From the Secretory Physiology Section, Gene Therapy and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, December 30, 2002, and in revised form, January 16, 2003
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ABSTRACT |
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The exact role of TRPC1 in store-operated calcium
influx channel (SOCC) function is not known. We have examined the
effect of overexpression of full-length TRPC1, depletion of endogenous TRPC1, and expression of TRPC1 in which the proposed pore region (S5-S6, amino acids (aa) 557-620) was deleted or modified by
site-directed mutagenesis on thapsigargin- and carbachol-stimulated
SOCC activity in HSG cells. TRPC1 overexpression induced channel
activity that was indistinguishable from the endogenous SOCC activity.
Transfection with antisense hTRPC1 decreased SOCC activity
although characteristics of SOCC-mediated current, ISOC,
were not altered. Expression of TRPC1 Activation of cell surface receptors, which are coupled to
inositol lipid signaling, results in phosphatidylinositol bisphosphate (PIP2)1
hydrolysis, generation of diacylglycerol and inositol
1,4,5-trisphosphate, release of Ca2+ from internal
Ca2+ stores, and activation of plasma membrane
Ca2+ influx channels (1, 2). Two types of plasma membrane
channels have been associated with this signaling mechanism;
store-independent channels that appear to be activated as a result of
PIP2 hydrolysis and store-operated Ca2+
channels, SOCC, that are activated primarily in response to depletion of Ca2+ from the internal Ca2+ stores (3, 4).
However, the mechanism(s) underlying activation and gating of these
plasma membrane calcium channels as well as their molecular composition
have remained elusive for more than a decade (5-7). The most well
characterized SOCC is the Ca2+ release activated
Ca2+ channel (CRAC) that mediates the store-operated
current ICRAC in mast cells and lymphocytes (8, 9). This
channel has been reported to have a very high selectivity for
Ca2+ and unique inactivation properties. SOCCs have also
been described in a number of other cell types. Many of these display
characteristics different from CRAC and from each other (7, 8). Based
on the distinct activities seen in different cell types, it is likely that the molecular composition, or regulation, of their SOCCs is different.
Members of the TRP superfamily of putative ion
channel proteins have been suggested as components of SOCCs (10).
Although it is possible that some TRPVs or TRPMs might form SOCCs
(11-13), there are sufficient data to demonstrate that the TRPC family is involved in agonist-stimulated Ca2+ signaling (3, 4,
14). It is now clear that some TRPCs, e.g. TRPC1 and TRPC4,
allow plasma membrane Ca2+ influx in response to depletion
of the internal Ca2+ store. Others, like TRPC3 and TRPC6,
appear to be activated by PIP2 hydrolysis in a number of
cell types. Compared with the other TRPCs, TRPC1 displays a relatively
wider tissue distribution (15, 16). Furthermore, it is endogenously
present and suggested to be involved in SOCE in several cell types as
follows: salivary gland cells (17, 18), endothelial cells (19, 20),
vascular smooth muscle cells (21), and DT 40 cells (22) based on the decrease in SOCE induced by transfection with antisense
TRPC1 (17, 19), addition of the TRPC1 antibody
extracellularly (20, 21), or knock-out of the TRPC1
gene (22). Although these previous studies provide convincing evidence
that TRPC1 has a critical role in SOCE, they do not elucidate whether
TRPC1 serves as an essential regulatory subunit that is required for
the assembly or regulation of the SOCCs in these cells or contributes
more directly to the channel function per se,
i.e. as a component of the pore-forming unit of the channel.
We have previously suggested (17) TRPC1 as a candidate for
store-operated Ca2+ entry mechanism in the human
submandibular gland cell line, HSG. We have also reported that the
TRPC1 C terminus mediates Ca2+-dependent
feedback regulation of SOCE in HSG cells (18, 23). To elucidate whether
TRPC1 is a regulatory subunit of SOCC or contributes more directly to
SOCC-mediated Ca2+ influx, here we have measured SOCE and
SOCC activity in control HSG cells and in cells overexpressing either
TRPC1 or TRPC1 with mutations in the proposed pore region. The data
demonstrate that TRPC1 is required for generation of functional SOCC in
HSG cells and that specific acidic aa residues in the proposed pore
region of TRPC1 contribute to Ca2+ influx.
HSG Cell Culture, Transfection, and Crude Membrane
Preparation--
HSG cells were cultured and stably transfected as
described before (17, 18, 23). For transient transfection, cells were detached, diluted to desired the concentration, and allowed to attach
to tissue culture plates overnight and then co-transfected with
pcDNA3.1 encoding the required TRPC1 (1 µg) and
pcDNA3.1 encoding GFP (0.5 µg) or with the GFP plasmid
alone. Cells were cultured on coverslips or tissue culture plates for
24 h, and GFP-positive cells were selected for activity measurements.
Site-directed Mutagenesis--
The expression plasmid
pcDNA3.1 containing the TRPC1 gene was
used as a template for PCR. Oligonucleotides were synthesized as
required for the mutagenesis, and PCR was performed using the QuickChange Mutagenesis kit (Stratagene). Following PCR, the parental DNA template was removed by DpnI endonuclease digestion, and
the remaining PCR product was transformed into Escherichia
coli XL1-Blue cells. Colonies were selected, and each mutation was
confirmed by sequencing.
Electrophysiology--
Cell attached patch clamp measurements
were performed as described earlier (24). Pipette solution contained
100 mM Na/HEPES, 1 mM MgCl2, 2 mM CaCl2 (pH 7.2) (HCl). Standard bath solution containing (mM) 145 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES ((pH 7.4) with NaOH) was replaced by a high
KCl solution containing (mM) 145 KCl, 5 NaCl, 1 MgCl2, 2 CaCl2, 10 HEPES ((pH 7.4) with KOH)
after the seal was formed. Liquid junction potentials (8.0, 4.3, and
4.2 mV with Ca2+, Na+, and Ba2+,
respectively) were calculated using P-Clamp7 software. Currents were
recorded using an Axopatch 200A amplifier and digitized with Digidata
1200 (Axon Instruments) at a rate of 10 kHz with filtering at 1 kHz.
P-Clamp 7 (Axon instrument) and Origin 6 (Microcal) were used for data
analyses. The open probability of the channel (NPo) was defined as the ratio of open channel
area to the total area in an all-point amplitude histogram. The mean
unitary amplitudes of the single channel currents were determined from
all-point amplitude histogram fit to a sum of Gaussian distributions.
Mean open times were calculated by maximum likelihood estimation using pClamp7 software.
The store-operated inward Ca2+ current (ISOC)
was measured using the whole cell patch clamp technique as described
before (23, 25) with some modifications. The external solution
contained 135 mM sodium glutamate, 1 mM
MgCl2, 2 mM CaCl2, 10 mM glucose, 10 mM HEPES (pH 7.4) (NaOH). The
pipette solution contained 135 mM cesium glutamate, 3 mM MgCl2, 3 mM ATP (Mg), 10 mM HEPES (pH 7.2) (CsOH), 10 mM EGTA, and 3.5 mM CaCl2 (final [Ca2+] = 100 nM). Cells were held at 0 mV and subjected to voltage ramps
( Immunoprecipitation and Western Blotting--
Crude membranes
were prepared as described previously (26). Protein concentration was
determined by using the Bio-Rad protein assay (microassay procedure).
Membrane solubilization and immunoprecipitation were performed as
described previously (26, 27). Immunocomplexes were pulled down with
protein A beads (anti-HA was used at 1:100 dilution for IP). Proteins
were released with SDS buffer and visualized by the ECL reaction after
Western blotting. Anti-HA (Roche Molecular Biochemicals, 3F10) and
anti-TRPC1 (16) were used at 1:1000 dilution.
Confocal Microscopy--
HSG cells expressing TRPC1 [Ca2+]i Measurements--
Fura2
fluorescence in single cells (cultured overnight on glass bottomed
microwell dishes, MatTek Corp.) was measured as described earlier (23)
by using an SLM 8000/DMX 100 spectrofluorimeter or Polychrome 1V (TILL
Photonics) attached to an inverted Nikon Diaphot microscope with a
Fluor 40× oil-immersion objective. Images were acquired using an
enhanced CCD camera (CCD-72, DAGE-MTI) and either Image-1 or Metafluor
software (Universal Imaging Corporation, PA). In cells that were
transiently transfected, fura2 fluorescence was measured in
GFP-positive cells. Analog plots of the fluorescence ratio (340/380)
are shown, and peak values were used for analysis.
SOCC Activity in HSG Cells Expressing TRPC1--
SOCC activity was
measured in control HSG cells and TRPC1-overexpressing HSG cells
(TRPC1-cells) by using cell attached patch clamp technique as we have
described previously (24). Tg and CCh induced burst-like (Fig.
1, A and C) and
flickery (E and G) SOCC activities, respectively,
in both sets of cells (membrane potential was held at
The I-V relationships obtained with 2 mM Ca2+ + 100 mM Na+ (Fig. 1, I and
J, values were obtained from the major current peak), or 2 mM Ca2+ + 100 mM
N-methyl-D-glucamine (data not shown), in the
pipette solution were similar in control and TRPC1-cells stimulated
either with CCh or Tg. In all cases, the current displayed inward
rectification and reversed at about +20 mV. The slope conductance of
SOCC with Ca2+ was 20 pS, which was similar in CCh- or
Tg-stimulated TRPC1-cells and control cells. Furthermore, as in control
cells, SOCC in TRPC1-cells was also blocked by Gd3+ (Fig.
1, I and J, 0/17 cells tested displayed
activity), La3+, and by 2-aminoethoxydiphenylborate
(data not shown). Additionally, the kinetics and unitary current
amplitudes with Na+ (2 pA) or Ba2+ (0.65 pA) as
charge carrying ions (Ca2+ was either removed from the
pipette solution or replaced with Ba2+) were similar in
control HSG and TRPC1-cells (membrane potential was held at Deletion of the TRPC1 Pore Region Decreased SOCC
Activity--
TRPC proteins have been suggested to have six
transmembrane domains. The aa sequence spanning the region between S5
and S6 includes a seventh hydrophobic domain and has been proposed to form the pore of the channel (3, 4), although the exact topology of
TRPC1 has not yet been confirmed. To elucidate the role of TRPC1 in
SOCC function, we have truncated TRPC1 after S5 (aa 567-793) thus
deleting the proposed pore region, S6, and the C terminus. Stable
expression of TRPC1
Fig. 2G shows that the levels of TRPC1, TRPC1 Effect of Mutating Acidic Amino Acid Residues in the Pore Region of
TRPC1 on SOCE--
To demonstrate directly the role of the TRPC1 pore
region in SOCC function, we made Asp
We could not determine whether Mut-pore interacted with endogenous
TRPC1 because the molecular weights were similar and anti-TRPC1 antibody recognized both proteins. However, to determine whether the
expressed Mut-pore can interact with other TRP monomers, HA-tagged Mut-pore was co-expressed with FLAG-tagged TRPC1. The two TRPC1 proteins were co-immunoprecipitated with each other (Fig.
3G). Because TRPC1
To analyze further the effect of Mut-pore on CCh- and Tg-stimulated
SOCC activity, we performed whole cell patch clamp experiments. Cells
were held at 0 mV and subjected to voltage ramps every second. The
traces shown in Fig. 4, A and
D, represent CCh- and Tg-stimulated currents that have been
reconstructed from the current amplitudes at
We also measured ISOC in the absence of external
Ca2+, in which case the inward current is carried by
Na+ (Fig. 4F, leak current measured prior to
stimulation of cells, trace not shown, has been subtracted). In control
HSG cells both CCh (data not shown) and Tg (Fig. 4F)
activated an inwardly rectifying current that reversed at 0 mV
(black trace, 9.2 ± 1.0 pA at Identification of Specific Amino Acid Residues in the TRPC1 Pore
Region That Are Involved in SOCC Function--
Fig. 5A
shows the alignment of aa residues in the pore region of TRPCs 1, 4, and 5. We made individual substitutions of two residues E576K and
E615K, which flank the proposed hydrophobic region (shaded
region) and are conserved in TRPC1, -4, and -5. Asp-581, which is
present only in TRPC1 and is closest to the pore, was also substituted
with Lys or Asn. Fura2 fluorescence and whole cell patch clamp
measurements were performed with cells expressing these mutant TRPC1
proteins (note that both stable and transient expression of these TRPC1
mutants yielded similar results). Fig. 5B summarizes the
Ca2+ influx data. E576K and D581K induced dominant
suppression of SOCE, similar to that seen by Mut-pore expression.
Although E615K and D581N (data not shown) induced lower Tg-stimulated
Ca2+ influx as compared with TRPC1-cells, it was not lower
than that in control HSG cells. Fig. 5C shows that the
expression levels of these mutant TRPC1 proteins were similar and do
not account for their distinct effects on SOCE. Current-voltage ramps
of ISOC generated by expression of these mutants are shown
in Fig. 5, D-F. Averages of the reversal potentials and
current densities in these cells are shown in Fig. 5, G and
H. Importantly, there was maximum left shift of the
Erev (shown by the arrows in
D-F) in D581K cells, from +10.8 ± 1.3 and +10.2 ± 0.9 mV in TRPC1-cells and control HSG cells, respectively to
Previous studies (17-23) from our laboratory, and others, provide
convincing evidence that TRPC1 has a critical role in SOCE. Based on
the presently available data two possible roles can be assigned to
TRPC1: (i) it is a regulatory subunit of SOCC that is required for its
activation or assembly, and (ii) it is a component of the SOCC pore
that directly contributes to SOCC-mediated Ca2+ influx. The
data presented above suggest that TRPC1 is a functional component of an
SOCC in HSG cells that contributes to Ca2+ influx in
response to stimulation by either agonist or Tg. We have shown that the
characteristics of SOCC activity generated by expression of TRPC1 in
HSG cells are similar to the endogenous SOCC activity in these cells.
Consistent with this, expression of TRPC1 in HSG cells induces an
increase in the amplitude of the SOCC-mediated current,
ISOC, without altering its characteristics (23) (Fig. 4).
We have also shown here and previously (17) that endogenous TRPC1 in
HSG cells is essential for SOCC activity. Tg- or CCh-stimulated SOCC
activity, ISOC, and Ca2+ influx, as well as
levels of endogenous TRPC1 in HSG cells are decreased by 80% or more
in cells transfected with antisense-TRPC1. More significant
is our finding that acidic amino acid residues in the proposed pore
region of TRPC1 contribute to SOCE. We have shown that expression of
TRPC1 with simultaneous mutations (Asp Previous studies have shown that Asp Although presently we do not have evidence for homo-multimerization of
endogenous TRPC1 in HSG cells, we have shown that the expressed mutant
TRPC1 is immunoprecipitated with endogenous TRPC1. This result has
major implications because it suggests that TRPC1 monomers interact
with each other to form SOCC. Our data are consistent with reports
suggesting that Drosophila TRP and TRPC1 In conclusion, the data presented here suggest that TRPC1 is a
component of the functional (pore-forming) unit of SOCC in HSG cells.
However, these data do not rule out the possibility that SOCC might be
a heteromer of TRPC1 with other TRPCs (5, 30, 33-35) or with other as
yet unknown protein(s). We have shown earlier that in HSG cells TRPC1,
like the Drosophila TRP (14, 36), is assembled in a
supramolecular protein complex with key proteins involved in the
Ca2+ signaling cascade that leads to SOCC activation (26).
We suggest that SOCC activity in any cell type will depend not only on
the proteins that constitute its pore-forming unit but also other regulatory proteins that might affect its function, assembly, or
localization. It will be important to determine whether differences in
the molecular composition of the channel per se, or its
regulation, account for the large variation in the characteristics of
SOCCs seen in different cell types.
567-793, but not
TRPC1
664-793, induced a similar decrease in SOCC activity.
Furthermore, TRPC1
567-793 was co-immunoprecipitated with endogenous
TRPC1. Simultaneous substitutions of seven acidic aa in the S5-S6
region (Asp
Asn and Glu
Gln) decreased SOCC-mediated Ca2+, but not Na+, current and induced a left
shift in Erev. Similar effects were induced by
E576K or D581K, but not D581N or E615K, substitution. Furthermore,
expressed TRPC1 proteins interacted with each other. Together, these
data demonstrate that TRPC1 is required for generation of functional
SOCC in HSG cells. We suggest that TRPC1 monomers co-assemble to form
SOCC and that specific acidic aa residues in the proposed pore region
of TRPC1 contribute to Ca2+ influx.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
100 to +100 mV at 2 mV/ms). Cells were stimulated by perfusion with
Tg- or CCh-containing medium. Data are presented as mean ± S.E.
or percentage of responding cells. Statistical analyses of the data
were performed using Student's t test or
2
test, as appropriate. p < 0.05 was considered significant.
567-793
were cultured on coverslips for 24 h. Cells were fixed,
permeabilized, and treated with the HA antibody at 1:100 dilution for
1 h. Cells were then washed and probed with rhodamine-linked
secondary antibody as described before (16). Confocal images were
collected using an MRC 1024 krypton/argon laser scanning confocal
equipped with a Nikon Optiphot II photomicroscope.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
40 mV). This
difference in the kinetics is due to modulation of the channel by the
[Ca2+] in the internal store and in the subplasma
membrane region near the channel (24). Both CCh- and Tg-induced SOCC
activities displayed unitary current amplitudes of about
0.85 pA,
with occasional overlapping channel openings corresponding to multiples
of the first level (i.e. about
1.7 pA, Fig. 1, B, D,
F, and H), although SOCC activity in TRPC1-cells was
higher than in control cells. The number of events in the major current
level was significantly increased from 1115 ± 145 (n = 9) to 2109 ± 237 (n = 12)
and 1278 ± 141 (n = 11) to 2318 ± 265 (n = 11) with Tg and CCh, respectively (12 s recording;
n = number of cells, p < 0.05 in both
cases). Average NPo values in TRPC1-cells
0.43 ± 0.045 with CCh and 0.33 ± 0.036 with Tg were
significantly higher than the values in control cells 0.26 ± 0.019 and 0.12 ± 0.015, respectively (p < 0.05 in both cases, n is given above). The mean open times of
SOCC (calculated from the activity at the major current level) with CCh
or Tg were 5.5 ± 0.4 ms or 11.6 ± 2.1 ms, respectively, in
TRPC1-cells and 3.8 ± 0.4 ms or 6.5 ± 1.2 ms in control
cells.
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Fig. 1.
Expression of TRPC1 in HSG cells increases
the endogenous SOCC activity. Tg- or CCh-activated single channel
activities were recorded in control HSG cells (A and
E) and cells expressing TRPC1 (C and
G) using the cell-attached patch clamp method. The tracings
show currents obtained at 40 mV (data were filtered at 200 Hz). The
corresponding all-point histograms are shown in B,
D, F, and H. Channel activity was monitored
between
80 and +80 mV to obtain the I-V relationships shown in
I and J (n = 7-9 cells in each
case).
40 mV).
The I-V characteristics of SOCC-mediated Na+ and
Ba2+ currents as well as the slope conductances (44 pS with
Na+ and 15 pS with Ba2+) were also similar in
control and TRPC1-cells (current recordings not shown). Note that the
Ca2+ conductance of SOCC determined here is close to that
estimated earlier for TRPC1 by noise analysis, 16 pS (28). In
aggregate, the data in Fig. 1 demonstrate that TRPC1 expression in HSG
cells generates channel activity that is indistinguishable from the endogenous SOCC activity. Additionally, and consistent with our previous studies (17), transfection with antisense TRPC1
(TRPC1-as, but not with antisense TRPC3 or TRPC4) eliminated SOCC
activity in 12/14 cells (86%). In 2/14 cells (14%) SOCC activity was
similar to that in control cells (current traces not shown, data are
summarized in Fig. 5). These data demonstrate that endogenous TRPC1 is
required for the Tg- and CCh-stimulated SOCC activity and SOCE in HSG cells.
567-793 exerted an inhibition of endogenous SOCC
activity. 8/9 cells (89%) failed to display any channel activity in
response to stimulation by either CCh or Tg at both negative and
positive membrane potentials (Fig. 2,
A and B, cells were held at either +40 or
40
mV). 1/9 cells (11%) displayed normal SOCC activity with both Tg and
CCh. The frequency of detection of channel activity was significantly reduced (p < 0.01,
2 test) as compared
with that in TRPC1-cells (23/31, 74%) and control cells (21/29, 72%).
In agreement with these single channel measurements, Tg- or
CCh-stimulated Ca2+ influx (Fig. 2, C-F) in
cells expressing TRPC1
567-793 was significantly reduced (>70%) as
compared with that in control cells and in cells expressing TRPC1
(p < 0.05, see figure legend for the average fluorescence values). Consistent with our earlier reports (18, 23) and
in contrast to the effect of TRPC1
567-793 on SOCE, expression of
TRPC1
664-793 induced an increase in SOCE (Fig. 2, E and
F). Thus, the region aa 567-664 is important for SOCE.
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Fig. 2.
SOCC activity in cells expressing
TRPC 1 567-793. A and
B, lack of CCh- and Tg-stimulated SOCC activity in 90% of
TRPC1
567-793-expressing cells. C, Tg-induced influx;
D, CCh-induced influx in TRPC1
567-793-cells and
TRPC1-cells. Time scale and [Ca2+] in the medium are
indicated in the figures. E and F, average
Ca2+ influx data (expressed as % of control cells) of Tg-
and CCh-stimulated Ca2+ influx in TRPC1-cells,
TRPC1
664-793- and TRPC1
567-793-expressing cells. Values
indicated by * are significantly different from each other and from
controls. G, detection of TRPC1 (lane 1),
TRPC1
664-793 (lane 2), and TRPC1
567-793 (lane
3) in crude membrane fractions by Western blotting using anti-HA
antibody. H, localization of TRP1
567-793 by confocal
imaging using anti-HA antibody and rhodamine-linked secondary antibody.
I, endogenous TRPC1 immunoprecipitates with
TRPC1
567-793. IP was done with anti-HA; blot was probed with
anti-TRPC1 antibody (top panel) and with anti-HA
(bottom panel).
664-793,
and TRPC1
567-793 were similar. Thus, the observed differences in
SOCE in cells expressing these proteins are not due to differences in
the levels of expression. Furthermore, deletion of the aa 567-793 of
TRPC1 did not affect its localization to the plasma membrane (Fig.
2H). Importantly, TRPC1
567-793 was immunoprecipitated
with endogenous TRPC1 (Fig. 2I). Together, these data
suggest that expressed TRPC1 proteins associate with the endogenous
TRPC1, a component of the endogenous SOCC in these cells.
Asn and Glu
Gln
substitutions of the seven negatively charged residues present in the
region between S5 and S6 of TRPC1 (aa 557-620, Fig.
3A) and stably expressed this
mutant TRPC1 (Mut-pore) in HSG cells. 13/15 cells (87%) expressing this mutant did not display detectable CCh- or Tg-stimulated SOCC activity (Fig. 3, B and C, respectively). This
represents a significant decrease in the frequency of channel detection
when compared with that in TRPC1-cells or control HSG cells
(p < 0.01,
2 test). 2/15 cells (13%)
displayed a few channel openings at very negative membrane potentials
(
80 mV or lower), and the amplitude of the current detected at
80
mV appeared to be lower than that seen in control HSG cells or in
TRPC1-cells (data not shown). Outward current was also decreased. Due
to the low frequency of detection, we have not yet characterized these
single channel characteristics in detail. These observations are in
contrast to cells transfected with antisense TRPC1, where 14% of the
cells displayed normal channel activity. Consistent with the effects of
Mut-pore on SOCC activity, fura2 fluorescence measurements demonstrated
that the CCh- or Tg-stimulated Ca2+ influx was
significantly reduced (by 70%) as compared with those in control HSG
cells and TRPC1-expressing cells (Fig. 3, D-F). Internal
Ca2+ release was not changed. Additionally, infection of
Mut-pore cells with adenovirus encoding TRPC1 (AdTRPC1) induced a 30%
recovery of Tg-stimulated Ca2+ influx. Infection with
AdTRPC3 did not increase Ca2+ influx in Mut-pore cells
(data not shown), suggesting that full-length TRPC1 can overcome the
effects of Mut-pore. Thus the extent of inhibition achieved by Mut-pore
depends on the level of expression relative to that of full-length
TRPC1.
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Fig. 3.
SOCC activity in cells expressing TRPC1 with
mutations in the pore region. A, aa substitutions made
in the putative pore region of TRPC1. B and C,
SOCC activity measured in Mut-pore-expressing cells stimulated with Tg
and CCh. D and E, fura2 fluorescence changes
induced by Tg and CCh. Time scale and [Ca2+] in the
medium are indicated in the figures. F, average SOCE,
Mut-pore value is expressed relative to that in control HSG cells. **
indicates values significantly different from the ratio in control HSG
cells, p < 0.05 (n = 200-300 cells in
each group from 6-8 experiments). G, Western blots
demonstrating the interaction of HA-tagged Mut-pore with FLAG-tagged
TRPC1. Cells were co-transfected with HA-Mut-pore or FLAG-TRPC1.
Antibodies used for IP and Western blotting are indicated in the
figure.
567-793 interacts with endogenous
TRPC1 (Fig. 2I) and Mut-pore interacts with FLAG-tagged
TRPC1, it is likely that Mut-pore can interact with the endogenous TRPC1.
40 mV obtained from
voltage ramps (
100 to +100 mV at 2 mV/ms). The individual ramps
obtained at time "I" (before stimulation of cell) are shown in Fig.
4B and at "II" (30 s after stimulation of cell) are
shown in Fig. 4, C and E. At time I,
i.e. before addition of CCh or Tg to the bath, a small
non-selective basal current was detected, which was not changed by
expression of TRPC1 or Mut-pore. Also note that there was no change in
the current for up to 10 min of recording, unless cells were stimulated
(data not shown). Both CCh and Tg stimulated ISOC, a
store-operated current that we have described previously (23, 25).
Under the experimental conditions used in this study, ISOC
displayed inward rectification and reversed at about +10 mV
(black trace in Fig. 4, C and E, the
basal currents have been subtracted from each trace, also see Fig.
5 for additional data). The inward
currents are primarily carried by Ca2+ because replacement
of Na+ in the medium with
N-methyl-D-glucamine did not alter this current (trace not shown). In TRPC1-cells the amplitude of the inward current
was significantly increased (11.7 ± 0.9 pA in control versus 17.1 ± 1.4 pA in TRPC1-cells at
40 mV,
red trace in Fig. 4, C and E).
However, the characteristics of ISOC were not altered. Importantly, in Mut-pore-expressing cells the amplitude of
ISOC was significantly lower than that in control HSG cells
and TRPC1-cells (green trace in Fig. 4, C and
E, also see details in Fig. 5H). Furthermore,
there was a significant left shift in the reversal potential of
ISOC (see inset in Fig. 4, C and
E, also see Fig. 5G for average values and
statistical analysis), suggesting a decrease in Ca2+
selectivity of the channel. Consistent with the single channel measurements, the effects induced by antisense TRPC1 on
ISOC were different from those seen with Mut-pore; the
amplitude of ISOC was significantly decreased, but the
reversal potential was not significantly changed (see Fig. 5,
G and H).
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Fig. 4.
Whole cell current recordings in cells
expressing Mut-pore. A, stimulation of ISOC
by CCh in Mut-pore (green traces), control HSG (black
traces), and TRPC1-cells (red traces). The current
traces have been reconstructed from the amplitudes measured at 40 mV
in voltage ramps and smoothed for the presentation using Origin6.
B and C, voltage ramps of currents recorded at
time points I (before stimulation) and II (30 s
after stimulation). C, inset, reversal potentials
of the currents in the three cells. D and E,
ISOC measured in Tg-stimulated cells. E,
inset, reversal potentials of the currents. F,
Tg-stimulated Na+ currents measured when Ca2+
is excluded from the external solution. Inset shows the
Na+ and Ca2+ currents in Mut-pore cells
relative to those in TRPC1-cells. **, the decrease in the
Ca2+ current was significantly different (p < 0.05) from the decrease in the Na+ current.
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Fig. 5.
SOCE in cells expressing TRPC1 with single
amino acid mutations. A, alignment of TRPC1, TRPC4, and
TRPC5. The putative pore (hydrophobic domain) is shaded, and
the acidic amino acids that were altered in TRPC1 are marked by
asterisks. B, average data of Tg-stimulated
Ca2+ influx, expressed relative to the value in control
cells. ** indicates values significantly different from all other
values; values marked * are different from the unmarked value and those
marked ** but not from each other. C, Western blot of crude
membranes isolated from TRPC1-, E576K-, D581K-, and E615K-transfected
cells. Protein levels were detected using anti-HA antibody.
D-F, voltage ramp data from cells expressing E576K, D581K,
and E615K mutations, respectively. The arrow in each case
indicates the Erev. G, average of the
reversal potentials. ** indicates values significantly different from
the unmarked values. Unmarked values are not significantly different
from each other. H, Tg-induced whole cell currents at 40
mV, shown as current densities. * indicates values that are
significantly different from other values in the figures but not from
each other. ** indicates values that are significantly different from
unmarked values and values marked *. (p < 0.05 was
considered significant, n = 4-6 cells in each
case.)
40 mV). This current
was significantly (p < 0.05) increased in TRPC1-cells (Fig. 4, F, red trace, 16.5 ± 1.2 pA at
40 mV). Note that TRPC1 expression induced the same increase
(1.5-2.0-fold) in the Ca2+ and Na+ currents,
which is consistent with the 1.5-2.0-fold increase in SOCE detected by
fura2 fluorescence measurements. In contrast to the Ca2+
current in Mut-pore cells, which was 67% lower than in TRPC1-cells and
52% lower than in control cells, the Na+ current was 17%
less than that in TRPC1-cells (green trace, Fig. 4F) and significantly larger than that in control cells. The
inset in Fig. 4F shows Mut-pore-mediated
Ca2+ and Na+ currents relative to that seen in
TRPC1-cells. Thus, mutation of the seven acidic aa residues in the
TRPC1 pore region induced a much larger decrease of SOCC-mediated
Ca2+ influx than SOCC-mediated Na+ influx.
These data strongly suggest that these mutations do not result in an
overall decrease in channel activity.
12.2 ± 2.5 mV. Erev was also
left-shifted in E576K cells but not to the same extent as in D581K
cells. E615K did not induce any shift in Erev.
Consistent with the Ca2+ measurements, the Ca2+
currents generated by Tg in D581K and E576K were significantly lower
than that in control cells (see Fig. 5H). D581K induced the
largest decrease in ISOC. ISOC in E615K,
although lower than in TRPC1 cells, was similar to that in control HSG
cells. These data demonstrate that two acidic amino acid residues in
the TRPC1 pore region, Glu-576 and Asp-581, are involved in
Ca2+ influx via SOCC. However, other amino acids in TRPC1
(which were not tested in this study) might also have a role in
Ca2+ influx. We have not presently examined changes in the
permeability of monovalent cations such as Cs+ (which is
present in the intracellular solution) in cells expressing D581K, which
might account for the negative reversal potential.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Asn and Glu
Gln) of
seven acidic aa residues in the proposed pore region suppressed
endogenous SOCC activity in more than 80% of the cells. In the few
cells expressing this mutant where channel activity was detected, there
was an apparent decrease in the Ca2+ conductance.
Importantly, the amplitude of ISOC in these cells was
decreased, and there was a left shift in the reversal potential, suggesting a decrease in the Ca2+ permeability of the
channel. Interestingly, the Na+ current mediated by
Mut-pore was similar to that seen in TRPC1-cells. Thus, mutations in
the acidic aa residues in the pore region do not appear to decrease
SOCC activity per se. We have also demonstrated that two of
these seven acidic amino acid residues, Glu-576 and Asp-581, are
involved in SOCC-mediated Ca2+ influx. Substitution of
either residue, E576K or D581K, induced a suppression of Tg-stimulated
SOCE and ISOC. Notably, Glu-576 and Glu-615 are conserved
in TRPC1, TRPC4, and TRPC5, although Asp-581 is present only in TRPC1.
Our data also rule out the possibility that a decrease in SOCC activity
in cells expressing mutant TRPC1 proteins is due to differences in
protein expression or altered localization. In aggregate, these data
support the proposal that TRPC1 is a component of the functional
(i.e. pore-forming) unit of SOCC, rather than an associated
regulatory protein. Although it is possible that a single mutation in
an extracellular domain of TRPC1 might alter its interaction with an as
yet unknown SOCC protein and lead to a decrease in Ca2+
influx via the channel, it is highly unlikely.
Asn substitution in the pore
region of TRPV4 decreased Ca2+ influx, although Asp
Lys substitutions completely blocked channel function (29).
Substitution of conserved hydrophobic residues (LFW) in the TRPC6 pore
region eliminated TRPC6-generated channel activity in HEK293 cells
(30). However, neither of these channels appear to be involved in
SOCE. While this manuscript was in preparation, it was reported (31)
that expression of TRPV6 with substitution of FEL to AAA in the
proposed pore region induced suppression of endogenous
ICRAC in Jurkat cells. However, it is not clear whether the
endogenous TRPV6 in Jurkat cells is required for ICRAC. Furthermore, TRPV6 displayed both store-operated and store-independent activities. The data we have presented above are significant because they demonstrate the following: (i) endogenous TRPC1 in HSG cells is
required for the endogenous SOCE detected in these cells; (ii) expressed TRPC1 is regulated by store depletion and is not
spontaneously active; and (iii) acidic aa residues in the pore region
of TRPC1 contribute to SOCE.
homomultimerize via N-terminal interactions (32, 33). We suggest that endogenous or
exogenously expressed TRPC1 monomers associate with each other to form
functional SOCCs. Thus, when full-length TRPC1 is expressed channel
activity is increased. When mutant TRPC1s are overexpressed relative to
the endogenous protein, the probability of mutant TRPC1 monomers
associating with each other or with endogenous TRPC1 monomers is
relatively high. As a result, aberrant SOCCs are formed which have
decreased permeability for calcium. In contrast, in cells
transfected with antisense TRPC1 or expressing
TRPC1
567-793, where either depletion of TRPC1 or the pore region is
deleted, respectively, there is a reduction in the total number of
functional channels rather than a change in the channel properties.
However, other proteins, including other TRPCs, might be co-assembled
with endogenous TRPC1 and required for functional SOCC in HSG cells. Studies using heterologous expression have shown that TRPC1 interacts with TRPC3 (33, 34), TRPC6 (30), and TRPC4 and TRPC5 (35). The latter
study also showed that endogenous heteromers of either TRPC1 and TRPC4
or TRPC1 and TRPC5 were co-immunoprecipitated from rat brain. TRPC1
expression altered the currents generated when either TRPC5 or TRPC4
was expressed alone, and furthermore, these currents were not activated
by store depletion. Although it is unclear why TRPC1 forms different
types of channels in different cells, it is important to note that the
characteristics of the channel formed by heteromeric TRPC1 channels
(35) appear to be considerably different from those of ISOC
measured in HSG cells. Further studies will be required to determine
which TRPC proteins are endogenously expressed in HSG cells and
interact with endogenous TRPC1.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Klaus Groschner, Martha Nowycky, and Michael Zhu for helpful comments during the preparation of this manuscript. We are also grateful to Dr. Mitchel Villereal for kindly providing us with antisense constructs for TRPC1, TRPC3, and TRPC4 and Dr. Craig Montell for the TRPC1-FLAG construct.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: Bldg. 10, Rm. 1N-113, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-5298; Fax: 301-402-1228; E-mail: indu.ambudkar@nih.gov.
Published, JBC Papers in Press, January 20, 2003, DOI 10.1074/jbc.M213271200
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ABBREVIATIONS |
---|
The abbreviations used are: PIP2, phosphatidylinositol bisphosphate; GFP, green fluorescent protein; SOCC, store-operated calcium influx channel; aa, amino acids; IP, immunoprecipitation; HA, hemagglutinin; Tg, thapsigargin; CCh, carbachol; CRAC, Ca2+ release activated Ca2+ channel; TRP, transient receptor potential protein; TRPC, TRP canonical family; TRPV, TRP vanalloid family; TRPM, TRP melastatin family.
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