Signaling Mechanism for Receptor-activated Canonical Transient
Receptor Potential 3 (TRPC3) Channels*
Mohamed
Trebak
,
Gary
St. J. Bird,
Richard R.
McKay,
Lutz
Birnbaumer, and
James W.
Putney Jr.
From the Laboratory of Signal Transduction, NIEHS, National
Institutes of Health, Research Triangle Park, North Carolina
27709
Received for publication, January 17, 2003, and in revised form, February 26, 2003
 |
ABSTRACT |
Canonical transient receptor potential 3 (TRPC3)
is a receptor-activated, calcium permeant, non-selective cation
channel. TRPC3 has been shown to interact physically with the
N-terminal domain of the inositol 1,4,5-trisphosphate receptor,
consistent with a "conformational coupling" mechanism for its
activation. Here we show that low concentrations of agonists that fail
to produce levels of inositol 1,4,5-trisphosphate sufficient to induce Ca2+ release from intracellular stores substantially
activate TRPC3. By several experimental approaches, we demonstrate that
neither inositol 1,4,5-trisphosphate nor G proteins are required for
TRPC3 activation. However, diacylglycerols were sufficient to activate TRPC3 in a protein kinase C-independent manner. Surface receptor agonists and exogenously applied diacylglycerols were not additive in
activating TRPC3. In addition, inhibition of metabolism of diacylglycerol slowed the reversal of receptor-dependent
TRPC3 activation. We conclude that receptor-mediated activation of
phospholipase C in intact cells activates TRPC3 via diacylglycerol
production, independently of G proteins, protein kinase C, or inositol
1,4,5-trisphosphate.
 |
INTRODUCTION |
In non-excitable cells, depletion of internal
Ca2+ stores activates store-operated channels
(SOCs)1 mediating calcium
entry across the plasma membrane, a process known as capacitative
calcium entry (CCE) (1-3). Despite considerable attention, the
molecular identity of SOCs and their gating mechanism(s) remain
unknown. Two major hypotheses for the mechanism of activation of CCE
have been proposed. The first involves the release of a soluble factor
from the endoplasmic reticulum (4, 5). The second, the
"conformational coupling" model, proposes a direct interaction of
inositol 1,4,5-trisphosphate (IP3) receptors in the
endoplasmic reticulum with SOCs in the plasma membrane (2, 6). The
latter hypothesis has gained widespread support in the last few years.
TRPC3 is a member of the canonical transient receptor potential (TRPC)
family of Ca2+-permeant, non-selective cation channels (7)
whose members have been hypothesized to form, or contribute to the
formation of the elusive SOC channel (7-9). TRPC3 and its close
structural homologs, TRPC6 and TRPC7, in many expression systems,
including HEK293 cells, behave as receptor-operated cation channels
(10-12) that can be activated by exogenous application of
diacylglycerols (DAG) independently of store-depletion (13, 14).
However, Kiselyov et al. (15, 16) reported that TRPC3
channels in excised patches from TRPC3-expressing HEK293 cells could be
stimulated with IP3 and IP3 receptors. These
authors suggested that TRPC3 activation involves interaction with
IP3 receptors in their ligand-bound state, consistent with
the conformational coupling hypothesis. Ma et al. (17) also
proposed a requirement of IP3 receptors for native SOC and
TRPC3 channel activation based on experiments with the
membrane-permeant IP3 receptor antagonist,
2-aminoethoxydiphenyl borane (2-APB). Finally, Boulay et
al. (18) provided biochemical evidence for TRPC3 interaction with
IP3 receptors by utilizing a glutathione
S-transferase pull-down strategy to map the interacting domains on these two proteins.
Thus, conflicting results from different laboratories have generated a
controversy as to the physiological mode of activation of TRPC3
channels. It is possible that different backgrounds of expression or
different expression levels may produce different behaviors of TRPC3
(for example, see Refs. 19 and 20). In addition, studies examining
single channel behavior in excised patches may suffer from loss of
cellular components essential for cellular signaling. Therefore, in the
current study, we have re-examined the role of IP3 in
regulation of TRPC3 channels, by utilizing whole cell patch clamp and
single cell microfluorimetry. We have also utilized two HEK293 cell
lines stably expressing TRPC3, one of which has been used in previous
studies by other groups to provide evidence for a role of
IP3 and the IP3 receptor. Our findings indicate
that receptor activation of TRPC3 in intact HEK293 cells does not
require activation of heterotrimeric G-proteins or the formation of
IP3. Rather, it appears that, in intact HEK293 cells,
receptor activation of TRPC3 is mediated by DAG in a protein kinase C
(PKC)-independent manner.
 |
MATERIALS AND METHODS |
Reagents--
Thapsigargin, methacholine,
1-oleoyl-2-acetyl-sn-glycerol (OAG), IP3,
inositol 2,4,5-trisphosphate ((2,4,5)IP3), phorbol
12-myristoyl 13-acetate (PMA), and DAG kinase inhibitor II, R59022,
were all purchased from Calbiochem. The
1-(
-glycerophosphoryl)-D-myo-inositol 4,5-bisphosphate (GPIP2) was purchased from Roche Molecular
Biochemicals, low molecular weight heparin and atropine from
Sigma, the DAG lipase inhibitor RHC-80267 from Alexis Biochemicals, and
epidermal growth factor (EGF) from Upstate Biotechnology Inc.
Cell Culture and Transfection--
HEK293 cells were obtained
from ATCC and were transfected, using Superfect reagent (Qiagen), with
pcDNA3 vector containing the green fluorescent protein (GFP) coding
sequence added in-frame to the C terminus of human TRPC3 (11). Cells
stably expressing TRPC3-GFP fusion protein (HEK-TRPC3) were selected
first by antibiotic resistance and second by GFP fluorescence by flow
cytometry. Cells were maintained in culture as described previously
(21). An HEK293 cell line expressing human TRPC3 with the hemagglutinin epitope (HA) fused to its C terminus (HEK-T3, clone 9) were generated and grown as previously described (10).
Measurement of Intracellular Calcium
Concentration--
Coverslips with attached cells were mounted in a
Teflon chamber and incubated at 37 °C for 30 min in culture media
(Dulbecco's modified Eagle's medium with 10% fetal bovine serum)
containing 1 µM Fura-2 AM (Molecular Probes). Cells were
then washed and bathed in Hepes-buffered saline solution (HBSS; in
mM, 140 NaCl, 4.7 KCl, 10 CsCl2, 2 CaCl2, 1.13 MgCl2, 10 glucose, and 10 Hepes, pH
7.4) for at least 15 min before Ca2+ measurements were made.
In some experiments, reagents were introduced into the cell through
patch pipettes (2-5 M
, Corning glass, 7052). The cells for these
experiments had membrane capacitances of 10-25 picofarads. The pipette
solution (P1) contained in mM: 140 K+
gluconate, 0.1 EGTA, 2 MgCl2, 10 Hepes, 2 MgATP, 0.075 Fura-2, pH 7.2, adjusted with KOH. Various concentrations of
IP3, IP3 analogues, or heparin were included in
the pipette when indicated. The bath solution was HBSS or (nominally
Ca2+-free) HBSS with no added Ca2+. Whole cell
mode was achieved by gentle suction and giga-ohm-seal quality was
monitored throughout the recordings using pCLAMP 8.0 software (Axon
Instruments, Foster City, CA). Single cell Ca2+
measurements in patch clamp experiments were made with a
photomultiplier-based system mounted on a Nikon Diaphot microscope
(×40 Neofluor objective). The cells were excited alternatively by 340 and 380 nm wavelength light from a Deltascan D101 (Photon Technology
International, Princeton, NJ) light source equipped with a light-patch
chopper and dual excitation monochromators. Fluorescence emission at
510 nm was recorded by a photomultiplier tube (omega Optical). A
constant holding potential of
60 mV was applied to the cells. All
experiments were conducted at room temperature. The data are expressed
as the ratio of Fura-2 fluorescence because of excitation at 340 nm to
that because of excitation at 380 nm.
For Ca2+ measurements after application of external stimuli
only (i.e. not involving patch clamp), fluorescence images
of the cells were recorded and analyzed with a digital fluorescence
imaging system (InCyt Im2, Intracellular Imaging Inc., Cincinnati, OH). Fura-2 fluorescence at an emission wavelength of 510 nm was induced by
exciting Fura-2 alternately at 340 and 380 nm. The 340/380 nm ratio
images were obtained on a pixel by pixel basis and converted to
absolute Ca2+ concentrations by in vitro
calibration. Average Ca2+ values per cell are reported.
For dose-response experiments with methacholine (Fig. 1),
Ca2+ measurements were performed on attached populations of
cells in polylysine-coated 96-well plates with a fluorometric imaging plate reader (FLIPR384, Molecular Devices, Inc., Sunnyvale, CA). Cells
were prepared, loaded with 4 µM Fluo-4 AM in Dulbecco's modified Eagle's medium for 45 min at 37 °C, and analyzed as
previously described (21). All experiments were performed at room temperature.
Electrophysiology--
For whole cell TRPC3 current
measurements, coverslips of TRPC3-expressing HEK293 cells were prepared
and mounted in a perfusion chamber. Electrophysiological measurements
were made at room temperature 24-72 h after cells have been seeded on
coverslips. The cells studied had membrane capacitances of 10-25
picofarads. Patch pipettes (Corning glass, 7052) with a resistance of
2-5 M
were generated using an automatic micropipette puller (model
P-97, Sutter Instruments, Novato, CA). An agar bridge served as the
electrical connection between the bath and the signal ground. Whole
cell currents were elicited by voltage stimuli lasting 250 ms,
delivered every 2 or 10 s, with voltage ramps from
120 to +80
mV. Data were sampled at 2 KHz, filtered at 1 KHz, recorded with an
Axopatch 200B amplifier (Axon Instruments), and analyzed using pCLAMP
8.0 software (Axon Instruments). The internal pipette solution (P2) was
buffered to 125 nM free Ca2+ and contained (in
mM): 140 CsCl2, 10 BAPTA (cesium salt), 5 CaCl2, 2 MgATP, 0.1 GTP (sodium salt), 10 Hepes, pH 7.2, with Tris base. The bath solution was (in mM): 140 NaCl,
4.7 KCl, 10 CsCl2, 2.0 MgCl2, 10 glucose, 2.0 CaCl2, and 10 Hepes, pH 7.4, with NaOH. The osmolarity of
these solutions was adjusted to 290-310 milliosmole with
glucose. Bath solution change was accomplished by perfusion and the
time required for a complete solution change was around 2 s. All
voltages were corrected for liquid junction potential.
Microinjection--
Cells were microinjected with
(2,4,5)IP3 together with Fura-2 (as a marker for
injection), or with Fura-2 alone as described previously (22).
 |
RESULTS |
Low Concentrations of G
q-linked Receptor Agonists
Activate TRPC3--
TRPC3 channels expressed in HEK293 cells have been
shown in a number of laboratories (10, 12) including our own (11, 21)
to behave as a receptor or second messenger operated cation channel,
which is not activated by store-depletion (but see Ref. 15). However,
the exact mechanism through which agonists activate TRPC3 remains
controversial. To gain further insights into the mechanism of TRPC3
activation, we explored the concentration dependence of TRPC3
activation by the muscarinic receptor agonist methacholine, which
activates PLC-
through a heterotrimeric
G-protein-dependent pathway. HEK293 cells express
muscarinic receptors, and respond to muscarinic agonists with a robust
IP3-mediated release of Ca2+, and subsequent
store-operated Ca2+ entry (23). In these experiments, an
automated real-time fluorescence plate reader system was utilized (see
"Materials and Methods"), and background controls (i.e.
Ca2+ additions in the absence of agonist) were carried out
in parallel and subtracted from the conditions with agonist. Gadolinium
(Gd3+, 5 µM) was included in the media in
some wells to inhibit endogenous agonist-induced Ca2+
entry, as described previously (21). As shown previously (21), TRPC3
expressing cells showed a larger influx of Ca2+ than did
wild-type cells in response to muscarinic receptor activation. This
TRPC3-dependent influx was unaffected by Gd3+,
whereas it was completely blocked in the wild-type cells. The concentration-effect relationships for release of Ca2+ in
both cell types, as well as for the entry of Ca2+ in the
wild-type cells, were very similar. However, interestingly, the
relationship for TRPC3-dependent Ca2+ entry was
left-shifted, such that concentrations of methacholine that produced
little or no Ca2+ store release were able to substantially
activate TRPC3-dependent Ca2+ entry (Fig.
1). This is not likely because of a
temporal delay in accumulation of IP3, because this should
have resulted in a leftward shift of the wild-type cells as well. This
result means either that, unlike SOCs, TRPC3 is sensitive to very low
concentrations of IP3, or that IP3 is not
involved in TRPC3 activation. To address this issue, we next decided to
directly introduce IP3 into cells and determine its effect
on TRPC3-mediated Ca2+ entry.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1.
Low concentrations of methacholine activate
TRPC3. HEK-TRPC3 or wild-type cells were seeded in 96-well plates
for measurement of [Ca2+]i signals associated
with agonist-induced Ca2+ release and entry. For the
HEK-TRPC3 cells, 5 µM Gd3+ was added to block
endogenous CCE. Release (top) was assessed as the peak
fluorescence following addition of 0.01-300 µM
methacholine in the absence of Ca2+, and entry
(bottom) was assessed as the maximum increase following
restoration of Ca2+. Constitutive permeability of the two
cell lines was determined in parallel without addition of methacholine,
and subtracted from the data. The data represent mean ± S.E. from
three independent experiments, with 2 wells per condition in each
experiment.
|
|
IP3 Does Not Activate TRPC3--
TRPC3-mediated
Ca2+ entry in response to introduction of IP3
into stable TRPC3-GFP-expressing HEK293 cells (HEK-TRPC3) was assessed by Fura-2 imaging of single cells as indicated under "Materials and
Methods." Five µM Gd3+ was used throughout
these experiments to fully inhibit the endogenous CCE pathway in
response to store depletion induced by intracellular introduction of
IP3 or external application of methacholine. Surprisingly, IP3 did not activate TRPC3-mediated Ca2+ entry
over a wide range of concentrations (2-400 µM). Even at very high concentrations (400 µM; Fig.
2a), IP3 failed to
activate TRPC3 while subsequent addition of methacholine (100 µM) consistently activated TRPC3 in the same cells
(n = 58). The Ca2+ rise seen after
methacholine addition resulted entirely from Ca2+ entry
rather than release from internal stores, because when we added
methacholine after IP3 introduction into the cell in the
absence of external calcium, we observed no additional Ca2+
release (Fig. 2b). Thus, these concentrations of
IP3 were sufficient to deplete intracellular
Ca2+ stores completely (n = 10; Fig.
2b). Similar concentrations of IP3 in the
absence of external Gd3+ maximally activated CCE in
wild-type cells, as methacholine did not induce a further increase of
Ca2+ entry, and also induced a complete depletion of
internal Ca2+ stores in wild-type HEK293 cells (data not
shown).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 2.
IP3 does not activate TRPC3.
a, TRPC3-GFP-expressing HEK293 cells (HEK-TRPC3) were
incubated in the absence of added Ca2+. 5 µM
Gd3+ was present throughout the experiment to inhibit
endogenous CCE. 400 µM IP3 in solution P1
(see "Materials and Methods") was included in the patch pipette and
the whole cell mode was established. After the Ca2+ release
phase, 2 mM external Ca2+ and 100 µM methacholine (MeCh) were sequentially added
as indicated. b, similar experiment to a, except
that external application of methacholine was performed before
Ca2+ addition. c, similar to a,
except that 1 mM (2,4,5)IP3 was included in the
patch pipette instead of 400 µM IP3.
d, similar to a, except that 1 mM
GPIP2 was included in the patch pipette instead of 400 µM IP3. e, similar to
a, using HEK293-T3-9 cells instead of HEK-TRPC3 cells.
f, similar to e, except that external application
of methacholine was performed before Ca2+ addition.
|
|
To rule out possible complications because of metabolism of
IP3, we also used high concentrations of
(2,4,5)IP3 and GPIP2, two non-metabolized
analogues of IP3 (22, 24). When 1 mM
(2,4,5)IP3 (Fig. 2c) or 1 mM
GPIP2 (Fig. 2d) were introduced into HEK-TRPC3 cells, no TRPC3-mediated Ca2+ entry was seen when
Ca2+ was restored to the external medium, while again
methacholine (100 µM) activated TRPC3 in the same cells.
These findings are clearly inconsistent with the previously published
findings of Kiselyov et al. (15, 16). These authors observed
activation of TRPC3 single channel currents in the cell-attached configuration after stimulation with carbachol. These TRPC3 currents declined when the patch was excised, and were restored upon addition of
IP3 and the IP3 receptor to the patch (but not
the IP3 receptor alone), leading to the conclusion that
IP3 was the mediator of TRPC3 activation. These authors
used a clonal HEK293 cell line stably expressing TRPC3 (T3-9) (10). To
ensure that our apparently discrepant findings could not be attributed
to the use of a different cell line, we examined the activation of
TRPC3 in response to introduction of IP3 into T3-9 cells
using the same protocol in Fig. 2a. As shown in Fig.
2e, 400 µM IP3 did not induce any
TRPC3-mediated Ca2+ entry in T3-9 cells, whereas
methacholine substantially activated TRPC3 in the same cells
(n = 9). This concentration of IP3 was sufficient to induce complete store depletion in T3-9 cells, showing that the Ca2+ signal in response to methacholine was
because of entry rather than Ca2+ release from internal
stores (n = 4, Fig. 2f).
To ensure that the failure of IP3 to activate TRPC3 was not
because of the loss of some necessary co-factor because of diffusion from the patch-clamped cells, we also microinjected
(2,4,5)IP3 into TRPC3-GFP expressing HEK293 cells as
described earlier (22). In TRPC3 cells, (2,4,5)IP3
activated Ca2+ entry in the absence of Gd3+,
indicating that microinjection of this poorly metabolized inositol trisphosphate efficiently discharges stores and activates CCE, but
fails to activate TRPC3 channels (data now shown).
We next investigated the regulation of TRPC3 expressed in HEK293 cells
by examining ionic currents with the whole cell patch clamp technique.
After establishing the whole cell configuration, cell membrane
potential was clamped to a holding potential of +30 mV, and 250-ms
voltage ramps from
120 to +80 mV were applied every 2 s.
Following break-in, the whole cell currents measured at
60 mV were
greater in TRPC3 expressing cells than in wild-type (Fig.
3a). Addition of methacholine
(100 µM) resulted in the development of larger inward
currents in TRPC3-expressing cells, whereas control cells showed no
detectable response to the same concentration of methacholine (Fig.
3b). The failure to detect agonist-activated currents in
wild-type cells allows us to examine the activation of TRPC3 in the
absence of the CCE blocker, Gd3+. A typical current-voltage
relationship curve (I-V curve) before and after addition of
methacholine to a HEK-TRPC3 cell is shown in Fig. 3c. We
examined the ability of IP3 to induce TRPC3 current in
HEK-TRPC3 cells by including 400 µM IP3 in
the patch pipette. Consistent with the Fura-2 experiments in Fig. 2, no
current developed after IP3 introduction into HEK-TRPC3
cells, and the steady-state current was not significantly different
from TRPC3 cells without IP3 (Fig. 3a). Again,
100 µM methacholine induced large inward currents in the
IP3-treated cells (Fig. 3d). A lower, perhaps more physiological concentration of IP3 (20 µM), as used by Kiselyov et al. (16), was also
inefficient in activating TRPC3 currents (data not shown, and see Fig.
6c). Methacholine increased inward and outward whole cell
currents as seen in the I-V curves (Fig. 3, c and
e). These effects were highly reproducible; IP3
introduction into HEK-TRPC3 cells never resulted in the development of
current, whereas methacholine consistently yielded a substantial
increase in TRPC3 current amplitude.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
TRPC3 conductance is not increased by
IP3. a, mean whole cell inward currents
measured at 60 mV in wild-type cells, TRPC3-expressing cells, and
TRPC3-expressing cells with 400 µM IP3 in the
patch pipette. b, representative inward current (at 60 mV)
from HEK293-Wt and HEK-TRPC3-GFP cells, before and after application of
100 µM methacholine (MeCh), are plotted as a
function of time. Only the TRPC3-expressing cell shows inward current
developing after MeCh addition. Basal currents (as summarized in
a) have been subtracted. c, representative
currents elicited by 250-ms voltage ramps every 2 s ranging from
120 to +80 mV from wild-type cells, and from HEK-TRPC3 cells before
and after addition of 100 µM MeCh. d, inward
current (at 60 mV) from a TRPC3-expressing HEK293 cell after internal
application of 400 µM IP3 and subsequent
external addition of 100 µM MeCh, is plotted as a
function of time. e, representative currents elicited by
250-ms voltage ramps ranging from 120 to +80 mV, after introduction
of IP3 and after external application of MeCh. The data
were derived from a total of 9 (wild-type), 25 (TRPC3), and 17 (TRPC3 + IP3) experiments. The experiments in
b-e are representative of at least 8 separate
experiments.
|
|
IP3 Receptor Activation Is Not Required for TRPC3
Activation by Methacholine--
One point of evidence supporting the
requirement of IP3 receptors in TRPC3 activation was the
sensitivity of TRPC3 to the membrane permeant IP3 receptor
antagonist 2-APB (17). 2-APB was subsequently shown to block CCE in
response to store depletion by ionomycin in the DT40 B-cell line, and
this compound was equally effective on DT40 cells lacking the three
isoforms of the IP3 receptor (25). Furthermore, 2-APB was
shown to be more efficient from the outside of the cell suggesting that
it is likely inhibiting SOC channels through a direct action (26).
Heparin is a competitive IP3 receptor antagonist that has
been previously reported to inhibit Ca2+ release and
Ca2+ entry in response to agonist or to direct introduction
of IP3 into the cell, but has no effect on CCE induced by
passive depletion of Ca2+ stores by thapsigargin or
ionomycin (22, 25). Unlike 2-APB, heparin is membrane-impermeant and so
we introduced it into the cell through the patch pipette. An effective
concentration clearly entered the cell, because ~300 s after forming
the whole cell mode, external application of methacholine failed to
release stored Ca2+ or activate CCE in wild-type HEK293
cells (Fig. 4a). In HEK-TRPC3 cells, Ca2+ release because of methacholine was also
blocked (Fig. 4, b and d), whereas
thapsigargin-induced release was unaffected (Fig. 4d). The
same protocol was used to examine the effect of heparin on TRPC3
activation in HEK-TRPC3 and T3-9 cells. In these experiments, 5 µM Gd3+ was included in all solutions to
inhibit endogenous CCE. In HEK-TRPC3 (Fig. 4b) and T3-9
(Fig. 4c) cell lines, introduction of heparin (10 mg/ml)
into the cell completely blocked Ca2+ release in response
to external application of methacholine. However, TRPC3-mediated
Ca2+ entry in both cell lines was unaffected. These results
show that neither IP3 receptor activation, nor discharge of
intracellular Ca2+ stores is involved in agonist-induced
activation of TRPC3.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4.
IP3 receptor activation is not
required for TRPC3 activation. a, 10 mg/ml heparin was
introduced into a HEK293 wild-type cell (HEK-Wt) incubated
in Ca2+-free HBSS. After ~300 s, 100 µM
methacholine (MeCh) and 2 mM Ca2+
were sequentially added as indicated. b, similar protocol to
a was applied to a TRPC3-GFP-expressing HEK293 cell
(HEK-TRPC3), except that 5 µM Gd3+
was included throughout the measurements to inhibit endogenous CCE.
c, similar protocol to b was used with the
TRPC3-expressing HEK293 cell line, T3-9. d, HEK-TRPC3 cell
with a similar protocol to b, except that 2 µM
thapsigargin was added between MeCh and Ca2+ additions as
indicated.
|
|
Growth Factor Receptor Activates TRPC3--
To determine whether
heterotrimeric G proteins play an obligatory role in TRPC3 activation,
we tested whether TRPC3-mediated Ca2+ entry can be
activated through the tyrosine kinase-PLC-
pathway. We serum-starved
HEK-TRPC3 cells to boost EGF receptor expression. Stimulation of the
EGF receptor by EGF activates PLC-
through receptor tyrosine kinase
(27). Application of EGF (50-200 ng/ml) to wild-type HEK293 cells
induced neither Ca2+ release nor Ca2+ entry
upon restoration of external Ca2+. When higher
concentrations of EGF were applied (1 µg/ml), only 4 cells of 45 cells studied (~8%) showed Ca2+ release and subsequent
Ca2+ entry (data not shown). Stimulation of HEK-TRPC3 cells
with 1 µg/ml EGF also resulted in Ca2+ release in a small
fraction of the cells studied (6%; n = 96). However,
all cells showed substantial TRPC3-mediated Ca2+ entry when
Ca2+ was restored to the external medium regardless of
Ca2+ release (Fig.
5a). The extent of
TRPC3-mediated Ca2+ entry was similar when lower
concentrations of EGF (100 ng/ml) were used (data not shown). The
control experiment performed in the absence of EGF is shown in Fig.
5b. These results demonstrate that TRPC3 can be activated in
a G protein-independent manner. Fig. 5, a and b,
shows the results of individual imaging experiments showing average
data from 29 and 19 cells, respectively; S.E. values were very small,
and not visible at this graphic resolution.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
EGF receptor stimulation activates
TRPC3. a, HEK-TRPC3 cells were incubated in the absence
of added Ca2+. 5 µM Gd3+ was
present to inhibit endogenous CCE. 1 µg/ml EGF was added followed by
sequential addition of 2 mM external Ca2+ and
100 µM methacholine (MeCh) when indicated.
b, HEK-TRPC3 cells were treated in a similar way to the
protocol in a, except that EGF was omitted.
|
|
Methacholine and OAG Cause Non-additive Activation of
TRPC3--
Our results to this point would seem to essentially rule
out IP3 as the signal for TRPC3 activation.
Membrane-permeant analogues of DAG have been shown to stimulate TRPC3
and TRPC6 expressed in Chinese hamster ovary cells, providing a
possible mechanism of activation of these channels by PLC-linked
receptors (13). Thus, we tested whether receptor activation and OAG
activation of TRPC3 occur through independent pathways. Stimulation of
HEK-TRPC3 cells with methacholine in the presence of external
Ca2+ and 5 µM Gd3+ leads to an
[Ca2+]i rise that reaches a stable plateau,
corresponding to sustained Ca2+ entry through TRPC3
channels. Addition of OAG to these methacholine-activated cells did not
induce any further increase of Ca2+ entry, rather, a slight
but reproducible decrease was consistently observed after addition of
OAG (n = 274; Fig.
6a). Application of
methacholine to OAG-stimulated HEK-TRPC3 cells induced Ca2+
release, but did not produce any further increased Ca2+
entry (n = 177; Fig. 6b). Fig. 6,
a and b, are individual imaging experiments
showing average data and S.E. of 50 and 27 cells, respectively; S.E.
values are smaller than the line thickness. Patch clamp experiments
showed that whole cell currents elicited by methacholine were not
further increased by OAG and vise versa (Fig. 6, c and
d; in the experiment shown, 20 µM
IP3, which did not affect current, was present in the
pipette; similar results were obtained in the absence of
IP3, data not shown). The current-voltage relationship
after establishing the whole cell configuration with 20 µM IP3 in the patch pipette after subsequent
addition of methacholine (100 µM) and after further
addition of OAG (300 µM) to the same HEK-TRPC3 cell (Fig.
6d) shows that methacholine and OAG have no additive effect
on TRPC3 current amplitude. These results suggest that the same TRPC3
channels are activated by OAG and through PLC-coupled receptors. The
lack of additivity is also consistent with a mechanism whereby DAG,
formed as a consequence of PLC activation, is the signal for TRPC3
activation.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
Methacholine (MeCh) and OAG
activate TRPC3 non-additively. a, HEK-TRPC3
cells were incubated in the presence of 2 mM
Ca2+. 5 µM Gd3+ was present
throughout the experiment to inhibit endogenous CCE. 100 µM MeCh was added to the cells and when a sustained
plateau of Ca2+ entry was reached, 300 µM OAG
was added as indicated. b, HEK-TRPC3 cells were treated in a
similar protocol to a, with OAG added first followed by MeCh
as indicated. c, inward TRPC3 currents, measured every
10 s (at 60 mV) are plotted as a function of time. 20 µM IP3 was included in the patch pipette and
100 µM MeCh was added to the cells followed by 300 µM OAG (red trace) or inversely, OAG was added
before MeCh (blue trace). A control trace (black)
where only MeCh was added is also shown. d, representative
currents elicited by 250-ms voltage ramps ranging from 120 to +80 mV,
after the whole cell mode was established with IP3 in the
pipette (black), after MeCh addition (red), and
after OAG addition (blue).
|
|
Inhibitors of DAG Metabolism Activate TRPC3 and Slow the
Inactivation of Agonist-activated TRPC3--
To assess the role of
endogenously generated DAG in activation of TRPC3, we treated TRPC3
expressing cells with a combination of two inhibitors of DAG
metabolism: DAG kinase inhibitor II (28) and the DAG lipase inhibitor,
RHC80267 (29, 30). As shown in Fig.
7a, the combination of
inhibitors caused a rise in [Ca2+]i. This
increase was not seen in wild-type cells (Fig. 7a, red
trace), and was not seen in the absence of extracellular Ca2+ (data not shown). Interestingly, in wild-type cells,
the inhibitors substantially inhibited both release and entry because
of methacholine, likely because of PKC activation that commonly
inhibits the PLC pathway (31, 32). However, addition of 100 µM methacholine to the inhibitor-treated TRPC3 cells
resulted in an increase in [Ca2+]i beyond that
seen with the inhibitors alone, consistent with the finding that only
minimal PLC activation is required to stimulate TRPC3 (i.e.
Fig. 1). Subsequent addition of 50 µM atropine to block
the muscarinic receptor caused Ca2+ to decline to a level
similar to that seen with the inhibitors alone. The rate of decline was
considerably slower than that for the [Ca2+]i
signal in methacholine-stimulated cells that had not been treated with
the inhibitors (Fig. 7b), as shown by the normalized data
from the two experiments (Fig. 7e). This effect was not
because of changes in Ca2+ pumping across the plasma
membrane, because the rate of decline following removal of
extracellular Ca2+ was not affected by the inhibitors (Fig.
7, c, d, and f).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 7.
Inhibitors of DAG metabolism activate TRPC3
and slow the off-rate of activated TRPC3. a, a
combination of DAG kinase inhibitor II (30 µM) and DAG
lipase inhibitor (RHC-80267, 75 µM) was added to
HEK-TRPC3 cells, followed by 100 µM methacholine
(MeCh), and 50 µM atropine (Atro,
a and b), or the medium was changed from one
containing Ca2+ to a Ca2+-free medium
(0-Ca2+, c and d) as
indicated. The time course of the [Ca2+]i changes
in the absence of the inhibitors is shown in b and
d. 5 µM Gd3+ was present
throughout. In e and f, the time course of the
decline in [Ca2+]i after atropine addition
(e) or Ca2+ removal (f) has been
scaled between 100% (immediately before atropine addition or
Ca2+ removal) and 0% (value at the end of the experiment).
In a, also shown is the response to the inhibitors (or lack
thereof) in wild-type cells (in the absence of Gd3+,
red trace).
|
|
OAG Activation of TRPC3 Is PKC-independent--
We next tested
whether the effect of OAG on TRPC3 activation is mediated through PKC.
TRPC3 activation by OAG was completely blocked by the PKC activator,
PMA (n = 311; Fig.
8a), suggesting rather that
PKC negatively regulates TRPC3 channels. This result is consistent with
data in Fig. 6a showing a decrease of methacholine-induced activation of TRPC3 after OAG addition; this decrease may be because of
slight activation of PKC by OAG. OAG apparently does not maximally activate PKC, because it does not block Ca2+ release due to
muscarinic receptor activation (i.e. see Fig. 6b)
while phorbol ester drugs do (data not shown). Down-regulation of PKC
isoforms by treatment with 1.6 µM PMA for 20 h did
not prevent OAG-induced activation of TRPC3 but did prevent blockade of
the response by PMA (n = 244; Fig. 8b).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 8.
TRPC3 activation is PKC-independent.
a, HEK-TRPC3 cells were incubated in HBSS containing 2 mM Ca2+. Cells were pretreated with 1.6 µM PMA 10-15 min before starting the recordings. Control
cells were not PMA-treated. 5 µM Gd3+ was
present throughout the experiment, added where indicated. 300 µM OAG was added where indicated. Average of 50 cells
from a single imaging experiment; results from 3 other experiments were
similar. b, PKC down-regulation was achieved by incubation
of HEK-TRPC3 for 20 h in culture medium containing 1.6 µM PMA. Control cells were not treated. Before starting
the experiment, both groups of cells were incubated in HBSS containing
2 mM Ca2+ and 5 µM
Gd3+. 300 µM OAG and 100 µM
MeCh were added where indicated. Average of 50 cells from a single
imaging experiment; results from 3 other experiments were
similar.
|
|
 |
DISCUSSION |
In the present study, we have shown that when expressed in HEK293
cells, TRPC3 activation through PLC-coupled receptors is not mediated
by IP3; intracellular application of IP3 did
not activate TRPC3, and maximally effective concentrations of the IP3 receptor antagonist, heparin, had no effect on the
activation of TRPC3 by methacholine. G
q-linked receptors
or tyrosine kinase receptors, activating PLC-
or PLC-
,
respectively, both increase TRPC3 channel activity. External
application of a DAG analogue (OAG) or an increase in cellular DAG by
use of DAG metabolism inhibitors stimulates TRPC3 activity. TRPC3
channel activation through receptor stimulation cannot be further
increased by external application of DAG analogues. Reciprocally,
DAG-mediated activation of TRPC3 cannot be augmented by agonist
stimulation. The turn-off of receptor-activated TRPC3 following
application of a receptor antagonist is significantly slowed by
inhibitors of DAG metabolism. We conclude that receptor-mediated
activation of PLC activates TRPC3 through DAG production in a PKC- and
G protein-independent fashion.
These results were unexpected giving the substantial published evidence
that IP3 receptors liganded with IP3 are
required for TRPC3 activation (15-17). Kiselyov et al.
(15-17) studied IP3 effects on single channels in excised
patches, from cells previously stimulated with carbachol, whereas our
findings were obtained in intact cells stimulated with a wide range of
intracellular IP3 concentrations. Thus, it may be that
TRPC3 channels can be activated by IP3 receptors in some
cells, under some conditions. However, we conclude that the
physiological mechanism in HEK293 cells involves signaling through DAG.
The pharmacological evidence provided by Ma et al. (17) was
based on experiments with the IP3 receptor antagonist 2-APB
shown, in subsequent studies to be a relatively nonspecific inhibitor
of other ion channels (25, 26, 33-36). A report by Venkatachalam
et al. (12) demonstrated that receptor activation of TRPC3
in DT40 B-lymphocytes was unimpaired in a line lacking IP3
receptors, a result consistent with the findings in this study.
TRPC3 is structurally similar to TRPC7 and TRPC6 and the three
constitute a subfamily of the TRPC family. All three members of this
subfamily have been shown to be DAG-activated channels (13, 14, 37).
Furthermore, TRPC6 could not be activated when IP3 was
included in the patch pipette (13, 37). The efficacy of DAG analogues
and drugs that cause an increase in intracellular DAG in activating
TRPC3-mediated Ca2+ entry, the inability of DAG analogues
to have an additive effect on receptor-activated TRPC3, and the ability
of DAG metabolism inhibitors to slow the reversal of
agonist-dependent TRPC3 activation suggest that DAG
generated by stimulation of PLC-coupled receptors is the activator of
TRPC3. DAG activation of TRPC6 and TRPC7 was shown to be
PKC-independent (13, 14). This is consistent with our results obtained
with TRPC3. In light of these findings, it is very likely that members
of the TRPC3/6/7 subfamily share the same mode of activation, through
DAG production independently of IP3.
Agonist concentrations insufficient to produce IP3-induced
Ca2+ release from stores induce significant TRPC3-mediated
Ca2+ entry. Furthermore, TRPC3 could be activated through
receptor tyrosine kinase activation of PLC-
, even in the absence of
detectable IP3-induced Ca2+ release from
stores. It seems therefore that TRPC3 is activated by relatively small
amounts of DAG generated from low PLC activity, probably in a
membrane-delimited fashion, as previously proposed by Hofmann
et al. (13).
In conclusion, receptor-activated TRPC3 channels are activated by DAG
production independently of IP3 and G proteins. These data
support a similar activation mechanism for the TRPC3/6/7 subfamily and
call into question the previously proposed role of IP3 in
activating TRPC3 channels, as well as the proposed conformational coupling mechanism for this ion channel. Accumulating experimental evidence suggests that members of the TRP family of ion channels are
requisite components of native Ca2+-permeant channels
activated through physiological stimuli. Clearly, further work is
needed to understand how TRP channels are gated and regulated and how
their activation participates in complex physiological functions.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge generous advice and
discussions by Drs. Jerel Yakel, Franz-Josef Braun, Elizabeth Murphy,
and David Armstrong.
 |
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.
To whom correspondence should be addressed: NIEHS, National
Institutes of Health, P. O. Box 12233, Research Triangle Park, NC
27709. Tel.: 919-541-1129; Fax: 919-541-1898; E-mail:
trebak@niehs.nih.gov.
Published, JBC Papers in Press, February 26, 2003, DOI 10.1074/jbc.M300544200
 |
ABBREVIATIONS |
The abbreviations used are:
SOC, store-operated
channel;
CCE, capacitative calcium entry;
IP3, inositol
1,4,5-trisphosphate;
TRPC, canonical transient receptor potential;
DAG, diacylglycerol;
2-APB, 2-aminoethoxydiphenyl borane;
PKC, protein
kinase C;
OAG, 1-oleoyl-2-acetyl-sn-glycerol;
(2, 4,5)IP3, inositol 2,4,5-trisphosphate;
PMA, phorbol
12-myristate 13-acetate;
GPIP2, 1-(
-glycerophosphoryl)-D-myo-inositol-4,5-bisphosphate;
GFP, green fluorescent protein;
HBSS, Hepes-buffered saline solution;
PLC, phospholipase C;
EGF, epidermal growth factor;
BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid;
[Ca2+]i, intracellular
[Ca2+].
 |
REFERENCES |
1.
|
Putney, J. W., Jr.
(1986)
Cell Calcium
7,
1-12[Medline]
[Order article via Infotrieve]
|
2.
|
Berridge, M. J.
(1995)
Biochem. J.
312,
1-11[Medline]
[Order article via Infotrieve]
|
3.
|
Putney, J. W., Jr.
(1997)
Capacitative Calcium Entry
, Landes Biomedical Publishing, Austin, TX
|
4.
|
Putney, J. W., Jr.
(1990)
Cell Calcium
11,
611-624[Medline]
[Order article via Infotrieve]
|
5.
|
Randriamampita, C.,
and Tsien, R. Y.
(1993)
Nature
364,
809-814[CrossRef][Medline]
[Order article via Infotrieve]
|
6.
|
Irvine, R. F.
(1990)
FEBS Lett.
263,
5-9[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Birnbaumer, L.,
Zhu, X.,
Jiang, M.,
Boulay, G.,
Peyton, M.,
Vannier, B.,
Brown, D.,
Platano, D.,
Sadeghi, H.,
Stefani, E.,
and Birnbaumer, M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
15195-15202[Abstract/Free Full Text]
|
8.
|
Putney, J. W., Jr.,
and McKay, R. R.
(1999)
Bioessays
21,
38-46[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Elliott, A. C.
(2001)
Cell Calcium
30,
73-93[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Zhu, X.,
Jiang, M.,
and Birnbaumer, L.
(1998)
J. Biol. Chem.
273,
133-142[Abstract/Free Full Text]
|
11.
|
McKay, R. R.,
Szmeczek-Seay, C. L.,
Lièvremont, J.-P.,
Bird, G.,
St, J.,
Zitt, C.,
Jüngling, E.,
Lückhoff, A.,
and Putney, J. W., Jr.
(2000)
Biochem. J.
351,
735-746[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Venkatachalam, K.,
Ma, H.-T.,
Ford, D. L.,
and Gill, D. L.
(2001)
J. Biol. Chem.
276,
33980-33985[Abstract/Free Full Text]
|
13.
|
Hofmann, T.,
Obukhov, A. G.,
Schaefer, M.,
Harteneck, C.,
Gudermann, T.,
and Schultz, G.
(1999)
Nature
397,
259-262[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Okada, T.,
Inoue, R.,
Yamazaki, K.,
Maeda, A.,
Kurosaki, T.,
Yamakuni, T.,
Tanaka, I.,
Shimizu, S.,
Ikenaka, K.,
Imoto, K.,
and Mori, Y.
(1999)
J. Biol. Chem.
274,
27359-27370[Abstract/Free Full Text]
|
15.
|
Kiselyov, K.,
Xu, X.,
Mozhayeva, G.,
Kuo, T.,
Pessah, I.,
Mignery, G.,
Zhu, X.,
Birnbaumer, L.,
and Muallem, S.
(1998)
Nature
396,
478-482[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Kiselyov, K.,
Mignery, G. A.,
Zhu, M. X.,
and Muallem, S.
(1999)
Mol. Cell
4,
423-429[Medline]
[Order article via Infotrieve]
|
17.
|
Ma, H.-T.,
Patterson, R. L.,
van Rossum, D. B.,
Birnbaumer, L.,
Mikoshiba, K.,
and Gill, D. L.
(2000)
Science
287,
1647-1651[Abstract/Free Full Text]
|
18.
|
Boulay, G.,
Brown, D. M.,
Qin, N.,
Jiang, M.,
Dietrich, A.,
Zhu, M. X.,
Chen, Z.,
Birnbaumer, M.,
Mikoshiba, K.,
and Birnbaumer, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14955-14960[Abstract/Free Full Text]
|
19.
|
Vazquez, G.,
Lièvremont, J.-P.,
Bird, G.,
St, J.,
and Putney, J. W., Jr.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
11777-11782[Abstract/Free Full Text]
|
20.
|
Ma, H.-T.,
Venkatachalam, K.,
Li, H.-S.,
Montell, C.,
Kurosaki, T.,
Patterson, R. L.,
and Gill, D. L.
(2001)
J. Biol. Chem.
276,
18888-18896[Abstract/Free Full Text]
|
21.
|
Trebak, M.,
Bird, G.,
St, J.,
McKay, R. R.,
and Putney, J. W., Jr.
(2002)
J. Biol. Chem.
277,
21617-21623[Abstract/Free Full Text]
|
22.
|
Bird, G.,
St, J.,
Rossier, M. F.,
Hughes, A. R.,
Shears, S. B.,
Armstrong, D. L.,
and Putney, J. W., Jr.
(1991)
Nature
352,
162-165[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Luo, D.,
Broad, L. M.,
Bird, G.,
St, J.,
and Putney, J. W., Jr.
(2001)
J. Biol. Chem.
276,
5613-5621[Abstract/Free Full Text]
|
24.
|
Bird, G.,
St, J.,
Obie, J. F.,
and Putney, J. W., Jr.
(1992)
J. Biol. Chem.
267,
17722-17725[Abstract/Free Full Text]
|
25.
|
Broad, L. M.,
Braun, F.-J.,
Lièvremont, J.-P.,
Bird, G.,
St, J.,
Kurosaki, T.,
and Putney, J. W., Jr.
(2001)
J. Biol. Chem.
276,
15945-15952[Abstract/Free Full Text]
|
26.
|
Braun, F.-J.,
Broad, L. M.,
Armstrong, D. L.,
and Putney, J. W., Jr.
(2001)
J. Biol. Chem.
276,
1063-1070[Abstract/Free Full Text]
|
27.
|
Margolis, B.,
Rhee, S. G.,
Felder, S.,
Mervic, M.,
Lyall, R.,
Levitzki, A.,
Ullrich, A.,
Zilberstein, A.,
and Schlessinger, J.
(1989)
Cell
57,
1101-1107[Medline]
[Order article via Infotrieve]
|
28.
|
de Chaffoy de Courcelles, D.,
Roevens, P.,
and Van Belle, H.
(1985)
J. Biol. Chem.
260,
15762-15770[Abstract/Free Full Text]
|
29.
|
Sutherland, C. A.,
and Amind, D.
(1982)
J. Biol. Chem.
257,
14006-14010[Free Full Text]
|
30.
|
Broad, L. M.,
Cannon, T. R.,
and Taylor, C. W.
(1999)
J. Physiol. (Lond.)
517,
121-134[Abstract/Free Full Text]
|
31.
|
Ozawa, K.,
Yamada, K.,
Kazanietz, M. G.,
Blumberg, P. M.,
and Beaven, M. A.
(1993)
J. Biol. Chem.
268,
2280-2283[Abstract/Free Full Text]
|
32.
|
Brown, K. D.,
Blakeley, D. M.,
Hamon, M. H.,
Laurie, M. S.,
and Corps, A. N.
(1987)
Biochem. J.
245,
631-639[Medline]
[Order article via Infotrieve]
|
33.
|
Bakowski, D.,
Glitsch, M. D.,
and Parekh, A. B.
(2001)
J. Physiol. (Lond.)
532,
55-71[Abstract/Free Full Text]
|
34.
|
Prakriya, M.,
and Lewis, R. S.
(2001)
J. Physiol. (Lond.)
536,
3-19[Abstract/Free Full Text]
|
35.
|
Gregory, R. B.,
Rychkov, G.,
and Barritt, G. J.
(2001)
Biochem. J.
354,
285-290[CrossRef][Medline]
[Order article via Infotrieve]
|
36.
|
Iwasaki, H.,
Mori, Y.,
Hara, Y.,
Uchida, K.,
Zhou, H.,
and Mikoshiba, K.
(2001)
Recept. Channels
7,
429-439[Medline]
[Order article via Infotrieve]
|
37.
|
Inoue, R.,
Okada, T.,
Onoue, H.,
Hara, Y.,
Shimizu, S.,
Naitoh, S.,
Ito, Y.,
and Mori, Y.
(2001)
Circ. Res.
88,
325-332[Abstract/Free Full Text]
|
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.