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Cell Calcium 42 (2007) 213–223
TRPC1: The link between functionally distinct
store-operated calcium channels
Indu S. Ambudkar∗, Hwei Ling Ong, Xibao Liu,
Bidhan Bandyopadhyay, Kwong Tai Cheng
Secretory Physiology Section, GTTB, NIDCR, NIH, Bethesda, MD 20892, USA
Received 25 January 2007; received in revised form 30 January 2007; accepted 31 January 2007
Available online 12 March 2007
Abstract
Although store-operated calcium entry (SOCE) was identified more that two decades ago, understanding the molecular mechanisms that
regulate and mediate this process continue to pose a major challenge to investigators in this field. Thus, there has been major focus on
determining which of the models proposed for this mechanism is valid and conclusively establishing the components of the store-operated
calcium (SOC) channel(s). The transient receptor potential canonical (TRPC) proteins have been suggested as candidate components of
the elusive store-operated Ca2+ entry channel. While all TRPCs are activated in response to agonist-stimulated phosphatidylinositol 4,5,
bisphosphate (PIP2) hydrolysis, only some display store-dependent regulation. TRPC1 is currently the strongest candidate component of
SOC and is shown to contribute to SOCE in many cell types. Heteromeric interactions of TRPC1 with other TRPCs generate diverse SOC
channels. Recent studies have revealed novel components of SOCE, namely the stromal interacting molecule (STIM) and Orai proteins. While
STIM1 has been suggested to be the ER-Ca2+ sensor protein relaying the signal to the plasma membrane for activation of SOCE, Orai1 is
reported to be the pore-forming component of CRAC channel that mediates SOCE in T-lymphocytes and other hematopoetic cells. Several
studies now demonstrate that TRPC1 also associates with STIM1 suggesting that SOC and CRAC channels are regulated by similar molecular
components. Interestingly, TRPC1 is also associated with Orai1 and a TRPC1-Orai1-STIM1 ternary complex contributes to SOC channel
function. This review will focus on the diverse SOC channels formed by TRPC1 and the suggestion that TRPC1 might serve as a molecular
link that determines their regulation by store-depletion.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: Transient receptor potential canonical 1; Store-operated calcium entry; Endoplasmic reticulum; Calcium signaling; Regulatory protein complex
1. The conundrum of store-operated calcium entry
The endoplasmic reticulum (ER) provides a large intra-
cellular Ca2+ store from which Ca2+ can be released under
a number of different conditions. The ER, as well as plasma
membrane (PM) in resting cells have low permeability to
Ca2+ which increases following stimulation of cells by a
wide variety of extracellular ligands as well as physical and
chemical stimuli. Store emptying is most often evoked by
an increase in the level of inositol trisphosphate (IP3)by
∗Corresponding author at: Building 10, Room 1N-113, NIH, Bethesda,
MD 20892, USA. Tel.: +1 301 496 5298; fax: +1 301 402 1228.
E-mail address: indu.ambudkar@nih.gov (I.S. Ambudkar).
stimulation of a variety of cell surface receptors that are
associated with the activation of PLC and hydrolysis of the
plasma membrane lipid phosphatidylinositol bisphosphate,
PIP2.IP
3induces release of Ca2+ from the ER via activa-
tion of IP3receptor, IP3R[1–4]. While it was recognized
almost two decades ago that this Ca2+ signaling process is
accompanied by activation of plasma membrane Ca2+-entry,
understanding the molecular basis of this mechanism remains
somewhat of a conundrum. This Ca2+ entry pathway, which
was first identified in non-excitable cells, was termed store-
operated Ca2+ entry (SOCE) since it is stimulated in response
to depletion of Ca2+ from intracellular Ca2+ stores in ER
(and inactivated by refilling of these stores), and not due to
PIP2hydrolysis per se. SOCE has been subsequently found
0143-4160/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ceca.2007.01.013
214 I.S. Ambudkar et al. / Cell Calcium 42 (2007) 213–223
to be present in neuronal as well as muscle cells. Although
it is clear that other Ca2+ entry pathways are also activated
by agonist-stimulated PIP2hydrolysis, via direct effects of
diacylglycerol or PIP2, this review will focus on SOCE. It is
important to note that other mechanisms can also induce inter-
nal Ca2+ store release and activate Ca2+ entry [3,4]. Recently,
protein synthesis has been associated with release of ER
Ca2+ and activation of SOCE. Termination of protein syn-
thesis and release of nascent polypeptide is associated with
a change in the ion permeability of the ribosome-translocon
complex resulting in Ca2+ release from the ER [5–7]. Thus,
depletion of ER by several different conditions, converge on
the activation of SOCE. In addition to regulating a num-
ber of neurotransmitter-dependent physiological processes,
e.g. T-lymphocyte activation, smooth muscle contraction,
endothelial cell permeability, salivary gland fluid secretion,
etc., SOCE, therefore, also sustains optimal levels of protein
synthesis by maintaining ER-[Ca2+]. It can be suggested that
SOCE is a mechanism devised by the cell to ensure optimal
refilling of the internal Ca2+ stores which is critical for protein
synthesis, cell growth, and proliferation.
SOCE is mediated via the activation of specific plasma
membrane channels, termed store-operated calcium (SOC)
channels [1,2,4], which are ubiquitously expressed in all cell
types. However, and somewhat uniquely, the channel char-
acteristics are diverse in different cell types. The first SOC
channel to be identified, calcium-release-activated calcium
(CRAC) channel, mediates a highly Ca2+-selective calcium
current detected in T-lymphocytes and RBL cells [3,8]. Based
on the methodology and criteria used for measuring and iden-
tifying CRAC channel function, SOC channels with different
biophysical characteristics were subsequently identified in
various cell types [1–3,9,10]. These channels range from non-
selective for Ca2+ to those that are relatively selective. While
the molecular basis for these channels is not clear, it can be
suggested that type of channel formed in a cell is determined
by the physiological function that it serves. An important
question that arises from these significant findings is how all
these diverse channels sense internal Ca2+ store depletion;
i.e. are they all activated by the same ER-PM signal?
Understanding the mechanism by which the status of Ca2+
in the ER is transmitted to the PM, to activate or inacti-
vate SOC, has presented a major challenge to investigators
in this field. Among the many models proposed to describe
this elusive pathway, three have received most attention [4].
(i) Conformational coupling hypothesis which suggests that
ER signal is relayed to the SOC via a direct interaction of the
channel with IP3R in the ER. (ii) The diffusible factor hypoth-
esis suggests that a factor is released or generated in response
to internal Ca2+ store depletion. (iii) The secretion coupling
model proposes regulated recruitment of channels by fusion
of intracellular vesicles. A major roadblock towards under-
standing this mechanism has been the lack of knowledge of
the channel or accessory regulatory components. Over the
past decade, several essential components of SOCE havebeen
identified, including members of transient receptor potential
canonical (TRPC) sub-family of cation channels that have
emerged as central players in this process (further discussed
below) [1,3,11]. However, studies within the last 2 years have
identified new components of SOCE that provide insight into
the possible mechanism by which this process is regulated
and account for the diversity in SOC channels.
2. TRPC1: a molecular component of diverse SOC
channels
Studies with Drosophila TRP channel, which is activated
by a phospholipase C (PLC)-dependent mechanism, led to the
search for mammalian TRP channels, which were suggested
as candidate components of the elusive SOC, store-operated
channel. Consistent with the initial prediction, seven mam-
malian channels (TRPC1–TRPC7) were identified which
were activated in response to stimulation of PIP2hydroly-
sis by stimulation of various plasma membrane receptors in
different tissues [3,11]. One of the major criteria for identifi-
cation of SOC channels has been activation by ER depletion
per se; in absence of receptor-mediated PIP2hydrolysis [3,4].
Thus, only some TRPCs consistently display store-dependent
regulation while others have been suggested to primarily con-
tribute to agonist-stimulated non-store-operated Ca2+ entry
mechanisms. It is important to recognize that there is consid-
erable conflict regarding the mode of activation of a number
of TRPC channels. Part of this discrepancy is due to the use
of heterologously expressed proteins in different cell types
where the background SOC channels could confound the
observations. Relatively less information is available regard-
ing the status of endogenous TRPC channels.
TRPC1, the first mammalian TRPC protein to be identi-
fied, is widely expressed in neuronal as well as non-neuronal
tissues including muscle [3,11–14]. Based on all the studies
reported until now, data demonstrating endogenous TRPC1
as a SOC component have been most consistent. TRPC1 has
been reported to contribute to SOCE in a variety of cell types,
including salivary gland, keratinocytes, platelets, smooth,
skeletal, and cardiac muscle, DT40, HEK293, neuronal,
intestinal, and endothelial cells [15–25]. In contrast, some
studies, primarily with the heterologously expressed pro-
tein describe store-independent regulation of TRPC1 [3,11].
TRPC1 forms diverse SOCs ranging from relatively selective
to non-selective (Ca2+ versus Na+)[26,27]. Some TRPC1-
SOCs display strong anomalous mole-fraction behavior for
Ca2+/Na+permeability which renders them more permeable
to Ca2+ under physiological conditions. Such diversity is pro-
posed to be a result of differences in the composition of the
SOC channels generated by homomeric or heteromeric inter-
actions of TRPC1 with other TRPC monomers. Although
there is no conclusive evidence so far of a native homo-
meric TRPC1 channel, selective interactions of TRPC1 with
other TRPCs, e.g. TRPC4/TRPC5 or TRPC3/TRPC7 have
been reported [23,25,27–32]. TRPC1–TRPC1 multimers are
generated by interaction of the N-terminal coiled–coiled
I.S. Ambudkar et al. / Cell Calcium 42 (2007) 213–223 215
domain interactions [33] while TRPC1–TRPC3 heteromers
are formed by interaction of the first ankyrin repeat region
in TRPC1with an as yet unknown region in the TRPC3
N-terminus [27]. Of these, only interaction with TRPC4,
TRPC3, or TRPC7 has been linked with generation of SOC
channels. There is convincing evidence to show that het-
eromeric TRPC3 + TRPC1 channels are involved in SOCE
in HSY cells [27] and hippocampal neuronal cells [34];
TRPC1 + TRPC4 channels mediate SOCE in endothelial
cells [28] and TRPC1 + TRPC3 + TRPC7 in HEK-293 cells
[27]. These studies provide a molecular basis by which
the previously described diversity in SOC channels can be
explained. Although the exact molecular signal(s) that dic-
tates the interaction of TRPC1 with other TRPCs is not yet
known, it can be suggested that this is likely determined by
the type of channel that is required by the cell to fulfill a
specific physiological function.
In the following section, we will discuss well-studied
TRPC1-SOC channels in various cell types (see Table 1)
including salivary gland cells, platelets, endothelial cells,
smooth and skeletal muscle cells. The voluminous amount of
data reported in the last 8–10 years which describe function
of heterologously expressed TRPC, and the controversies in
those observations, will not be discussed here due to inherent
problems associated with such expression systems [13,35].
2.1. TRPC1-SOC in salivary gland cells
SOCE has been suggested to be critical for salivary fluid
secretion since [Ca2+]iincreases regulate ion channels that
drive water flow out of the gland. Studies regarding the role
of TRPC1 in the SOCE mechanism of salivary gland cells
have primarily been reported by us over the last 8–10 years.
Presently most of the studies have been done with cell lines
and primary cells. We first used a knockdown strategy to
show that endogenous TRPC1 contributes to SOCE in the
human submandibular gland cell line, HSG [19]. We fur-
ther identified a TRPC1-SOC channel in these cells that is
distinct from the CRAC channel in that it is less selective
for Ca2+ (Ca2+/Na+permeability was 40 for SOC versus
400 for CRAC). Activation of this channel by depletion of
internal Ca2+ stores with either IP3or inhibition of SERCA
pump by thapsigargin generates relatively inwardly rectify-
ing currents (Erev = +20 mV). The channel displays strong
anomalous mole fraction behavior for Ca2+ versus Na+per-
meability demonstrating it is relatively more permeable to
Ca2+ under physiological conditions [10]. Single channel
measurements demonstrated single channel conductance of
20 pS. The contribution of TRPC1 to the SOC channel was
confirmed by (i) demonstrating that exogenously expressed
TRPC1 generated channel activity similar to the endogenous
activity, (ii) more importantly, by mutating the proposed-pore
region of the protein, confirmed by topology studies [36],
which resulted in change in the calcium permeability of the
channel, (iii) deletion of the pore region which induced a
dominant suppression of the endogenous current [37], and
(iv) by expressing TRPC1 encoded by an adenovirus in vivo
in rat salivary gland which not only increased salivary flow
in the animal but also increased SOCE in the salivary gland
cells [38]. Yuan et al. [39] also reported a similar TRPC1-
dependent SOC current in HSG cells and further identified
a regulation of TRPC1 by IP3R and the scaffolding protein,
Homer. Together, these important findings provided strong
evidence that TRPC1 is a critical component of the SOC
channel in HSG cells and contributes to the Ca2+ selectivity
of the channel. Whether this is a TRPC1-monomeric channel
or not is not yet known. However, TRPC4 and TRPC3 do not
appear to contribute to SOC channel function in these cells.
Surprisingly, but consistent with the ability of TRPC1 to
form diverse SOC channels, we identified a non-selective
Table 1
Presence and function of TRPC1-SOC channels
Cell type Channels Functions References
Salivary gland C1-C1 (?) Ion channel regulation/fluid secretion [10,19,27,37,38]
C1-C3
Endothelial C1-C1 (?) Permeability/proliferation/inflammation [20,23,28,40,41,43,45–47]
C1-C4
Smooth muscle C1-C1 (?) Contraction, proliferation [15,17,50–53]
C1-C5
Neuronal C1-C3 Post-synaptic signaling/growth/apoptosis [18,30,34]
C1-C1 (?)
C1-C5 (?)
Keratinocytes C1-? Wound healing/proliferation/differentiation/ER stress [16,73]
Cardiac myocytes C1-? Hypertrophy [57]
Skeletal myocytes C1-? Role in muscular dystrophy [24]
Intestinal epithelial cells C1-? Wound healing [22]
HEK-293 C1-C3-C7 ? [25,65–68]
DT-40 C1-? ? [21]
Platelets C1-C4 (?) ? [58–60,62–64]
216 I.S. Ambudkar et al. / Cell Calcium 42 (2007) 213–223
SOC channel in human parotid gland cell line, HSY [27].In
this case, TRPC1 interacts with TRPC3 via its N-terminal
domain. Expression of either the N-terminus of TRPC1 or
TRPC3 in these cells induces dominant suppression of SOC
channel function. In addition to minimal selectivity of Ca2+
versus Na+or Na+versus Cs+, this channel does not display
anomalous mole fraction behavior for Ca2+ versus Na+. The
relative amount of Na+or Ca2+ permeating via this chan-
nel is not yet determined. Neither is it clear why cells that are
functionally quite similar require such diverse SOC channels.
Mouse submandibular gland cells also display a linear ISOC,
although TRPC1 contributes to this channel other TRPC com-
ponents have not yet been identified (Liu et al. unpublished
observations).
2.2. TRPC1-SOC in endothelial cells
Store-depletion-activated Ca2+ entry is a critical deter-
minant of endothelial permeability [23,40–44]. Endothelial
cells express almost all TRPC proteins, specifically TRPC1
and TRPC4 have been identified in all the cells studied thus far
including: HUVEC, human umbilical vein endothelial cells;
HPAEC, human pulmonary artery endothelial cells; HMEC,
human dermal microvascular endothelial cell line; RPAEC,
rat pulmonary artery endothelial cells; BAEC, bovine aor-
tic endothelial cells; MAEC, mouse aortic endothelial cells;
MLEC, mouse lung endothelial cells. There is consider-
able evidence for the contribution of TRPC1 to SOCE in
endothelial cells. TRPC1 is shown to form SOC channels
in HUVEC and HMEC. Increased TRPC1 expression in
HMEC increases thrombin-induced Ca2+ influx and IP3-
sensitive store-operated cationic current [45]. In HUVEC,
VEGF-activated Ca2+ entry is blocked by anti-TRPC1 anti-
body directed against the external pore domain of the protein,
suggesting a role for TRPC1 in regulation of endothelial per-
meability [46]. TRPC1 expression is regulated by TNF-␣
and this accounts for the enhanced Ca2+ influx in response
to thrombin in HPAEC [45]. Further, this overexpression of
TRPC1 is associated with modulation of the permeability
change induced by thrombin. Thus, TRPC1 expression in
microvascular endothelial cells may contribute to pulmonary
edema associated with acute respiratory distress syndrome.
Groschner et al. [47] have demonstrated the expression
of TRPC1, TRPC3, and TRPC4 in HUVEC utilizing RT-
PCR and on the basis of the inhibition of SOCE induced
by the N-terminal fragment of hTRPC3 have suggested
that SOCE is mediated via TRPC1 + TRPC3 heteromer. A
role for TRPC1 in SOC has also been reported in the
A549 cell line as well as pulmonary artery endothelial cells
[26].
Heteromeric TRPC1 + TRPC4 SOC channels have also
been described in endothelial cells. Brough et al. [26]
have reported that transfection of a TRPC1 specific anti-
sense oligonucleotide in HPAEC markedly reduced the
thapsigargin-induced increase in [Ca2+]iand store-operated
Ca2+ entry current. This current is inwardly rectifying
(Erev = +30 mV), suggesting the presence of a relatively Ca2+
selective channel. This channel is reported to be generated
by heteromeric association of TRPC1 and TRPC4. It is sen-
sitive to changes in actin status since it is anchored to the
cytoskeleton by an interaction of TRPC4 to protein 4.1 [48]
which in turn is tethered to spectrin. SOCE in aortic and lung
endothelial cells is decreased in TRPC4−/−mice [49] sug-
gesting that TRPC1 cannot function alone and compensate
for the loss of TRPC4. Interestingly, TRPC4 has been asso-
ciated with generation of a channel with high selectivity for
Ca2+ (highest of all known TRPCs) which does not display
any increase in outward currents at mV more positive than
the Erev. Although, a similar highly Ca2+-selective current is
detected in aortic endothelial cells, it is unclear whether it is
mediated via a homomeric TRPC4 channel.
2.3. TRPC1-SOC in smooth muscle cells
TRPC1 is expressed in most types of smooth muscle cells
with the exception of a few [3,13,15] and in various species
such as human, dog, mouse, rabbit, and rat. An antibody to the
pore region of the protein as well as TRPC1-siRNA decrease
SOCE and arterial smooth muscle function, both in primary
and cultured cells. A role for TRPC1 in SOCE has been shown
in smooth muscle cells of caudal, basal, pulmonary artery, and
cerebral arterioles. However, the effect of TRPC1 on SOCE
is not always correlated with changes in function. TRPC1
regulates smooth muscle cell proliferation since TRPC1-
antisense RNA decreased pulmonary artery smooth muscle
cell growth [50]. Furthermore, TRPC1 is upregulated under
conditions that initiate smooth muscle regulation and prolifer-
ation [51,52]. TRPC4 also appears to contribute to the SOCE
together with TRPC1 in pulmonary artery myocytes in cul-
ture [53]. The likelihood of interaction between these TRPC
proteins has been demonstrated convincingly by FRET-based
assays. It has also been suggested that TRPC4 might facilitate
the trafficking and surface expression of TRPC1 [54]. TRPC6
knockdown decreases SOCE in rat pulmonary arterial smooth
muscle cells [55], however, the available data do not support
a heteromeric TRPC6 + TRPC1 SOC channel. How TRPC6
contributes to this SOCE is not known, although it is impor-
tant to note that TRPC6 (−/−) mice have been associated
with increased TRPC3 expression and exhibit functional sec-
ondary effects due to TRPC3-dependent channel activity.
Thus, the role of TRPC6 in SOCE needs to be examined
in further detail.
Rat aortic smooth muscle cell line, A7r5 express TRPC1,
C4, and C6 [56]. Knockdown of TRPC1 decreased ISOC in
these cells suggesting that TRPC1 contributes to the SOC
channel in these cells. Notably, ISOC in these cells was quite
similar to that seen in HSG and smooth muscle cells; i.e. rel-
atively inwardly rectifying. While the role of TRPC4 in this
SOC cannot be ruled out, this study rules out a contribution of
TRPC6 based on the differences in the currents that are asso-
ciated with the two proteins. It is important to note that TRPCs
are not uniformly distributed in all types of SMCs. Further,
I.S. Ambudkar et al. / Cell Calcium 42 (2007) 213–223 217
TRPC1 transcript is not expressed in some of them (e.g. from
mouse and rat cerebral artery, human trachea, canine pul-
monary artery, mouse and canine colon, and rat mesenteric
artery). Thus, it will be important to determine what other
TRPC contribute to SOCE in these cells. In terms of the
properties, SOC channels in smooth muscle cells vary from
highly selective to relatively non-selective. The SOC current
in rabbit pial arteriole smooth muscle has been recently been
proposed to be generated via a TRPC1 + TRPC5 heteromeric
channel based on the characteristic outward rectification pre-
viously described for this heteromer. This current is quite
distinct from the inwardly rectifying found in A7r5 cells
[56] as well as the non-selective currents reported earlier
in rabbit and rat aortic smooth muscles [9]. Recently roles
for TRPC1 and TRPC3 have been reported in skeletal and
cardiac muscle, respectively [24,57].
2.4. TRPC1-SOC in platelets
Studies by Sage and co-workers strongly suggest the
involvement of TRPC1 in the SOCE mechanism in platelets
[58–60]. Activation of platelets has been associated with
increased interaction of TRPC1 with IP3Rs. In addition,
conditions that disrupt this interaction have been shown to
attenuate SOCE. SOCE is also directly linked with regulation
of platelet aggregation induced by a number of different stim-
uli. However, there is some discrepancy in the data reported
for platelets which primarily arises due to the differences
observed in the localization and expression of different TRPC
proteins in platelets by two groups. While Sage and co-
workers have suggested plasma membrane localization of
TRPC1, Authi reported that TRPC1 is primarily localized
intracellularly [61]. Since TRPC1 trafficking to the plasma
membrane as well as its retention there is shown to depend
on several factors [61], it is unclear whether differences in
any of these can account for the observed discrepancies.
TRPC6 is expressed in platelets in relatively high abundance
and forms DAG-activated channels, although it is unclear
whether it contributes to SOC. Thus, the present data sug-
gest the SOC channels in platelets are made up of TRPC1,
possibly together possibly with other TRPCs, e.g. TRPC4,
[59–64]. There is presently no information on the biophys-
ical properties of this channel, which could provide some
insight and potential resolution of the controversies regarding
its components.
2.5. TRPC1-SOC in HEK293 cells
Despite the fact that this is the most widely used cell
for study of TRP channel function, there is little data
regarding its endogenous SOC channels. This is possi-
bly an important contributing factor to the contradictory
findings in studies using heterologous expression of var-
ious TRPCs in these cells. The available data regarding
the molecular components of endogenous SOCE in HEK-
293 cells has been reported primarily by Villereal and
co-workers [25,65–67]. In a systematic study using a knock-
down approach they have demonstrated that TRPC1, TRPC3,
and TRPC7 contribute to the SOCE in these cells [25]. The
role of TRPC1 + TRPC3 in generating a non-selective SOC
was suggested earlier by Klaus Groschner’s group [68].On
the other hand over expression systems have not always
been consistent with these findings. TRPC3, when expressed
at relatively low levels, TRPC2, as well as TRPC7 have
been reported to generate SOC channels in these cells [3].
It is unclear whether these functions are dictated by the
interaction of the expressed TRPCs with an endogenous
protein; e.g. TRPC1. This should be given serious consid-
eration since other studies have shown that heterologously
expressed TRPCs can co-IP with each other and although
TRPC1/TRPC4/TRPC5 or TRPC3/TRPC6/TRPC7 form two
groups whose members seem to associate with each other,
exceptions have been reported. Importantly, such exceptions;
e.g. TRPC1/TRPC3/TRPC7 appear to require the presence of
TRPC1 in the channel [30].
2.6. TRPC1-SOC in neuronal cells
Several studies demonstrate a role for TRPC1 in neuronal
cells. A role for TRPC1 in the proliferation of embryonic
rat neural stem cells was recently reported. Store deple-
tion generated a non-selective current that was attenuated
by antisense-TRPC1 [18]. While TRPC2, C3, C4, C6 tran-
scripts were detected in these cells, their involvement in
SOC channel function was not assessed. A TRPC1 + TRPC3-
SOC channel was suggested in proliferating hippocampal
neurons [34]. In these cells, expression of TRPC1- and
TRPC3-mRNA and protein dramatically increased, while the
expression of TRPC4 and TRPC7 mRNA and protein dramat-
ically decreased in parallel with a 3.4-fold increase in the level
of SOCE. siRNA-induced knockdown of TRPC1 and TRPC3
indicate they are involved in mediating SOCE, TRPC4 and
TRPC7 were not involved. Inhibition of SOCE also decreased
cell proliferation suggesting a role for TRPC1 + TRPC3-
SOC channel in cell proliferation. TRPC1 also contributes
to SOCE in SH-SY5Y human neuroblastoma cells which
have been associated with a pro-survival effect on the
cells [69,70] and mediates mGluR1-dependent Ca2+ entry
[71].
2.7. TRPC1-SOCs in other cell types
In addition to the role of TRPC1 in SOCE in the cell
types discussed above, it is also shown to generate store-
dependent non-selective cation channels in CHO cells [72].
In gingival keratinocytes [16,73,74] where SOCE regulates
cell differentiation, two SOC currents have been described
which are activated sequentially by store depletion; an ICRAC-
like Ca2+ selective current that is initially activated followed
by a larger non-selective cation current. The former has been
associated with TRPC4 and the latter with TRPC1. Thus, in
these cells TRPC1 and TRPC4 appear to form independent
218 I.S. Ambudkar et al. / Cell Calcium 42 (2007) 213–223
SOC channels. TRPC1 is also involved in SOCE in LNCaP
human prostate cancer epithelial cells [75–77]. The currents
associated with TRPC1 in these cells are relatively inwardly
rectifying and dependent on IP3Rs as well as the cytoskele-
tal status. There is only one report showing that TRPC1
contributes to ICRAC. This study in DT40 cells reveals that
deletion of TRPC1 protein expression induces almost com-
plete attenuation of the current in about 80% of the cells,
and normal SOCE is restored in these cells by expression
of hTRPC1. In the remaining 20% cells, loss of TRPC1 is
not associated with any changes in ICRAC [21]. The reason
for this diversity in the cell function is unclear. Interestingly,
we have recently reported that TRPC1 is not endogenously
involved in ICRAC in RBL-2H3 cells [78]. However, heterolo-
gous expression of TRPC1 in these cells alters the properties
of CRAC, shifting the Erev to the left. Mutant TRPC1 lacking
the pore region exerts dominant suppression of ICRAC. These
data suggest that TRPC1 can interact heteromerically with
the CRAC channel components in RBL-2H3 cells.
Together the studies discussed above provide strong evi-
dence that endogenous TRPC1 contributes to SOC channels.
Further, they illustrate the remarkable diversity in the char-
acteristics and functions of TRPC1-SOC channels. TRPC1
forms SOC channels by interactions with TRPCs that are sug-
gested to form SOC (e.g. TRPC1 and TRPC4) as well as those
which are not typically associated with generation of SOCs
(e.g. TRPC3, TRPC7). The involvementof the latter is consis-
tent with a previous suggestion that novel interaction between
TRPC proteins requires the presence of TRPC1. An important
question that arises is whether typically store-independent
TRPCs gain store-dependent regulation by interacting with
TRPC1; i.e. is TRPC1 the molecular link in diverse SOC
channels? (see Fig. 1). To address this question, the mecha-
nisms involved in store-dependent activation of TRPC1 have
to be considered.
Fig. 1. Contribution of TRPC1 to diverse SOC channels. Homomeric and
heteromeric SOC channels can be generated by interaction of TRPC1 with
other TRPC monomers, as indicated in the Figure. Some of these TRPCs
are ER-coupled in that they interact with STIM1 while others are not. The
latter can still contribute to generation of SOC via interaction with a TRPC
monomer that is regulated by store-depletion, e.g. TRPC1. For sake of keep-
ing the figure simple, only dimeric channels have been depicted and other
proteins implicated in TRPC1 function; such as IP3R, HOMER, caveolin,
etc., have not been shown.
3. Store-dependent regulation of TRPC1-SOC
The mechanism(s) involved in regulation of SOC chan-
nels continues to be the focus of a large number of studies.
The activity of TRPC1-SOC channels are determined by store
depletion and refilling, by feedback inhibition of Ca2+ entry
which modulates Ca2+ entry after it has been activated, and
by regulation of its surface expression. Plasma membrane
localization of TRPC1 is dependent on its interactions with
other TRPCs (e.g. TRPC1, TRPC4) [54], or other proteins
such as caveolin-1 [79–81],-tubulin (microtubule struc-
tures) [82], and RhoA (remodeling of the cytoskeleton) [20].
TRPC1 interaction with caveolin-1 and RhoA mediates its
localization in caveolar lipid raft domains. Disruption of
these domains abrogates SOCE leading to the suggestion that
TRPC1-SOC channels are assembled within such domains.
A number of reports show that TRPC1 is assembled in a
signalplex that contains key Ca2+ signaling proteins such
PLC, G␣q/11 and IP3R[12,79,81] and depending on the cell
type, neurotransmitter and growth factor receptors, such as
bradykinin [83] and FGF receptors [18], and mGluR [71]
which are involved upstream events, leading to PIP2hydrol-
ysis, IP3generation, and release of Ca2+ from the ER; i.e.
events that result in activation of Ca2+ entry. Despite exten-
sive studies, the mechanism that is involved in the final step
of SOC activation, i.e. sensing ER depletion and transmis-
sion of the signal to the PM, is not yet established. IP3R
was the first ER protein to be proposed as a candidate pro-
tein for this function and thus a major focus has been placed
on possible interactions between IP3Rs and TRPC1 [4].In
fact the “conformational coupling” model for activation of
SOCE was based on the ryanodine receptor-dependent regu-
lation of voltage-gated Ca2+ channel in excitation-contraction
coupling in muscle cells which is reminiscent of ER–PM
interactions during regulation of SOCE. Consistent with this,
TRPC channels including TRPC1 have been shown to inter-
act with IP3R. Further, there is convincing evidence that IP3R
regulates TRPC1 function. The scaffolding protein HOMER
was shown to mediate interaction between TRPC1 and IP3R
and disruption of this interaction was associated with TRPC1
activation by ER-Ca2+ depletion [39]. In contrast, an increase
in association of IP3R with TRPC1 has been seen in platelets
and endothelial cells suggesting that the IP3R-TRPC1 inter-
action is required for SOCE [20,84]. CaM, which mediates
Ca2+-dependent feedback inhibition of TRPC1 has also been
proposed to be involved in the regulation of channel function
by IP3R. IP3R and CaM have been proposed to have antag-
onistic effects in the activation of TRPC1, with CaM being
inhibitory and IP3R activating the channel by displacing CaM
[85].
TRPC1 trafficking to the membrane and its assembly in
aCa
2+ channel complex is key to its function and regula-
tion. Within this complex, TRPC1 interacts with scaffolding
as well as regulatory proteins [86–88]. Plasma membrane
expression of TRPC1 is increased following stimulation of
endothelial cells, via a mechanism that is mediated via local
I.S. Ambudkar et al. / Cell Calcium 42 (2007) 213–223 219
cytoskeletal changes. In endothelial cells, channel activa-
tion is also associated with recruitment of TRPC1, IP3R,
and RhoA in a complex [20]. Further, specialized plasma
membrane domains appear to be involved in regulating the
assembly, function, as well as trafficking of TRPC1 [1]. These
domains, lipid raft domains, are enriched in PIP2as well as
sphingolipids, both of which have been implicated in regula-
tion of TRPC channels. Cholesterol and cholesterol-binding
proteins are also concentrated in lipid raft domains. This
lipid structure in the plasma membrane provides a platform
for assembly of TRPC1 channel complexes and facilitates
the regulation of these channels [81]. Local remodeling of
cytoskeleton has also been suggested occur in these regions
since actin-binding proteins such as ezrin are found here.
Thus, despite significant progress in our understanding of
SOC channels and identification of several “candidates” for
the mechanism of regulation, how TRPC1-SOC channels are
actually gated still remains unclear.
4. New findings: insights into old mechanisms?
As discussed above, a considerable amount of data provide
strong evidence that TRPC1 is a component of SOC chan-
nels in a large variety of cells. However, the fact that none
of the TRPCs generate ICRAC has been a source of concern.
The exceptions, as noted above, are TRPC4 in aortic endothe-
lial and smooth muscle cells [42,49] and keratinocytes [74],
TRPC1 in DT40 cells [21], and TRPC3 in T lympho-
cytes [89]. Thus, considerable effort has been made towards
identifying the components of the CRAC channel. Two lab-
oratories using siRNA strategies independently identified
stromal interacting protein1, STIM1, a single transmembrane
domain protein found both in the plasma membrane and
the ER, as a critical protein involved in SOCE, including
CRAC channel function [90,91] Although overexpression of
the protein does not increase SOCE, knockdown effectively
decreases SOCE in several cell types. STIM1 has an EF-
hand domain at its N-terminal end that is localized within
the ER lumen. Thus it has been proposed that STIM1 can
function as a sensor for ER-[Ca2+][92–94]. Depletion of
ER-Ca2+ causes a relocation of STIM1 from a diffuse ER
localization to clusters in peripheral ER-plasma membrane
junctions which have been proposed as the centers where
SOCE occurs [95]. Importantly, TRPC1 also interacts with
STIM1 [63,78,96]. Huang et al. report that the mechanism
involved in STIM1-dependent activation of SOCE and CRAC
channels are identical to those involved in the binding and
activation of STIM1 and TRPC1 [96]. Activity of STIM1
requires an ERM domain, which mediates the selective bind-
ing of STIM1 to TRPC1, 2 and 4, but not to TRPC3, 6 or 7.
Further, TRPC1 forms clusters that coincide with the STIM1
clusters in the sub-plasma membrane region after internal
Ca2+ store depletion. In platelets, store depletion induces an
increase in TRPC1-STIM1 association which is required for
activation of SOCE and depends on the cytoskeletal changes
[63]. Our recent studies [78] are consistent with these find-
ings. We have shown that TRPC1-C terminus interacts with
the C terminus of STIM1 in HSG cells and that this interaction
is involved in regulating TRPC1-SOC function. TRPC1 and
STIM1 co-localize and can be co-immunoprecipitated from
extracts of HSG, mouse submandibular gland cells, HEK-
293 cells, and A7r5 cells. Association between the proteins
is increased by thapsigargin treatment. It is important to note
that IP3Rs also cluster in response to cell stimulation [97].
Whether the IP3R clusters also coincide with the STIM1 and
TRPC1 clusters is not yet known. These observations reveal
similar regulation of CRAC, and TRPC-SOC channels by
STIM1.
Similar siRNA screen as well as genetic linkage stud-
ies in SCID patients identified another protein family (Orai,
which presently has three members). These proteins were ini-
tially suggested as possible regulators of the CRAC channel
since knockdown of their expression decreased ICRAC. More
significantly, mutations in Orai1, found in T lymphocytes
from SCID patients, were associated with defective ICRAC
[92,98]. Subsequently, overexpression of Orai1 and STIM1
was shown to induce large increases in SOCE and ICRAC
and mutations in negatively charged aa residues in the trans-
membrane domains caused changes in the Ca2+ permeability
of CRAC channel [93,99–102]. Thus, Orai1 has been now
been suggested to be a pore-forming component of CRAC
channel. Further, it was reported that STIM1 and Orai1 are
sufficient for generation of functional CRAC channels. How-
ever, studies reported presently suggest that these proteins are
not exclusive to CRAC channels. In addition to the associa-
tion of TRPC1 with STIM1, we have now reported that Orai1
and STIM1 interact with and contribute to TRPC1-SOC chan-
nel. Treatment with siSTIM1 and siOrai1 reduced SOCE
and SOC channel function in HSG cells [78] and HSY cells
(Cheng et al., unpublished observations). Furthermore, we
show that TRPC1, Orai1, and STIM1 are colocalized in the
plasma membrane region of cells and form a ternary complex.
Association of the three proteins is increased upon ER-Ca2+
store depletion. These studies reveal that TRPC1, Orai1, and
STIM1 concertedly determine SOCE in cells where TRPC1 is
an essential pore-forming component of SOC channels. We
have not yet determined whether Orai1 also contributes to
the SOC channel pore. Interestingly, the effects of the Orai1
mutants on channel function are very similar to what we have
previously reported with TRPC1 mutants. Clearly, further
studies are required to asses the role of Orai1 and STIM1
in SOC channels in other cell types. Furthermore, it is quite
possible that other TRPCs might also associate with these
two proteins. In an interesting study, it was observed that
Orai1 can interact with TRPC3 and TRPC6 to mediate store-
dependent regulation of these channels (Liao et al., PNASc, in
press. Personal communication from Dr. Lutz Birnbaumer).
By measuring the effect of Orai1 on the single channel proper-
ties of TRPC3, these investigators have proposed that Orai1
acts as a regulatory, , subunit of TRPC channels. Similar
studies are required for TRPC1-SOC channels to resolve the
220 I.S. Ambudkar et al. / Cell Calcium 42 (2007) 213–223
functional interaction between TRPC1 and Orai1. Part of the
problem with the studies with Orai1 is they have all been car-
ried out with overexpression systems and furthermore, there
are no antibodies for detecting endogenous Orai1. Thus the
status of endogenous Orai1, e.g. the level of expression in
different cells, is yet unclear. Despite this, the compelling
effect of siOrai1 on TRPC1-SOC channels provides strong
evidence that these proteins converge on the same mecha-
nism. Whether Orai1 also contributes to the pore of the SOC
channels or acts as a regulatory subunit, such as those of
ionotropic GluR channels as well as voltage-dependent cal-
cium channels, is yet to be determined. It is interesting that
the subunits affect the trafficking as well as gating of the
“excitable” cell Ca2+ channels.
We propose (see Fig. 1) that TRPC1 is the STIM1 bind-
ing component in TRPC-containing SOC channels. While
TRPC2 and TRPC7 can also link plasma membrane SOC to
ER, TRPC7 is not found in majority of the SOC channels
and TRPC2 is not expressed in human cells. Furthermore,
SOC channels in which TRPC1 involvement has been con-
clusively excluded appear to be rare. Thus a reasonable
hypothesis for how diverse SOC channels are regulated by
the same intracellular signal (ER Ca2+ depletion) is that
TRPC1 links the plasma membrane SOC channel to the ER-
Ca2+ sensor, STIM1. We suggest that TRPC1 and STIM1
can provide a molecular basis for TRPC-dependent SOCE.
TRPC1, together with other TRPC monomers forms the pore-
forming component of the SOC channel while STIM1 serves
as the ER-Ca2+ sensor that relays the ER “signal” to the
channel. Whether this directly results in gating the SOC chan-
nel or other proteins are also involved in this relay is not
yet known. Furthermore, our suggestion does not exclude
the involvement of the other proteins (discussed above) in
SOCE although clearly further studies will be required to
establish the relative contributions of proteins such as IP3R,
HOMER, and STIM1 to SOC channel function. There are no
data available presently to establish whether Orai1 interacts
directly with either STIM1 or TRPC1. Thus, how Orai1 con-
tributes to TRPC1-SOC or even other SOC channels needs
to be further evaluated. We suggest the following possible
models for the role of TRPC1 and Orai1 in SOC channels
(see Fig. 2):
•Orai1 and TRPC1 form independent channels: This is not
supported by the activity seen in cells after TRPC1 or Orai1
knockdown (e.g. TRPC1 knockdown does not result in
residual ICRAC-like current); also the activities due to the
two proteins are not additive.
•Orai1 and TRPC1 contribute to a single SOC channel:
Conclusive evidence for this is lacking although the knock-
down data do suggest that both proteins contribute to
SOCE. How a 4-TM and 6-TM domain proteins can con-
tribute to one channel pore requires further clarification.
•Orai1 is the pore-forming subunit and TRPC1 is a
regulator: Mutations in the charged aa residues in the trans-
membrane domain alters selectivity of Orai1. Although
Fig. 2. Possible role of TRPC1 and Orai1 in SOCE. The figure illustrates
the four possibilities discussed in the text. (i) TRPC1 and Orai1 form inde-
pendent channel, (ii) TRPc1 and Orai1 contribute to the same channel, (iii)
TRPC1 is a regulator of Orai1-channel (indicated by orange arrow at the
top), and (iv) Orai1 is a regulator of TRPC1 channel.
these studies are promising further structure-functional
confirmation is needed to establish whether this is the pore-
region of Orai1. Regulatory subunits of other calcium
channels such as GluRs or VDCCs, which have 4-TMs,
regulate gating, current amplitudes, and trafficking of the
pore-forming ␣subunits. Thus detailed single channel
studies are required to resolve the role of Orai1.
•TRPC1 is the pore-forming subunit and Orai1 is the
regulator: There are considerable data defining the pore
properties of TRPC channels. The proposed topology of
TRPC1 is similar to that of other 6-TM channels. Topology
and mutational studies have identified the region spanning
the 5th and 6th TM domain to be the site of the pore.
Thus, there is considerable evidence presently available
to support TRPC1 as the pore-forming unit of SOC chan-
nels. However, the possibility that Orai1 regulates SOC
channels (as described above for TRPC3) needs to be
investigated more thoroughly.
Since STIM1 and Orai1 are recently described proteins,
there are relatively few reports. For example there is no infor-
mation on endogenous Orai1 other than the effects of siRNA
on ICRAC, even these are not substantiated by assessment of
the protein. Further, more detailed studies are required to
substantiate the suggestion that a direct interaction between
Orai1 and STIM1 is involved in generation of CRAC chan-
nel. Despite the lack of such information, identification of
these proteins has provided new directions in the study of
SOCE which could potentially lead to an understanding of
its regulation. There is no doubt that future studies will reveal
important aspects of the role of TRPC1, Orai1, and STIM1
in generation of SOC channel.
5. Concluding remarks
Much effort has been focused on answering the ques-
tion of whether TRPC1 is store-operated or not. Majority
of the studies reported until now suggest that TRPC1 is func-
I.S. Ambudkar et al. / Cell Calcium 42 (2007) 213–223 221
tional component of a variety of native SOCE pathways in
different cell types. TRPC1 generates SOC channels by inter-
action with other TRPC monomers. SOC channels in which
TRPC1 involvement has been conclusively excluded appear
to be rare. Thus, we propose that TRPC1 is a common com-
ponent of diverse SOC channels. Most likely STIM1 is the
ER-regulator of a number of different TRPC1-SOC channels,
whether it regulates all of them is not known. Furthermore,
Orai1 might also interact with TRPC1 and contribute to
its function. While the exact mode by which Orai1 affects
TRPC1-SOC channel function needs to be clarified it appear
that Orail is required for TRPC1 function. Knowledge that
TRPC1 is part of SOCE represents a significant advancement
in our understanding of SOCE. Future studies should address
the important question of how the signal linked to store deple-
tion is actually relayed to TRPC1 for activation of SOCE.
Conflict of interest
None.
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