed to study the effect of a synthetic peptide from the type 3 IP3R on whole cell currents and single channel activity.
Fig. 7A illustrates the effect
of introducing 50 µM of the NH2-terminal
peptide from the type 3 IP3R (IP3R-P1) into the
cell via the patch pipette. This peptide has been previously shown to
induce hTRPC3 channel activity in excised patches and bind to several
TRP channels, competing with CaM for this common binding site (21).
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Fig. 7.
A peptide from the IP3R modulate
single channel activity. A, whole cell inward currents
induced by the addition of peptide IP3R-P1 in the patch
pipette. The pipette (NA) solution (with 100 nM free
calcium) contained either 50 µM IP3R-P1
(filled circles) or IP3R-P2 (open
circles, scrambled peptide used as control). CON
represents the current induced in the absence of the peptides
(filled triangles). The solid bar indicates the
application of 200 nM TG. The vertical arrow
shows the point at which the plasma membrane was disrupted to form the
whole cell configuration (WCC). This point was considered
time 0 for all experimental conditions. The current amplitude was
measured at 100 mV every minute using the voltage ramp protocol
described in the legend to Fig. 1. B, mean ± S.D. of
the inward current amplitude (pA/pF) induced by 0.1, 1, 5, and 50 µM IP3R-P1. C, effect of 50 µM IP3R-P1 and subsequent addition of 100 µM IP3 on single channel
Po obtained in the inside-out configuration.
After channel Po reached steady state,
application of 10 µM CaM produced a typical channel
Po reduction. D, effect of 50 µM IP3R-P2 (scrambled peptide) and 100 µM IP3 on single channel
Po obtained in the inside-out configuration.
Under these conditions, application of 10 µM CaM resulted
also in typical channel Po reduction. Pipette
and bath solutions are those used in Figs. 4 and 5. The holding
potential was 100 mV.
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Introducing the IP3R-P1 peptide resulted in activation of
inward current in the absence of TG or Bk stimulation in 12 of 14 cells
explored (Fig. 7A). Application of 200 nM TG
after the IP3R-P1-induced inward current reached steady
state resulted only in a minor increment in current amplitude. Cells
not exposed to the IP3R-P1 or cells exposed to 50 µM of the scrambled peptide IP3R-P2 (used as
control) did not produce inward current until the application of 200 nM TG, which resulted in typical inward current activation.
The amount and time course of inward current induced by TG in the
presence of IP3R-P2 was indistinguishable from the current
activated in the absence of both peptides (Fig. 7A,
CON).
These results indicate that TG cannot increase further the current
activated by IP3R-P1, suggesting that this peptide and TG activate the
same single channel conductance. On the contrary, the
IP3R-P2 peptide failed to activate inward current.
Fig. 7B illustrates a dose-response curve for
IP3R-P1 showing that the maximum amount of current is
obtained with 50 µM of this peptide. Fig. 7C
shows the effect of IP3R-P1 on single channel activity from
excised inside-out patches. As illustrated in the figure,
IP3R-P1 increased channel Po from
0.12 ± 0.1 to 0.71 ± 0.11 (n = 6). Addition
of 100 µM IP3 in the continuous presence of
IP3R-P1 resulted only in a minor (not statistically
significant) increment of channel Po to
0.76 ± 0.13 (n = 6; p > 0.1).
Addition of 10 µM CaM, in the continuous presence of
IP3R-P1, resulted in rapid reduction of channel
Po from 0.76 ± 0.13 to 0.21 ± 0.09 (n = 6).
Similarly to the whole cell experiments, IP3R-P2 failed to
increase channel activity in inside-out patches as illustrated in Fig.
7D (control = 0.15 ± 0.12, 50 µM
IP3R-P2 = 0.16 ± 0.12, n = 6).
However, in the presence of IP3R-P2, 100 µM
IP3 increased channel activity to 0.69 ± 0.13 in 5 of
6 patches tested. Under these conditions, 10 µM CaM
produced a typical Po channel reduction from
0.7 ± 0.11 to 0.26 ± 0.13 (n = 6). These
results strongly suggest that part of the effects observed with
IP3 on SOC channel activity (Fig. 6) is mediated by the
IP3R. Furthermore, similarly to the results obtained with
IP3, the inhibitory effect of 10 µM CaM
prevailed even in the presence of 50 µM
IP3R-P1 peptide.
TRP1 Is an Essential Component of SOCE in CHO Cells--
In an
attempt to determine the TRP channels present in CHO cells and
responsible for the inward current activated after depletion of the ER,
we performed PCR experiments using degenerated oligonucleotides that
ensure the identification of several of the TRPC members ("Materials
and Methods"). We tested these oligonucleotides with a cDNA
library from rat retina and found PCR amplification of TRP1, TRP4,
TRP5, and TRP6. However, in five independent PCR reactions using
cDNA from CHO cells, we found exclusively the amplification of a
single product. The product obtained under these conditions was
sequenced and compared with the data base at the National Center for
Biotechnology Information using the Blast program. The results indicate
that the product obtained was over 93% identical to rat TRP1 at the
nucleotide level (accession number AF061266) and 99% identical at the
amino acid level. These results show the presence of a TRP1 homologue
in CHO cells, confirming previously published results showing
expression of TRP1 and TRP2 in this cell line (22).
Although these results provide evidence indicating that CHO cells
possess a TRP1 homologue, a direct correlation between this channel and
SOCE was necessary. To accomplish this, we took advantage of the fact
that introduction of dsRNA into a wide variety of cells and organisms
results in potent and highly specific gene silencing (23). This
phenomenon has been termed RNA interference or RNAi (24).
Experiments were carried out to evaluate the role of TRP1 in the Bk-
and TG-induced SOCE. For this purpose, dsRNA from TRP1 was synthesized
by standard techniques and introduced into CHO cells using
LipofectAMINE ("Materials and Methods"). A repetitive transfection
protocol was designed to ensure the transfection of the majority of
cells ("Materials and Methods"). Calcium measurements with cell
populations from TRP1-dsRNA-treated CHO cells showed that TG-induced
calcium influx was markedly reduced in these cells, without any
significant effect on the release of calcium from the ER (Fig.
8A).
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Fig. 8.
TRP1 is an essential component of SOCE in CHO
cells. Changes in intracellular calcium monitored in Fura-2-loaded
cell populations. Each experiment was performed with 1.5-2 million
cells in suspension. Experiments were initially performed with 1 nM free extracellular calcium obtained by the addition of
EGTA as indicated under "Materials and Methods" (Rel) and later 2 mM calcium was added to the bath solution (Inf).
Data shows the mean ± S.D. from several independent measurements.
A, control cells, and B, cells treated with dsRNA
from TRP1 ("Materials and Methods"). The area under the curve
(gray boxes) was integrated for each experimental condition
representing the release (Rel) and calcium influx
(Inf) and plotted in C for TG-, and D
for Bk-stimulated cells. Notice that the release was not affected by
treatment with dsRNA from TRP1 but the influx activated by TG and Bk
(B-Inf) was significantly reduced. E, Western
blotting experiments illustrating the effect of transfecting cells with
dsRNA on protein concentration. Plasma membranes from control and
dsRNA-treated cells were isolated as indicated under "Materials and
Methods." Ten micrograms of protein were loaded in each lane and
exposed to specific anti-Bk2R and anti-TRP1 antibodies. Lane
1, cells exposed to anti-Bk2R; lane 2, cells exposed to
anti-TRP1; lane 3, cells treated with dsRNA for Bk2R and
exposed to both antibodies; lane 4, cells treated with dsRNA
for TRP1 and exposed to both antibodies. Notice that in lane
3 the signal for Bk2R was significantly reduced. In lane
4 the signal for TRP1 was also significantly reduced by treatment
with dsRNA from this channel. F, representative whole cell
currents obtained under control conditions (before the addition of
thapsigargin in a control cell, TG) and after addition of
200 nM TG in a control cell (+TRP1) and a cell
exposed to dsRNA for TRP1 in the presence of 200 nM TG
(TRP1). G, mean ± S.D. of the whole cell
current density (WCI) obtained at 100 mV in control cells
exposed to 100 nM Bk, cells exposed to dsRNA for Bk2R, and
current induced with 200 nM TG (TG-Bk2R), cells
exposed to dsRNA for TRP1 and stimulated with 100 nM Bk
(Bk-TRP1) or 200 nM TG (TG-TRP1).
Notice the significant reduction in current density in response to Bk
and TG cells exposed to dsRNA for TRP1. The sequence used for dsRNA
from Bk2R was nucleotides 420-762, and the sequence for TRP1 was the
entire PCR fragment (450 bp; GenBankTM accession number
AF448492).
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The integral of calcium influx from control cells in response to TG was
360 ± 80 nM × s
1 (Fig.
8C, n = 32). In TRP1-dsRNA-treated CHO cells
the integral calcium influx was significantly reduced to 36 ± 28 nM × s1 (n = 23). Similarly
to the TG experiments, the calcium influx induced by Bk in
TRP1-dsRNA-treated CHO cells was also greatly reduced. The integral of
calcium influx in response to Bk in control cells was 440 ± 51 nM × s1 (Fig. 8D,
n = 24). This value was reduced to 50 ± 26 nM × s1 (n = 24) in
TRP1-dsRNA-treated CHO cells.
The reduction of TRP1 protein in dsRNA-treated CHO cells was confirmed
by Western blotting analysis. As illustrated in Fig. 8E, CHO
cells stably transfected with the Bk2R react with the specific
anti-Bk2R antibody showing a single band (Fig. 8E,
lane 1). The same cells exposed to the specific anti-TRP1
antibody also recognize a single product (Fig. 8E,
lane 1). Cells not transfected with Bk2R do not show a
specific band for this receptor; however, under these experimental
conditions, a single product is recognized by the specific anti-TRP1
antibody (Fig. 8E, lane 2).
The use of dsRNA for the Bk2R significantly reduced the amount of Bk2R
protein as illustrated in Fig. 8E, lane 3. This
treatment did not affect the expression of TRP1 (Fig. 8E,
lane 3). On the other hand, CHO cells transfected with Bk2R
and treated with dsRNA for TRP1 did not alter the expression of Bk2R
but greatly reduced the amount of TRP1 protein (Fig. 8E,
lane 4). These results indicate that the RNAi is specific
for the sequence used. Interfering with the expression of Bk2R did not
alter the expression of TRP1 protein and vice versa.
These observations are consistent with the calcium experiments, where
introduction of dsRNA for TRP1 reduced significantly the Bk- and
TG-induced calcium influx, but not the release from the ER (Fig. 8,
A-D). Similarly, introduction of dsRNA for Bk2R eliminated
the Bk-induced calcium mobilizations (both release from the ER and
calcium influx) without affecting the TG-induced calcium release from
the ER and calcium influx (data not shown). The lack of effect of Bk
stimulation in Bk2R-dsRNA-treated cells may represent the interference
of the dsRNA with the synthesis of Bk2 receptors in these cells,
because the IP3 cascade appears to be unaffected as
indicated by experiments in which the stimulation of endogenous CHO
cells purinergic receptors resulted in typical increments in the
intracellular calcium concentration (data not shown).
Electrophysiology experiments performed with TRP1 dsRNA-treated cells
showed a dramatic reduction of the Bk- and TG-induced inward current,
consistent with the results obtained from calcium measurements. Fig.
8F shows representative experiments of a control cell
(before the addition of TG), a cell exposed to 200 nM TG (+TG), and a cell treated with dsRNA from TRP1 (TRP1).
The current density obtained at 100 mV with 200 nM TG in
control cells (not exposed to TRP1 dsRNA) was 6.2 ± 0.95 pA/pF
(n = 14, Fig. 8G, TG-Bk2R). This
value was significantly reduced in TRP1 dsRNA-treated cells showing
current density of 0.21 ± 0.35 pA/pF under the same conditions
(n = 23, Fig. 8G, TG-TRP1). Similar results
were obtained after Bk stimulation, where control cells showed current
densities of 6.3 ± 0.82 pA/pF (n = 12, Fig. 8G, Bk) and TRP1 dsRNA-treated cells of 0.17 ± 0.5 pA/pF (n = 19, Fig. 8G,
Bk-TRP1). Similarly to the results obtained with calcium
measurements, the TG-induced activation of the inward current was not
affected in CHO cells exposed to dsRNA from the Bk2R (Fig.
8G,TG-Bk2R), indicating specificity of the dsRNA
sequence used in the interference experiments. All these results
indicate either that TRP1 channels mediate most of the SOCE in CHO
cells, or that the TRP1 protein is an essential component of SOCE.
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DISCUSSION |
Activation of calcium influx after depletion of intracellular
calcium storage compartments is a phenomenon observed in practically all cells explored from a wide variety of organisms (1). For many years
the molecular identity of the SOC responsible for the influx of calcium
after depletion of intracellular stores remained elusive. However,
recently several candidates have been proposed.
Our initial findings showed that the TRP from Drosophila
melanogaster encoded a thapsigargin-activated, calcium-permeable channel (3). Because of this study, several TRP homologues have been
identified in a wide variety of organisms from yeast to humans (4,
6).
The best characterized SOC is the calcium release-activated calcium
current (Icrac) (25). The recent finding showing
that the expression of CaT1 (a calcium channel related to the TRP
superfamily) produces channels that are indistinguishable from
Icrac (26) further supports the hypothesis that
some members from this large family of cationic channels may be
responsible for SOCE.
Although the identification of new TRP homologues has been very
successful, the clear demonstration that any of the members from this
new family of channels may be responsible for SOCE has not been so
fruitful (7). Expression of some TRP homologues results in augmented
SOCE, but these findings are not consistent from cell to cell or from
group to group (for review see Ref. 2). An explanation for this
inconsistency has not been found to the present time.
One possible explanation might rely on the assumption that introduction
of foreign channels in a given cell may not necessarily result in a
physiological interaction between the TRP channel and the mechanisms
modulating SOCE. To complicate things further, several TRP homologues
aggregate to form functional heteromultimers with novel properties. In
particular, it has been shown that TRP1 and TRP5 form novel cationic
channels in the mouse brain (27), while co-expression of
Drosophila TRP and TRPL result in SOC channels with novel
properties (28).
To avoid this complication we decided to study endogenous TRP1 channels
from CHO cells, a cell line that has been frequently used to express
TRP homologues and to study SOCE. Our results show the presence of a
TRP1 homologue in this cell line. We consistently isolated a fragment
of TRP1 using degenerated oligonucleotides from conserved regions of
several TRP mammalian homologues ("Materials and Methods"). This
result is in agreement with previously published data showing
endogenous expression of TRP1 and TRP2 in CHO cells (22).
The RNAi technique allowed us to study the contribution of endogenous
TRP1 channel to SOCE in CHO cells. We have shown here that introduction
of dsRNA from TRP1 results in specific reduction in the amount of TRP1
protein and potent inhibition of SOCE. These results strongly suggest
that TRP1 is a major component of SOCE in CHO cells. Most importantly,
no sequence homology was observed between the TRP1 fragment from CHO
cells used in the RNAi experiments and TRP homologues other than TRP1.
This is important because, if substantial sequence similarities
were to be found between TRP1 and other TRP homologues (TRP2, TRP3,
etc.), then the dsRNA from TRP1 could have interfered with these other
channels, making difficult the interpretation about the relative
contribution of TRP1 to SOCE.
We have used combined rapid confocal calcium and electrophysiological
measurements to explore the correlation between the first measurable
release of calcium from the ER and the activation of calcium current.
Although this technique cannot detect the first release of calcium from
the entire ER, because we are scanning from a section of the cell
10-µm thick, what we could measure was the first release of calcium
in that particular focal plane.
Using these techniques we have observed consistently a delay of around
900 ms between the first measurable intracellular calcium increment and
the activation of calcium current induced by Bk. What this result
indicates is that at least 900 ms separate the first detectable release
from the ER from the activation of calcium influx. A delay between
depletion of the ER and activation of SOC has been observed also for
Icrac in different cells (29, 30). The mechanism
underlying this phenomenon has not been satisfactorily explained yet.
Using combined confocal microscopy and the patch clamp technique we
have studied the contribution of CaM to the delay period between the
initial release of intracellular calcium from internal stores and the
activation of calcium current. In our previous work we have shown that
CaM inhibits the TG-activated calcium current in vascular endothelium
in a concentration dependent manner (11). In the present study we have
explored in more detail the mechanism by which CaM modulates SOC.
Our results show that CaM increases the delay period between the first
measurable intracellular calcium increment and the activation of
calcium current, whereas CaM antagonists significantly reduced the
delay period. In the presence of CaM the amount of calcium current was
60% from control, whereas CaM antagonists did not affect the current
amplitude. The effects of CaM were not observed when intracellular
calcium was maintained at 1 nM by the addition of EGTA.
These results indicate that CaM not only increases the period between
release of intracellular calcium and activation of calcium influx, but
also modulates the amount of calcium current activated after depletion
of the ER. CaM requires free calcium above 1 nM to exert
its effects on TRP1. We have previously shown that the inhibition of
SOC by CaM in vascular endothelium requires also free calcium above 1 nM (11). In Drosophila photoreceptors, the
CaM-calcium complex mediates termination of the light response in
vivo via modulation of TRP and TRPL channels (12). TRP possess one
CaM binding domain whereas TRPL has two, in both channels these CaM
binding domains are at their carboxyl-terminal ends. In
mammalian TRP channels at least one CaM binding site has been recently
identified also at the COOH-terminal region (9). All these results
suggest that CaM may play a conserved role as an inhibitor of TRP
channel activity. This conclusion is further supported by the single
channel experiments reported here, showing that the CaM-calcium complex
reduces drastically single channel open probability in a
concentration-dependent manner.
Single channel activity induced by either TG or Bk runs down rapidly in
excised patches, the addition of IP3 recovered the activity. There are at least two possible explanations for the effect
of IP3. One is that IP3 may directly bind to
the TRP1 protein; the second possibility is that IP3 may
bind to a regulatory protein associated to TRP1 and responsible for
channel activation.
An interesting candidate for the second hypothesis is IP3R.
It is possible that the excised patch of membrane may contain pieces of
ER with IP3Rs. In fact, functional interactions between human TRP3 and IP3R have been previously studied in this
patch clamp configuration (31). It has been shown that several members of the TRPC subfamily (TRP1-TRP7) have a common binding site for CaM
and peptides from the IP3R (9). This common binding site is
located at the COOH-terminal region of TRP channels, in particular amino acids 720-749 from human TRP1. This region is perfectly conserved in mouse, rat, and Xenopus laevis TRP1
proteins. Although we have not isolated yet the full-length cDNA
from CHO-TRP1, this amino acid region is most likely present also in
CHO-TRP1, which is over 93% identical to rat TRP1. Interestingly, it
has been reported that amino acids 664-793 from human TRP1 are
involved in the modulation of SOCE, by exerting an inhibitory effect
(32).
A recent study shows activation of human TRP3 by peptides from the
IP3R through displacement of inhibitory calmodulin from a
common binding domain (21). We have used a synthetic peptide (IP3R-P1) containing this sequence from the type 3 IP3R, which has been previously shown to interact with TRP
channels displacing CaM from a common binding domain. The results
presented here show that introduction of the IP3R-P1
peptide into the cell via the patch pipette activates the calcium
current in CHO cells in the absence of any stimulation with TG or Bk.
After current activation with the peptide, addition of TG induced only
a minor increment in current amplitude, indicating that TG and
IP3R-P1 activate the same channel. The scrambled peptide
IP3R-P2, which contains the same amino acids found in
IP3R-P1 but in random order, failed to activate calcium
current, however, the subsequent addition of TG produced typical
current activation.
In excised inside-out patches, the IP3R-P1 peptide induced
channel activity with similar conductance to the channel activity induced by TG and Bk. Subsequent addition of 100 µM
IP3 failed to increase channel activity induced by the
IP3R-P1 peptide. These results show that the
IP3R-P1 peptide mimics the effect of IP3 in
terms of recovering channel activity after run-down, strongly suggesting that at least part of the effects produced by
IP3 in single channels is mediated by the
IP3R.
The results presented here are consistent with a previous report (21)
showing an antagonistic effect of IP3R and CaM for the
activation of TRP3 channel activity. However, we show here that this
competitive interaction between IP3R and CaM is sufficient to explain the activation of TRP1 channels and SOCE, after depletion of
the ER in CHO cells. Our results also show that the inhibitory effect
of 10 µM CaM on TRP1 channel activity prevails even at saturating IP3 concentrations or with 50 µM
IP3R-P1 peptide. These results suggest that CaM has a
dominant effect on channel activity, which may play a role in
preventing calcium influx under nonstimulated resting conditions.
Whereas the competitive interaction between IP3R and CaM
may be sufficient to explain the mechanism by which agonist stimulation leads to SOCE activation, less clear is the mechanism induced by
calcium ATPase inhibitors. We found that TG and Bk activate similar
calcium currents and single channel conductance, and that this
conductance is eliminated by the introduction of dsRNA from TRP1.
Furthermore, introduction of IP3R-P1 peptide into the cell produced current activation, under these conditions; subsequent addition of TG resulted in only a minor increment in current amplitude. All these results strongly suggest that the mechanism by which TG
induces SOC activity involves the IP3R. One possible
explanation may involve changes in ER intraluminal or cytosolic calcium
induced by TG, which may result in changes in IP3R
activity. Changes in IP3R activity may modulate
protein-protein interactions between IP3R and TRP1, as
proposed in a recent review (33).
One way of testing this hypothesis would be monitoring the simultaneous
activity of IP3R and TRP1 channels in response to TG and
Bk. Finding a correlate between IP3R and TRP1 channel
activities has proven very difficult so far. We are currently working
in the design of experimental procedures, which may allow us to monitor the simultaneous activity of these two channels in real time.
and
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Departamento de
Biología Celular, Instituto de Fisiología Celular,
UNAM, Ciudad Universitaria, D.F. 04510, México. Tel.:
525-622-5654; Fax: 525-622-5611; E-mail: lvaca@ifisiol.unam.mx.
