A Calmodulin/Inositol 1,4,5-Trisphosphate (IP3) Receptor-binding Region Targets TRPC3 to the Plasma Membrane in a Calmodulin/IP3 Receptor-independent Process*
Barbara J. Wedel,
Guillermo Vazquez,
Richard R. McKay,
Gary St. J. Bird and
James W. Putney, Jr.
From the
Laboratory of Signal Transduction, NIEHS, National Institutes of Health,
Department of Health and Human Services, Research Triangle Park, North
Carolina 27709
Received for publication, April 14, 2003
, and in revised form, May 1, 2003.
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ABSTRACT
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Conformational coupling with the inositol 1,4,5-trisphosphate
(IP3) receptor has been suggested as a possible mechanism of
activation of TRPC3 channels and a region in the C terminus of TRPC3 has been
shown to interact with the IP3 receptor as well as calmodulin
(calmodulin/IP3 receptor-binding (CIRB) region). Here we show that
internal deletion of 20 amino acids corresponding to the highly conserved CIRB
region results in the loss of diacylglycerol and agonist-mediated channel
activation in HEK293 cells. By using confocal microscopy to examine the
cellular localization of Topaz fluorescent protein fusion constructs, we
demonstrate that this loss in activity is caused by faulty targeting of
CIRB-deleted mutants to intracellular compartments. Wild type TRPC3 and
mutants lacking a C-terminal predicted coiled coil region downstream of CIRB
were targeted to the plasma membrane correctly in HEK293 cells and exhibited
TRPC3-mediated calcium entry in response to agonist activation. Mutation of
conserved YQ and MKR motifs to alanine within the CIRB region in TRPC3-Topaz,
which would be expected to interfere with IP3 receptor and/or
calmodulin binding, had no effect on channel function or targeting.
Additionally, TRPC3 targets to the plasma membrane of DT40 cells lacking all
three IP3 receptors and forms functional ion channels. These
findings indicate that the previously identified CIRB region of TRPC3 is
involved in its targeting to the plasma membrane by a mechanism that does not
involve interaction with IP3 receptors.
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INTRODUCTION
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Calcium entry in nonexcitable cells is most commonly triggered by the
activation of a receptor stimulating phospholipase C to cleave
phosphatidylinositol 4,5-bisphosphate into diacylglycerol
(DAG),1 which
stimulates protein kinase C, and inositol 1,4,5-trisphosphate
(IP3), which binds to the IP3 receptor to release
calcium from intracellular stores
(1). TRPC3, a member of the
transient receptor potential family of ion channels, has been shown to be
activated by agonist activation of plasma membrane G-protein-coupled
receptors, by synthetic diacylglycerols, and by store depletion in some cell
types
(24).
A conformational coupling model physically linking TRPC3 with the
IP3 receptor has been suggested, and a region in the cytosolic C
terminus of TRPC3 has been shown to co-immunoprecipitate with regions of the
IP3 receptor (5,
6). An overlapping region of
TRPC3 binds to calmodulin (CaM), which is hypothesized to be tethered to the
channel and to be displaced competitively by the activated IP3
receptor; the region that commonly binds both IP3 receptor and
calmodulin has been termed the calmodulin/IP3 receptor-binding
(CIRB) region (7). However,
binding of CaM to the isolated CaM-binding region of TRPC3 is dependent on the
calcium concentration, which is inconsistent with a model of CaM being
tethered to the channel independently of the calcium concentration. Calmodulin
binding to a CIRB homologous region in all members of the TRPC family
(TRPC17) has been demonstrated
(8). A second CaM-binding
region has been identified in other TRPs. For TRPC4, CaM binds in a region
unique to the splice variant TRPC4
, which is not present in the splice
variant TRPC4
(8,
9). This region has also been
shown to interact with the C terminus of IP3 receptors in a yeast
two-hybrid screen and glutathione S-transferase pull-down assays
(10). Binding to both
CaM-binding domains of TRPC4 occurs only above 10 µM
Ca2+ with an apparent Kd of
100200 nM CaM
(9). For TRPC1 a second
CaM-binding domain has also been reported that overlaps with a predicted
coiled coil region common to the C terminus of all TRPCs
(11). This second CaM-binding
region has been shown to be involved in calcium-dependent inactivation of
TRPC1 (11). Our goal was to
identify functional roles of the CIRB and other domains of TRPC3 using N- and
C-terminal truncation mutants of TRPC3.
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MATERIALS AND METHODS
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Plasmid ConstructionTRPC3 (provided by Lutz Birnbaumer,
NIEHS, National Institutes of Health), hemagglutinin-tagged at the C terminus
via an AscI site in pcDNA3, was used as a template for the
construction of truncation mutants (TRPC3N
1TRPC3N
3 and
TRPC3C
4TRPC3C
8). For N-terminal truncation mutants
(TRPC3N
1TRPC3N
3), the sequence preceding the desired
start site was changed to a BamHI site followed by an ATG using the
QuikChangeTM site-directed mutagenesis kit. To remove the 5'
sequence including the original ATG, cDNAs were digested with BamHI,
which has a recognition sequence in the multicloning box of pcDNA3 5' of
the insert and at the newly introduced site, and religated. For C-terminal
truncation mutants (TRPC3
C4TRPC3
C8), an NheI
site was introduced into the sequence (GCTAGC) with the TAG stop
codon positioned at the designated end of the coding region. For TRPC3-Topaz
fusion constructs (T3T), a TRPC3 construct fused to the brighter YFP version
Topaz at the C terminus via an AscI restriction site was used as a
template (12). For N-terminal
truncation mutants (T3T-N
0 and T3T-N
1), the same strategy as
described for untagged mutants was used. For C-terminal deletions
(T3T-C
7 and T3T-C
8), the AscI recognition site 3'
of the stop codon was mutated keeping the AscI site fusing TrpC3 to
Topaz intact. An AscI site was introduced into the sequence at the
desired site of truncation of TrpC3. The mutants were digested with
AscI and religated to yield truncated TrpC3-Topaz fusions. For the
internal deletion mutant T3T-C
78 oligonucleotides
5'-cagcattctcaatcagggatccatgtatcagcagataatgaaaag and
5'-cttttcattatctgctgatacatggatccctgattgagaatgctg were used in the
mutagenesis reaction with Trp3-Topaz as template.
Cell Culture and TransfectionFor the production of pools of
cells stably expressing wild type and mutant TRPC3, HEK293 cells were
transfected with LipofectAMINE2000® at
80% confluency according to
the manufacturer's instructions. The day after transfection the cells were
split into selection medium containing 0.5 mg/ml G418 and grown for 4 weeks in
the continued presence of selection medium. In parallel, control cells
transfected only with the fluorescent marker pdsRedmito were selected for G418
resistance as above, and pdsRedmito expression was confirmed by their red
fluorescence (excitation, 558 nm; emission, 610 nm).
The chicken B lymphocyte cell line DT40 and the mutant variant in which the
genes for all three IP3 receptor types were disrupted were obtained
from the Institute of Physical and Chemical Research (RIKEN; Cell Bank code
RCB1464 and RCB1467). The cells were cultured essentially as described by
Sugawara et al. (13).
DT40 were transiently transfected by electroporation
(14) with the indicated
amounts of the human isoform of TRPC3 or deletion variants or its vector
(pcDNA3), along with EYFP-C1 vector (Clontech) as a marker for transfection.
The cells were co-transfected with the human M5 muscarinic receptor (50
µg/ml, in pcDNA3). The cells were assayed 1725 h post-transfection.
The fluorescence measurements were performed under the conditions indicated
with single enhanced yellow fluorescent protein (EYFP)-positive cells,
selected by their yellow/green fluorescence (excitation, 485 nm; emission, 520
nm). Under the conditions of measurement, EYFP expression did not contribute
significant fluorescence.
Flow CytometryPools of G418-resistant cells expressing wild
type or mutant T3T were subjected to flow cytometric analysis to enrich for
cells with high expression levels of fluorescent protein fusions. The cells
were treated with trypsin and analyzed on a FACSVantageSE flow cytometer
equipped with Cell Quest software (Becton-Dickinson, San Jose, CA).
Fluorescence was assayed using an excitation wavelength of 488 nm and an
emission wavelength of 530 nm.
Calcium MeasurementIntracellular calcium concentration was
measured with a real time fluorescence plate reader system (FLIPR-384;
Molecular Devices, Sunnyvale, CA); the cells were plated in
poly-D-lysine-coated, black-walled 96-well plates at
3040% confluency and incubated overnight at 37 °C to allow
attachment of the cells. The cells were loaded for 90 min at 37 °C with 4
µM Fluo4-AM and washed twice with nominally calcium free buffer
(20 mM HEPES, 120 mM NaCl, 5.4 mM KCl, 0.8
mM MgCl2, 11.1 mM glucose, pH 7.4).
GdCl3, methacholine, and CaCl2 at final concentrations
of 1 µM, 100 µM, and 1.8 mM,
respectively, were added at the time points indicated in the figure. For
measurement of 1-oleoyl-2-acetyl-sn-glycerol (OAG) activation, the
cells were loaded with Fluo4-AM as described above and washed twice with
calcium containing buffer (20 mM HEPES, 120 mM NaCl, 5.4
mM KCl, 0.8 mM MgCl2, 11.1 mM
glucose, pH 7.4, 1.8 mM Ca2+). OAG (final
concentration, 100 µM) in calcium-containing buffer was added at
the indicated times. The fluorescent dye was excited with an argon laser at
488 nm, and the resultant emitted light (540 nm) was detected by a cooled CCD
camera. When representative traces are shown, they are averages of multiple
wells within an experiment, and each experiment was repeated at least three
times with similar results.
For fluorescence measurements of DT40 cells, the fluorescence intensity of
multiple Fura-2-loaded EYFP-positive cells or EYFP-negative control cells was
monitored with a CCD camera-based imaging system (Universal Imaging) mounted
on a Zeiss Axiovert 35 inverted microscope equipped with a Zeiss 40x
(1.3 NA) fluor objective. A Sutter Instruments filter changer enabled
alternative excitation at 340 and 380 nm, whereas the emission fluorescence
was monitored at 510 nm with a Paultek Imaging camera (model PC-20) equipped
with a GenII-Sys intensifier (Dage-MTI, Inc.). The images of multiple cells
collected at each excitation wavelength were processed using the MetaFluor
software (Universal Imaging Corp., West Chester, PA) to provide ratios of
Fura-2 fluorescence from excitation at 340 nm to that from excitation at 380
nm (F340/F380).
Confocal MicroscopyThe fluorescence images were acquired
with a Zeiss LSM510 confocal laser scanning microscope (Carl Zeiss, Inc.,
Thornwood, NY) with an argon laser and excitation at 488 nm through a
100x (oil immersion) objective lens (optical slice thickness, 0.5
µm). The level of expression of TRPC3-Topaz and thus the brightness of the
images in DT40 cells were considerably less than in HEK293 cells. Therefore,
all of the images have been subjected to a software-driven equalization
procedure, scaling gray scale pixel values between the same minimum (black)
and maximum (white) values. This allows for maximal contrast and maximal
spatial information, which is required in the current study, but precludes
quantitative comparisons between different images.
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RESULTS
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The initial goal of the present studies was to define domains of TRPC3 that
are required for channel activation by truncating the cytosolic N and C
termini of TRPC3. We generated a series of deletion mutants (mutants
N
13 and C
48;
Fig. 1), all of which retained
the six predicted transmembrane domains (TM1TM6) and a putative pore
region between TM5 and TM6. In N
1 four predicted ankyrin repeats were
deleted (amino acids 1199), whereas in N
2 an adjacent predicted
coiled coil region was additionally deleted (amino acids 1330). Mutant
N
3 lacked virtually all of the cytosolic N terminus (amino acids
1367). The cytoplasmic C terminus includes the Trp signature motif
(EWKFAR), a highly conserved proline-rich motif (TLPXPF) and a region
that was shown in pull-down assays to be CIRB. This CIRB site is followed by a
second predicted coiled coil region. For C-terminal truncations amino acids
690, 730, 759, 775, or 790844 were deleted, resulting in a TRPC3
lacking one or more of these domains (C
48;
Fig. 1). All of the mutants
were stably expressed in HEK293 cells and tested in FLIPR (see
"Materials and Methods") for agonist-dependent
Ca2+ entry in the presence of 1 µM
Gd3+, which blocks the endogenous, store-operated
channels (15,
16). In contrast to control
cells expressing only the fluorescent marker pdsRedmito, wild type
TRPC3-expressing cells showed significant
Gd3+-insensitive Ca2+ entry
(Fig. 2). All N-terminal
mutants (N
13) lacked Ca2+ entry in
response to 100 µM methacholine, suggesting a crucial role for
ankyrin repeats in the proper folding, targeting, or anchoring of TRPC3
(Fig. 2). Of the C-terminal
truncation mutants C
8, which includes the CIRB site, retained agonist
dependent Ca2+ entry, whereas C
7, which excludes
this region, was inactive (Fig.
3).

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FIG. 3. Agonist-induced Ca2+ entry in TRPC3 C-terminal
truncation mutants. HEK cells stably expressing pcDNA3, TRPC3, or
C-terminal truncation mutants C 48 were assayed in a FLIPR as
described under "Materials and Methods" and as in the legend to
Fig. 1. The light emitted by
the fluorescent dye (cps) is recorded as a measure for
[Ca2+]i. Ca2+
release in response to 100 µM methacholine in the absence of
extracellular Ca2+ and Ca2+ entry
upon restoration of 1.8 mM extracellular Ca2+
were assessed in the presence of 1 µM
Gd3+, which blocks endogenous channels. Shown is a
representative experiment.
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To examine expression and targeting of the truncated proteins, we
constructed a subset of mutants in a TRPC3-Topaz fusion construct
(T3T-N
1, T3T-C
7, and T3T-C
8;
Fig. 4, top panel). We
also constructed two additional deletion mutants. A N-terminal splice variant
of TRPC6 had previously been shown to selectively lose OAG but not
agonist-stimulated Ca2+ entry (Ref.
17 and see
"Discussion"). Thus, we constructed a N-terminal deletion of the
first 27 amino acids upstream of the first ankyrin repeat (T3T-N
0). We
also constructed a C-terminal deletion lacking most of the CIRB region but
retaining the downstream coiled coil sequence (amino acids 775789,
T3TC
78); a similar TRPC3 construct lacking the CIRB region had
previously been demonstrated to have increased current activity following
exposure to a TRPC3-interacting IP3 receptor peptide
(7). We selected pools of cells
stably expressing T3T or T3T mutants with G418 and enriched for cells with
high expression levels using flow cytometry of G418-resistant cell populations
(18). The cells were tested
for Gd3+-insensitive Ca2+ entry in
response to agonist stimulation and Ca2+ entry in
response to OAG using a FLIPR. As shown in
Fig. 5,
Ca2+ entry of the truncated Topaz fusion constructs
corresponded to Ca2+ entry seen with their corresponding
untagged counterparts. T3T-expressing control cells exhibited
Gd3+-insensitive Ca2+ entry in
response to methacholine activation, as did T3T-C
8, whereas deletion
constructs missing the N-terminal ankyrin repeats (T3T-N
1) or the
coiled coil and CIRB region (T3TC
7) showed no
Ca2+ entry. The conservative N-terminal deletion mutant,
T3T-N
0, showed normal or partially reduced entry, whereas the CIRB
deletion, T3T-C
78, was inactive. The latter deletion is similar in
length to the active T3T-C
8, indicating that it is the specific
sequence of amino acids in the C
78 (CIRB) region that is important for
activity. All of the mutants that showed
Gd3+-insensitive Ca2+ entry in
response to methacholine could also be activated by OAG
(Fig. 6).

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FIG. 4. Structure of Topaz-tagged truncation mutants of TRPC3 and alignment of
C-Terminal Sequences of Trp channels. Top panel, structural
features of TRPC3 C-terminally tagged with Topaz are as described in the
legend to Fig. 1. The region(s)
deleted in the respective truncation mutant are indicated in the structure.
Bottom panel, C-terminal sequences including the CIRB region and
putative coiled coil region of TRPC17 are shown in alignment. The C14
peptide has been shown by Zhang et al.
(7) to co-precipitate with the
IP3 receptor and Ca2+/CaM, whereas the C8
peptide interacts solely with Ca2+/CaM. The positions of
truncation for TRPC3 mutants C 68 are indicated with
arrows. Amino acids mutated in T3T-YQ/AA and T3T-MKR/AAA are
gray and marked with dots.
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FIG. 5. Agonist-induced Ca2+ entry in Topaz-tagged TRPC3
truncation mutants. HEK cells stably expressing pcDNA3, T3T, or T3T
truncation mutants were assayed in a FLIPR as described under "Materials
and Methods" to determine Ca2+ release and entry
in response to treatment with 100 µM methacholine. The protocol
is as described in the legend to Fig.
3. The measurements were performed in the presence of 1
µM Gd3+ to block endogenous channels.
Shown is a representative experiment.
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FIG. 6. OAG-activated Ca2+ entry in Topaz-tagged TRPC3
truncation mutants. HEK cells stably expressing pcDNA3, T3T, or T3T
truncation mutants were assayed in a FLIPR. 100 µM OAG was added
to cells in the continued presence of 1.8 mM
Ca2+. A pcDNA3 control trace obtained in the same FLIPR
experiment as for the truncation mutants is repeated in each panel. Shown is a
representative experiment.
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To determine whether mutants that showed no Ca2+
entry were properly targeted to the plasma membrane, we examined the cellular
localization of the respective Topaz fusion proteins by confocal microscopy
(Fig. 7). As previously
reported for a GFP fusion of TRPC3
(18), wild type T3T was mostly
localized in punctate regions in the plasma membrane. There was additional
staining in intracellular compartments, possibly in the region of the
endoplasmic reticulum (ER) and Golgi apparatus, which may represent newly
synthesized protein (Fig. 7). A
similar pattern was observed for the conservative and physiologically active
deletion mutants, T3T-N
0 and T3T-C
8, although the labeling
appears more uniform and less punctate. The physiologically inactive
N-terminal truncation mutant T3T-N
1 and C-terminal truncation mutants
T3T-C
7 and T3T-C
78 appeared to be predominantly located in
intracellular compartments with no detectable labeling of the plasma membrane
(Fig. 7).

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FIG. 7. Confocal images of HEK cells stably expressing Topaz-tagged TRPC3
truncation mutants. HEK cells stably expressing pcDNA3, T3T, or T3T
truncation mutants were analyzed by confocal microscopy. Plasma membrane
localization of fusion constructs is indicated with an arrow.
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Because the CIRB region appeared to be crucial for plasma membrane
targeting of T3T, we also mutated conserved amino acids within the overlapping
IP3 receptor/CaM-binding domain to see whether mutations within
this region (T3T-YQ/AA and T3T-MKR/AAA;
Fig. 4, bottom panel)
could also cause loss of plasma membrane targeting as well as agonist and
OAG-induced Ca2+ entry seen with deletion of the entire
region (Fig. 4, bottom
panel). Mutation to alanine of the corresponding residues in L-type
Ca2+ channels was shown to reduce both CaM binding and
Ca2+-dependent inactivation
(19). All of the T3T mutants
were expressed in HEK293 cells, and G418-resistant cells with high expression
levels were selected using flow cytometry. However, these mutations within the
CIRB region (T3T-YQ/AA and T3T-MKR/AAA) did not reduce
Gd3+-insensitive Ca2+ entry in
response to methacholine or OAG as compared with wild type T3T
(Fig. 8, A and
B). Consistent with this phenotype T3T-YQ/AA was
detectable throughout the plasma membrane with some protein in cytosolic
compartments, possibly ER/Golgi (Fig.
8C).

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FIG. 8. Mutations within the CIRB region have no effect on agonist/OAG-induced
Ca2+ entry and subcellular localization of TRPC3.
HEK cells expressing T3T constructs mutated within the CIRB region (T3T-YQ/AA
and T3T-MKR/AAA; see Fig. 4)
were tested in a FLIPR for Gd3+-insensitive
Ca2+ entry in response to methacholinemediated
(A) and OAG-mediated (B) Ca2+ entry as
described under "Materials and Methods." C, confocal
images of T3T and T3T-YQ/AA expressing HEK cells show plasma membrane
expression as indicated by arrows.
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The findings to this point demonstrate that deletions of the CIRB region
block function of TRPC3 by preventing proper trafficking of the protein to the
plasma membrane. Because this region has been shown to interact with
IP3 receptors (20),
we considered the possibility that this interaction with IP3
receptors is involved in proper targeting of TRPC3. To examine the possible
role of IP3 receptors in targeting of TRPC3, we made use of a DT40
chicken B-lymphocyte cell line lacking all three types of IP3
receptors (DT40-KO). To be able to compare phenotypes of T3T and deletion
mutants between HEK cells and DT40-KO cells, we first set out to optimize
transfection levels of T3T in wild type DT40 cells, which are by experience
harder to transfect than HEK cells, aiming to reach similar expression levels
as in HEK cells. We accomplished this by two different strategies: first by
increasing the amount of plasmid used for transfection from 10 to 100 µg/ml
cells as described recently by Vazquez et al.
(21) and second by lowering
the incubation temperature after transfection to 30 °C. For DT40 cells
grown at 37 °C, no TRPC3-Topaz fluorescence was detectable when applying
the settings used for HEK cells in Figs.
7 and
8C (optical slice
thickness, 0.5 µm); rather, fluorescent images could be obtained only when
the optical slice thickness was maximized, such that the resulting images were
not confocal (data not shown). Confocal images of T3T expressing DT40 cells
grown at 30 °C, however, could be obtained using the same settings used
for HEK cells (Fig.
9A) and show a punctate pattern in the plasma membrane as
seen before in HEK cells. Previous functional studies on TRPC3 expressing
cells have suggested that IP3 receptors are not required for
agonist induced Ca2+ entry in this cell line
(21,
22). Consistent with the
functional data, IP3 receptors also do not appear to be required
for plasma membrane targeting of T3T because we observe plasma membrane
expression of the Topaz-labeled protein in both wild type and DT40-KO cells
(Fig. 9A). As for
HEK293 cells, deletion of the CIRB region (C
7) resulted in the loss of
the distinct punctate pattern of plasma membrane localization
(Fig. 9A). For
functional studies DT40-KO cells were transfected with cDNAs encoding the M5
muscarinic receptor under the control of the chicken
-actin promoter,
EYFP as a transfection marker, and the respective T3T construct
(Fig. 9B). DT4-KO
cells are known to respond to phospholipase C-coupled receptors with
generation of IP3, but they do not generate phospholipase C-linked
cytosolic Ca2+ signals
(13). The addition of
carbachol to DT40-KO cells expressing T3T and M5 receptors presumably causes
formation of IP3 and DAG but no release of
Ca2+ from stores because of the lack all three types of
IP3 receptors. Despite this lack of IP3 receptors, T3T
is able to function in a receptor-operated mode as revealed by significant
entry of Ba2+ (Fig.
9B). When T3T-C
7 and T3T-C
8 were expressed
in DT40-KO cells, we obtained the same phenotypes as in HEK293 cells; whereas
the predicted C-terminal coiled coil region could be deleted in T3T without
loss of agonist induced entry, additional deletion of the CIRB region results
in loss of function, a function that is independent of the presence of
IP3 receptors.
Because the CIRB region of TRPC3 has been shown to bind to both the
IP3 receptor and CaM, we next set out to test the role of CaM in
targeting of TRPC3. We therefore overexpressed T3T in DT40-KO cells together
with wild type CaM or a dominant negative CaM mutant
(CaMEF14mut), in which all four EF hand motifs are mutated
(7). We observed no difference
in plasma membrane targeting of T3T in CaM versus
CaMEF14mut expressing cells (not shown). Thus, in aggregate,
our data demonstrate that CIRB (or elements contained therein) is necessary
for proper targeting of TRPC3 to the plasma membrane, but neither CaM nor the
IP3 receptor, the binding partners so far shown to interact with this region,
appear to play a role in this regard.
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DISCUSSION
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In recent years several genetic diseases have been linked to mutations
within ion channel genes (23).
Some of theses mutations interfere with proper trafficking of the channel to
the plasma membrane, for example, the most common mutation of the cystic
fibrosis transport regulator,
F508
(24,
25). There seems to be an
important trafficking checkpoint in the ER; membrane proteins such as
channels/channel subunits may contain an ER retention signal, which is masked
by another protein or a part of the same protein. Truncation of a channel or
lack of a corresponding subunit can unmask the ER retention signal and thus
prevents nonfunctional or improperly assembled channels from reaching the
plasma membrane (26). This has
recently been demonstrated for a variety of channels including the L-type
calcium channel, HERG channel, KATP channel,
N-methyl-D-aspartate receptor, and cyclic-nucleotidegated
channel
(2731).
In this study we show that deletion of the CIRB region in TRPC3 (mutants
T3T-C
78 and T3T-C
7) results in trapping of the channel in
intracellular compartments possibly ER/Golgi. A predicted C-terminal coiled
coil region, which is also conserved in all TRPC channels, is not required for
targeting or for OAG and agonist-dependent Ca2+ entry.
It is interesting to note that C-terminal truncation of TRPC1 up to the start
of the transmembrane domains results in functional ion channels that are
regulated by store depletion
(32). It is possible that
truncation of the CIRB region in TRPC3 results in exposure of an upstream ER
retention signal keeping the channels from trafficking to the plasma membrane.
A dibasic motif, either KK or RR, located close to the C terminus has been
identified as an ER retention signal for several misassembled membrane
proteins (33). The ER
retention signal appears to be RKR for KATP, RGR for HERG, and RRR
for the NMDA receptor. We considered a RRRR sequence (R4) in TRPC3
downstream of the transmembrane domains, upstream of the CIRB region as a
candidate ER retention sequence. However, mutation of R4 to
A4 in T3T-C
7 T3T did not rescue plasma membrane targeting
(data not shown). More extensive studies will be necessary to identify a
putative ER retention signal. Mutation of the ER retention signal would allow
the functional study of mutants that would otherwise be trapped in the
ER/Golgi and would provide a means to exclude defective trafficking as a
reason for lack of function. It is intriguing that the TRP-related protein,
PKD2, appears to be localized in the ER, whereas a PKD2 mutant lacking 34
C-terminal amino acids is now detectable in the plasma membrane
(34).
Calmodulin plays a crucial role in the activation, inactivation, or
modulation of a variety of ion channels
(35). CaM has been shown to
bind to the CIRB region of TRPC17 with different affinities
(8), although its effects on
channel activity appear to vary among different isoforms. In this study we
show that deletion of the CIRB region in TRPC3 (mutants T3T-C
78 and
T3T-C
7) results in trapping of the channel in intracellular
compartments possibly ER/Golgi. Interestingly, a TRPC1 mutant lacking the CIRB
region did not alter Ca2+-dependent feedback inhibition
of SOC in a human submandibular cell line, and from the data presented it is
not clear whether there is a phenotype associated with this mutant
(11). It is therefore tempting
to speculate that this mutant does not target properly to the plasma membrane
as is the case for the corresponding Topaz-tagged TRPC3 mutant T3T-C
78
(Fig. 7). We considered the
possibility that CaM binding to CIRB was a prerequisite for targeting TRPC3 to
the plasma membrane because it has been shown for
Ca2+-activated K+ channels that CaM regulates
the trafficking and surface expression of these ion channels
(36). To test this hypothesis,
we applied two strategies; we mutated conserved residues within the CIRB
region that might be expected to interfere with CaM and/or IP3
receptor binding (T3TYQ/AA and T3T-MKR/AAA;
Fig. 8), and we co-expressed
T3T with a dominant negative CaM mutant to see whether we could detect an
effect on T3T targeting (see "Results"). However, neither of these
strategies disrupted plasma membrane targeting of T3T.
A recent study showed that a coiled coil region in the N terminus of TRPC1,
but not a region containing the ankyrin repeats, was able to homodimerize
based on a yeast two-hybrid screen
(37). Thus, the function of
ankyrin repeats in TRPC channels is unclear. As we show here ankyrin repeats
appear to be required for targeting of TRPC3 to the plasma membrane
(T3T-N
1), whereas N-terminal deletion of TRPC3 up to the ankyrin
repeats yields functional and plasma membrane-targeted ion channels
(T3T-N
0). A similar trafficking defect has been described for a TRPC6
mutant lacking 131 N-terminal amino acid residues that include the first
ankyrin repeat (38). Ankyrin
repeats are found in a wide spectrum of proteins, including plant potassium
channels, TRPC, and vanilloid TRP subfamilies, and are thought to mediate
protein-protein interactions
(39). However, ankyrin repeats
are able to accommodate a variety of target molecules, making it hard to
predict a possible binding partner.
It has been shown that members of the TRPC 3/6/7 subfamily as well as the
Drosophila isoforms TRP and TRP-like receptor can be activated by the
diacylglycerol analog OAG (40,
41). The exact mechanism of
this activation is not known, but OAG may mimic the effects of phospholipase
C-induced generation of DAG. Recently Zhang and Saffen
(17) discovered a splice
variant of TRPC6, TRPC6B, which appeared to be activated in response to
agonist but not OAG activation. The TRPC6A splice variant that contains 54
additional amino acids at the N terminus was activated by both OAG and
receptor stimulation. These authors
(17) concluded that these 54
amino acids are crucial for OAG activation. However, none of the other TRP
isoforms that are OAG-activated possess this extended N terminus. In agreement
with that, Jung et al.
(42) have recently reported
OAG activation for the respective TRPC6 splice variant. Similarly, we show
here that the N terminus of TRPC3 can be truncated up to the start of the
ankyrin repeats without loss of proper targeting, OAG activation, or receptor
activation (Figs. 5,
6,
7, see mutant
T3T-N
0). It remains unknown whether DAG and OAG exert
their effect by acting on the TRPC3 channel directly. The well described DAG
target protein kinase C does not seem to be involved in activation, because
protein kinase C inhibitors do not block OAG activation of TRPC3 or TRPC6
(40,
43). However, protein kinase C
is not the only effector of DAG, and several newly described targets of DAG
should be investigated as possible intermediates between DAG and TRPC3 channel
activation (44,
45).
Conformational coupling has been suggested as a mechanism of activation of
TRPC3, and a N-terminal, IP3-binding fragment of the IP3
receptor has been shown to activate TRPC3 channels
(5). The CIRB region of TRPC3
was subsequently identified as a region that not only interacts with CaM but
also with two sequences within the cytoplasmic N terminus of the
IP3 receptor (7,
8). However, TRPC3-mediated
Ba2+ entry into DT40 lacking all three types of
IP3 receptors has been reported
(21,
22), excluding an absolute
requirement for the IP3 receptor in TRPC3 activation. We show here
that T3T targets to the plasma membrane of DT40-KO cells, which suggests that
the dependence of plasma membrane targeting of TRPC3 on the CIRB region is
unrelated to the interaction with the IP3 receptor. We also
demonstrate that the truncation mutants T3T-C
7 and T3T-C
8
exhibit comparable phenotypes when expressed in HEK cells or DT40-KO cells.
However, we cannot rule out the possibility that ryanodine receptors can
partially assume the function of IP3 receptors in DT40-KO cells.
However, the sequences of the IP3 receptor, which have been shown
to interact with TRPC3 do not appear to be conserved in ryanodine receptors.
In this context it is also interesting to note that the L-type
Ca2+ channel and the ryanodine receptor can directly
interact via their respective calmodulin-binding regions
(46).
 |
FOOTNOTES
|
---|
* The costs of publication of this article were defrayed in part by the
payment of page charges. This 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, P.O. Box 12233, Research
Triangle Park, NC 27709. Tel.: 919-541-1420; Fax: 919-541-7879; E-mail:
Putney{at}niehs.nih.gov.
1 The abbreviations used are: DAG, diacylglycerol; TRPC, canonical transient
receptor potential; ER, endoplasmic reticulum; IP3, inositol
1,4,5-trisphosphate; OAG, 1-oleoyl-2-acetyl-sn-glycerol; CIRB,
calmodulin/IP3 receptor-binding; CaM, calmodulin; EYFP, enhanced
yellow fluorescent protein. 
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Jeff Reece for help with confocal microscopy and to Carl
Bortner for assistance with flow cytometry. We thank Drs. Mariel Birnbaumer
and David Armstrong for critically reading the manuscript and Dr. Lutz
Birnbaumer for providing CaM expression constructs. We are indebted to Rebecca
Boyles for technical assistance.
 |
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