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PKC-dependent coupling of calcium permeation
through transient receptor potential canonical 3
(TRPC3) to calcineurin signaling in HL-1 myocytes
Michael Poteser
a
, Hannes Schleifer
a
, Michaela Lichtenegger
a
, Michaela Schernthaner
a
, Thomas Stockner
b
,
C. Oliver Kappe
c
, Toma N. Glasnov
c
, Christoph Romanin
d
, and Klaus Groschner
a,1
a
Institute of Pharmaceutical Sciences, University of Graz, 8010 Graz, Austria;
b
Institute of Pharmacology, Medical University of Vienna, 1090 Vienna, Austria;
c
Institute of Chemistry, University of Graz, 8010 Graz, Austria; and
d
Institute of Biophysics, University of Linz, 4040 Linz, Austria
Edited* by Lutz Birnbaumer, National Institute of Environmental Health Sciences, Research Triangle Park, NC, and approved May 17, 2011 (received for review
April 21, 2011)
Cardiac transient receptor potential canonical (TRPC) channels are
crucial upstream components of Ca
2+
/calcineurin/nuclear factor of
activated T cells (NFAT) signaling, thereby controlling cardiac tran-
scriptional programs. The linkage between TRPC-mediated Ca
2+
sig-
nals and NFAT activity is still incompletely understood. TRPC
conductances may govern calcineurin activity and NFAT transloca-
tion by supplying Ca
2+
either directly through the TRPC pore into
a regulatory microdomain or indirectly via promotion of voltage-
dependent Ca
2+
entry. Here, we show that a point mutation in the
TRPC3 selectivity filter (E630Q), which disrupts Ca
2+
permeability
but preserves monovalent permeation, abrogates agonist-induced
NFAT signaling in HEK293 cells as well as in murine HL-1 atrial myo-
cytes. The E630Q mutation fully retains the ability to convert phos-
pholipase C-linked stimuli into L-type (Ca
V
1.2) channel-mediated
Ca
2+
entry in HL-1 cells, thereby generating a dihydropyridine-
sensitive Ca
2+
signal that is isolated from the NFAT pathway. Pre-
vention of PKC-dependent modulation of TRPC3 by either inhibition
of cellular kinase activity or mutation of a critical phosphorylation
site in TRPC3 (T573A), which disrupts targeting of calcineurin into
the channel complex, converts cardiac TRPC3-mediated Ca
2+
signal-
ing into a transcriptionally silent mode. Thus, we demonstrate a di-
chotomy of TRPC-mediated Ca
2+
signaling in the heart constituting
two distinct pathways that are differentially linked to gene tran-
scription. Coupling of TRPC3 activity to NFAT translocation requires
microdomain Ca
2+
signaling by PKC-modified TRPC3 complexes.
Our results identify TRPC3 as a pivotal signaling gateway in Ca
2+
-
dependent control of cardiac gene expression.
Ca
2+
homeostasis
|
NFATc1 transactivation
|
transient receptor potential
canonical
|
divalent permeation
As a universal and versatile second messenger, calcium (Ca
2+
)
governs a multitude of cellular effector functions in the
heart including transcriptional programs and cellular remodeling
processes (1). Coordinated control of cardiac functions by Ca
2+
re-
quires efficient segregation of Ca
2+
signals into regulatory micro-
domains, resulting in specificity of coupling between Ca
2+
sources
and Ca
2+
-dependent effector systems. So far, the molecular com-
position and architecture of Ca
2+
signaling microdomains for
control of cardiac transcriptional programs is incompletely un-
derstood. Cation channels of the transient receptor potential ca-
nonical (TRPC) family constitute a ubiquitous signal transduction
machinery for Ca
2+
entry and have recently been identified as ion
channels that trigger pathophysiological activation of nuclear factor
of activated T-cell (NFAT)-mediated gene transcription and hy-
pertrophic remodeling in the heart (2–4). TRPC proteins form
Ca
2+
permeable plasma membrane channels that are typically ac-
tivated in response to hormonal stimuli linked to phospholipase
C signaling (5). These channels lack, or display only modest, se-
lectivity for Ca
2+
over monovalent cation (6) and are able to gen-
erate increases in cytosolic Ca
2+
via multiple mechanisms including
indirect initiation of Ca
2+
entry via voltage-gated Ca
2+
channels (7)
or the sodium calcium exchanger (NCX) because of modulation of
membrane potential and/or local Na
+
gradients (8). TRPC chan-
nels are expected to contribute divergently to Ca
2+
signaling in
nonexcitable and in excitable cells, which provide a certain reper-
toire of voltage-dependent Ca
2+
transport systems.
TRPC3 is a lipid-regulated member of the TRPC subfamily and
a potential player in cardiac pathophysiology (9). For homomeric
TRPC3 channels, a Ca
2+
/Na
+
permeability ratio of ≈1.6 was
determined (10) and functional crosstalk of TRPC3 channels with
cardiac voltage-gated Ca
2+
channels and NCX1 has been sug-
gested (7, 11). Representing a typical nonselective cation channel,
TRPC3 controls cellular processes by either Ca
2+
permeation
through its pore and generation of a local Ca
2+
signal at the TRP
channel signalplex or by remote effects on voltage-gated Ca
2+
channels or electrogenic transporters. According to a paradigm of
cardiac (patho)physiology, beat-to-beat Ca
2+
cycling (E-C cou-
pling) in the heart is separated from Ca
2+
signaling events that
control gene expression (12). Interestingly, TRPC3 has been
suggested to govern gene transcription by mechanisms involving
a linkage to voltage-dependent Ca
2+
entry (7), which is also es-
sential for E-C coupling. However, the Ca
2+
entry mechanism,
which links cardiac TRPC3 activity to gene expression is elusive
and it is unclear whether nonselective TRPC channels serve as
dual Ca
2+
signaling units that control segregated Ca
2+
pools. To
address these questions, we set out to engineer the TRPC3 cation
permeation pathway and generated a single point mutation
(E630Q) that lacks divalent- but retains monovalent permeability
and, thus, the potential to control voltage-dependent Ca
2+
entry.
This mutant was found capable of functional coupling to cardiac
Ca
V
1.2 signaling but not to NFAT activation. We present evi-
dence for a tight link between TRPC3 channel activity and NFAT
nuclear translocation based on Ca
2+
permeation through the
TRPC3 pore, generating a local Ca
2+
signaling event that is
sensed by the downstream effector calcineurin (CaN), which is
targeted to cardiac TRPC3 channels. Moreover, we demonstrate
that protein kinase C-dependent modulation of the channel
enables switching between transcriptionally active and inactive
TRPC3-signaling modes.
Results and Discussion
Identification of E630 as a Critical Residue in the TRPC3 Selectivity
Filter. In an attempt to obtain TRPC mutants with altered ion
selectivity, we initially generated a structural model of the TRPC3
pore region by using a recently developed alignment strategy (13)
Author contributions: M.P. and K.G. designed research; M.P., H.S., M.L., M.S., and T.S.
performed research; C.O .K., T.N.G., and C.R. co ntributed new reagents/a nalytic tools;
M.P., H.S., M.L., M.S., and T.S. analyzed data; and M.P., H.S., and K.G. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
1
To whom correspondence should be addressed. E-mail: klaus.groschner@uni-graz.at.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1106183108/-/DCSupplemental.
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and structure information available for KcsA as well as a Kv1.2-
Kv1.3 chimeric pore. Our hypothetical pore model is illustrated in
Fig. S1Aalong with the sequence comparison of TRPC3 and
template pore structures (Fig. S1B). Three of the five negative
residues were predicted to be accessible and exposed to the per-
meation pathway. According to our molecular model, only one
glutamate (E630) was localized within the central part of the
permeation pathway, whereas the other two residues (E616 and
D639) were predicted as part of the extracellular vestibule. Mu-
tagenesis and functional analysis confirmed a critical role of the
central glutamate in position 630. Charge inversion (E630K)
yielded a nonfunctional channel (Fig. S2), whereas neutralization
(E630Q) produced a channel that displayed moderately altered
current to voltage (I-V) relation in normal extracellular (Na
+
plus
Ca
2+
containing; Fig. 1 Aand C) solution with a relative increase
in the conductance at neutral potential. The I-V relation of the
TRPC3-E630Q mutant was virtually insensitive to changes in
extracellular Ca
2+
(Fig. S3). Inspection of I-V relations with Ca
2+
as the sole extracellular cationic charge carrier and BAPTA in the
pipette solution to eliminate indirect, Ca
2+
-mediated currents
revealed that the E630Q mutation profoundly reduced Ca
2+
permeation although the channel complex (Fig. 2 Band D). In-
ward currents were essentially small or lacking with Ca
2+
as
a charge carrier even at large hyperpolarizing potentials, and re-
versal potentials were difficult to determine in most experiments.
Nonetheless, from six experiments a mean of −79.6 ±6.3 mV was
calculated, demonstrating a substantial shift in reversal potential
compared with wild-type channels (1.9 ±1.4; n= 7). The Ca
2+
/
Cs
+
permeability ratio was reduced from 4.2 to less than 0.02 in
the mutant. Thus, our experiments identified a key amino acid
within the cation permeation structure of TRPC3. The negative
charge in position 630 is apparently essential for divalent per-
meation but not for transition of monovalent cations through the
TRPC3 pore. Our finding is in line with previous reports on the
role of negatively charged residues in Ca
2+
transition through
TRP pores (14–17) and, specifically, with a prediction of critical
residues in the TRPC selectivity filter obtained by Liu et al. in a
study with the prototypical Drosophila TRP channel (14). It is of
note that the identified negatively charged residue is conserved
in the TRPC3/6/7 subfamily but absent in more distant relatives of
TRPC3. Therefore, Ca
2+
permeation in within the TRPC family
of cation channels may involve distinctly different molecular
mechanisms. Our results support the notion that monovalent and
divalent permeation through nonselective TRP cation channels
may involve separate, specific interaction sites within the pore,
which combine to a “nonselective”pathway that conducts both
types of charge carriers. Based on our observation that a single
point mutation within the TRPC3 pore causes specific elimination
of Ca
2+
permeation, we set out to use this genetically engineered
cation channel to explore the cellular impact of the TRPC3-
mediated monovalent conductance and to identify downstream
signaling pathways that are specifically linked to either the
monovalent transport or to Ca
2+
entry through the channel pore.
Because the TRPC monovalent conductance is considered of
particular importance in excitable cells, and because recentstudies
have demonstrated the relevance of TRPC channels in cardiac
pathophysiology (2, 4, 7, 18) we focused on the cardiac system,
using the HL-1 murine atrial cell line. Initially, we compared basic
properties of TRPC3 signaling in HL-1 cells with those in the
well characterized electrically nonexcitable HEK293 system.
TRPC3 Mediates Agonist-Induced Ca
2+
Signals in HEK293 and HL-1
Cells by Divergent Mechanisms. So far, the relative contribution
of direct Ca
2+
permeation through the TRPC3 pore and indirect
mechanisms involving TRPC-mediated changes in membrane
potential and voltage-dependent signaling partners such as NCX1
has not been evaluated in HEK293 cells. Expression levels of
endogenous voltage-gated Ca
2+
channels are below the detection
threshold, and NCX1 expression is typically moderate to low.
Hence, only a minor fraction of the TRPC3-mediated Ca
2+
signal
is expected to involve indirect mechanisms in HEK293. Pharma-
cological characterization of the TRPC3-mediated Ca
2+
entry
pathway in HEK293 and HL-1 cells, determined by using a clas-
sical Ca
2+
readdition protocol, revealed that global Ca
2+
signals
were based on distinctly different mechanisms (Fig. S4). Elec-
trophysiological experiments confirmed that TRPC3 channels
were active when Ca
2+
was elevated after activation by agonist
administration in Ca
2+
-free solution (Fig. S5). TRPC3 over-
expressing HEK293 as well as HL-1 displayed Ca
2+
entry that
was highly sensitive to inhibition by the TRPC3 blocker Pyr3 (19).
KB-R7943, an inhibitor of NCX reverse mode operation, sup-
pressed Ca
2+
entry only moderately in HEK293 cells and lacked
inhibitory effects in HL-1 cells. Block of voltage-gated, L-type
Ca
2+
channels by nifedipine strongly suppressed Ca
2+
entry
into endothelin-stimulated HL-1 cells but had no effect on the
Ca
2+
signal in HEK293 cells (Fig. S4). These results indicate that
TRPC3 is effectively linked to voltage-gated Ca
2+
signaling in
cardiac cells and are able to produce large global cytosolic Ca
2+
rises via promotion of Ca
2+
entry through Ca
V
1.2 channels. The
observed lack of NCX-mediated Ca
2+
entry into HL-1 cells may
be explained by predominant forward mode operation in these
cardiac cells, based on a tight functional coupling to voltage-gated
Ca
2+
entry channels as well as more negative membrane poten-
tials compared with HEK293 cells. Thus, HL-1 myocytes repre-
sent an electrically excitable cell type that displays functional
cross-talk and signaling partnership between nonselective TRPC
conductances and voltage-gated Ca
2+
channels. Thereby, TRPC3
signaling in HL-1 cells is distinctly different from that in the
nonelectrically excitable HEK293 cell system.
As a next step, we aimed to delineate the cellular role of direct
Ca
2+
permeation through TRPC3 channels in these cells by char-
acterizing coupling between Ca
2+
entry and the NFAT down-
stream effector system for wild type and pore mutants (E630Q and
E630K) of TRPC3.
Ca
2+
Permeation Through the Pore of TRPC3 Channels Is Essential for
Activation of the NFAT Pathway. Carbachol-induced Ca
2+
entry as
well as NFAT translocation was strongly reduced by expression of
either the Ca
2+
permeation-deficient mutant (E630Q) or a pore-
dead mutant(E630K) (Fig. 2). Basal Ca
2+
entry into nonstimulated
cells was reduced when expressing either of the TRPC3 pore
mutants to the level of vector-transfected controls (Fig. 2B). As
expected from the proposed NCX1-mediated Ca
2+
entry contri-
Fig. 1. Neutralization of E630 in TRPC3 (E630Q) eliminates Ca
2+
but not
monovalent permeability. Representative ramp protocol recordings from
HEK293 cells transfected by either TRPC3-WT (Aand B) or the mutant
channel E630Q (Cand D) in the presence of 140 mM extracellular Na
+
and
2mMCa
2+
(Aand C), or in absence of extracellular Na
+
and presence of
10 mM Ca
2+
using 10 mM BAPTA in the pipette solution (Band D), before
(black) and after stimulation with 100 μM carbachol (+CCh, red).
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CELL BIOLOGY
bution, TRPC3-E630Q didnot fully eliminate the Ca
2+
entry signal.
Ca
2+
entry into cells expressing TRPC3-E630Q remained signifi-
cantly higher than in cells transfected to express TRPC3-E630K.
Nonetheless, NFAT translocation in HEK293 cells was completely
suppressed with either pore mutation of TRPC3 (Fig. 2 Cand D).
This finding indicated that Ca
2+
permeation through the pore is
essential to initiate NFAT translocation, whereas indirect NCX-
mediated signaling was barely involved because the NCX inhibitor
KB-R7943 (5 μM) failed to prevent NFAT translocation (Fig. S6).
A possible dual Ca
2+
signaling function of TRPC3 was further
investigated in the electrically excitable cardiac HL-1 cell line. As
illustrated in Fig. 3, Ca
2+
entry into endothelin-stimulated cells
was slightly enhanced compared with vector-transfected controls
(Fig. 3A) by expression of either wild-type TRPC3 (Fig. 3B)or
TRPC3-E630Q (Fig. 3C) but reduced down to basal (non-
stimulated) level with TRPC3-E630K (Fig. 3D). Expression of
wild-type TRPC3 (Fig. 3B) or TRPC3-E630Q (Fig. 3C) generated a
Ca
2+
signal that was mainly based on voltage-gated Ca
V
1.2
channels as evident by its sensitivity to nifedipine (3 μM). In clear
contrast to the observed Ca
2+
signals, cells expressing TRPC3-
E630Q lacked endothelin-stimulated NFAT translocation (Fig.
3C). Thus, the TRPC3 mutant with impaired divalent conductance
(E630Q) is able to initiate a large global Ca
2+
signal via promotion
of voltage-gated Ca
2+
entry, but this signal is not translated into
NFAT activation. Importantly, even endogenous TRPC3 channels
appear sufficient to exert a significant impact on NFAT trans-
location as evident from vector-transfected controls displaying
higher translocation than cells expression the pore-dead domi-
nant-negative E630K mutant. It is of note that, in contrast to
HEK293 cells, NFAT translocation in HL-1 cells was not signifi-
cantly promoted in response to depletion of intracellular stores
with thapsigargin (Fig. S7), indicating that the channels involved in
NFAT signaling of HL-1 are not classical store-operated Ca
2+
entry channels. Opening of TRPC3 channels resulted in NFAT
activation, but also generation of an additional intracellular Ca
2+
signal mediated by voltage-gated Ca
2+
channels that was fairly
well segregated from the NFAT pathway. Notably, TRPC3-
mediated NFAT activation can occur at barely detectable global
Ca
2+
changes such as in the presence of nifedipine (3 μM) in
control cells or cells overexpressing TRPC3 (Fig. 3 Aand B),
therefore we hypothesized that the triggering Ca
2+
elevation is
likely to take place in a restricted signaling microdomain at the
TRPC3 channel complex, containing essential downstream sig-
naling components such as calmodulin and calcineurin (CaN) to
allow specific transduction of this local Ca
2+
signal. Indeed, direct
association of TRPC3 with CaN have been demonstrated (20, 21)
and the existence of a dynamic TRPC/CaN signaling complexes
have been proposed.
Coupling of Cardiac TRPC3 Signaling to Activation of the NFAT
Pathway Involves PKC-Dependent Phosphorylation. Previous inves-
tigations demonstrated assembly of CaN along with immuno-
phyllins (FKBP12) into TRPC6 signalplexes and dependency of
this process on protein kinase C-mediated phosphorylation (21).
PKC-mediated phosphorylation of TRPC3 appears essential for
both recruitment of CaN into TRPC complexes and inhibitory
regulation (22, 23). This result prompted us to hypothesize that
suppression of PKC phosphorylation may disrupt the functional
TRPC3/CaN signaling unit without preventing channel function.
Consequently, we set out to test whether TRPC3 linkage to NFAT
nuclear translocation depends on regulation of the channel
complex by PKC. To suppress TRPC3 phosphorylation by PKC
isoenzymes, we performed experiments with GF109203X, a com-
pound that inhibits conventional PKC isoforms including PKC-γ,
as one essential player in the control of TRPC3 channels (23).
Because CaN has been shown to associate with TRPC3/6 channels
in a manner dependent on phosphorylation by PKC (21), we
speculated that prevention of PKC phosphorylation may disrupt
TRPC/CaN complexes. Indeed, immunoprecipitation experi-
ments in HEK293 cells confirmed that the PKC inhibitor prevents
association of CaN into TRPC3 complexes along with reduction
of threonine phosphorylation of the channel protein (Fig. 4).
Alternatively, a mutant that is defective in PKC-γ–mediated in-
hibitory modulation, i.e., T573A corresponding to the murine
TRPC3-moonwalker (Mwk) mutation, was expressed in HEK293
and HL-1 cells. It is of note that overexpression of the phos-
phorylation-deficient TRPC3-Mwk by itself was barely tolerated
by the cells, presumably due to a gain in function leading to Ca
2+
overload. Therefore, we transfected cells to overexpress the
T573A mutant along with wild-type TRPC3 (DNA ratio 3:1). This
transfection resulted in a heteromeric TRPC3 conductance larger
than that generated by wild-type TRPC3 alone (Fig. S8) but es-
sentially tolerated by the host cells. The derived heteromers
appeared correctly targeted into the membrane as indicated by
fluorescence microscopy. Interestingly, currents through TRPC3-
Mwk/TRPC3-WT channels were rather stable during continuous
agonist stimulation, most likely due to lack of inhibitory regula-
tion of the channels by PKC and recordings in Na
+
-free extra-
cellular solution confirmed Ca
2+
permeability of the channels.
Either treatment of cells expressing TRPC3-WT with
GF109203X (2 μM) or transfection of cells with TRPC3-Mwk/
TRPC3-WT produced a similar cellular phenotype, displaying
BCDA
Fig. 2. Receptor-stimulated Ca
2+
influx as well as NFAT translocation are impaired in HEK293 cells expressing the Ca
2+
impermeable TRPC3-E630Q or the
impermeant TRPC3-E630K, compared with cells expressing wild-type TRPC3. (A) Representative traces of fura-2 Ca
2+
-imaging experiments. Cells were stim-
ulated by 100 μM carbachol (arrow). (B) Mean Δratio values (±SEM, n>40) derived from fura-2 Ca
2+
-imaging. Black bars indicate the basal (unstimulated)
Ca
2+
entry at indicated transfections. (C) Mean nuclear/cytosol fluorescence intensity ratio (±SEM, n>11) of HEK293 cells expressing GFP-NFAT and the
respective channel protein after stimulation and application of the same protocol as used in fura-2 experiments. Black bar (basal) represents mean nuclear/
cytosol fluorescence intensity ratio in HEK293 cells transfected with GFP-NFAT only. (Band C) Asterisks indicate statistically significant of difference to TRPC3-
WT–expressing cells. (D) Representative fluorescence images recorded in GFP-NFAT translocation experiments shown at Left. Positions of nuclei are indicated
by arrowheads. Nucleus/cytosol fluorescence ratios of example images are indicated.
10558
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large-agonist–induced Ca
2+
entry signals that were not accom-
panied by significant NFAT nuclear accumulation (Fig. 5). This
“transcriptionally silent”Ca
2+
entry into HL-1 cells was for a large
part mediated by voltage-gated L-type Ca
2+
channels as indicated
by sensitivity to nifedipine (3 μM). Our results demonstrate dis-
ruption of transcriptional TRPC3 signaling in response to reduced
A
B
C
D
Fig. 3. Ca
2+
entry through TRPC3 is critical for activation of the calcineurin/NFAT pathway in HL-1 atrial myocytes. HL-1 cells were transfected with vector
control (A), TRPC3-WT (B), or the indicated pore mutant (Cand D). (Left) Representative traces of fura-2 imaging in cells at basal conditions (unstimulated +
Ca
2+
readdition) and stimulated with 100 nM endothelin (+ ET-1, arrow) in the absence or presence of 3 μM nifedipine (+ Nif). (Center Left) Mean fura-2 Δ
ratio values (±SEM, n>20). (Center Right) Mean nuclear/cytosolic NFAT-GFP fluorescence ratio (±SEM, n>8) in unstimulated HL-1 cells (basal), HL-1 cells
stimulated with 100 nM endothelin (+ ET-1) in the absence and presence of 3 μM nifedipine (+ Nif) and application of the same protocol as used in fura-2
experiments. Asterisks indicate statistically significant inhibition by nifedipine. (Right) Representative images of NFAT-localization before stimulation and
Ca
2+
readdition (control), after Ca
2+
readdition (basal), and after stimulation by endothelin and subsequent Ca
2+
readdition (+ ET-1) in the absence and
presence of 3 μM nifedipine (+ Nif). Positions of nuclei are indicated by arrows. Nucleus/cytosol fluorescence ratios of example images are indicated.
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PKC-dependent phosphorylation of the channel or as a conse-
quence of the TRPC3-Mwk mutation. This fact may be consid-
ered as part of the molecular mechanism underlying the moon-
walker pathophysiology (23).
Impaired PKC regulation of cardiac TRPC3 is shown to result in
uncoupling from the NFAT pathway without disrupting the link-
age between TRPC3 and global myocyte Ca
2+
via voltage-gated
Ca
2+
entry. We provide evidence that TRPC3-CaN/NFAT
sign aling takes place in a restricted microdomain and requiresboth
direct Ca
2+
permeation through the TRPC3 pore as well as CaN
targeting into the signal complex. Cardiac TRPC3 complexes are
shown to produce Ca
2+
signals both via direct Ca
2+
transport and
by control of voltage-dependent Ca
2+
entry. Our results demon-
strate the ability of cardiac TRPC3 channels to switch in a phos-
phorylation-dependent manner between a transcriptionally active
and a transcriptionally silent signaling mode (Fig. 6). Because
TRPC3 is likely to change in expression along with other TRPC
species during pathophysiologic stress, the formation of divergent
heteromeric TRPC3 channel complexes may be anticipated. The
observed dominantnegative effect of the phosphorylation-deficient
moonwalker mutation on CaN/NFAT activation suggests a prom-
inent role of phosphorylation in maintenance of transcriptionally
active TRPC complexes in the heart. Situations of hampered
PKC phopshorylation or promoted dephosphorylation may con-
vert TRPC complexes into a cardiac Ca
2+
signaling unit that is
functionally isolated from the CaN/NFAT activation pathway.
Our findings highlight the key role of nonselective TRPC
channels in the control of transcriptional programs and extend
this concept by demonstrating a pivotal role of Ca
2+
transport
trough the TRP pore structure along with a unique phosphory-
lation-dependent molecular switch that allows efficient control of
cardiac gene transcription by neurotransmitters and hormones.
Materials and Methods
Homology Modeling. For details on sequence alignment, homology modeling
and model evaluation, see SI Materials and Methods.
A
BC
Fig. 4. GF109203X inhibits phosphorylation of TRPC3
and its association with calcineurin. HEK-293 cells
expressing HA-tagged TRPC3 were incubated with or
without 2 μM GF109203X (GFX) and lysed and sub-
jected to SDS/PAGE and immunoprecipitation. (ALeft)
Total HEK-cell lysates were immunoprecipitated by
using an anti-HA antibody and immunoblotted with
an anti-phospho-threonine antibody. (A Right) Bars
representing the densitometric analysis of phospho-
threonine-immunoreactivity. Mean values are given
for carbachol-stimulated cells in the absence and pres-
ence of GFX (±SEM, n= 4). Asterisk indicates statisti-
cally significant differences. (B) Stripped membranes
were immunoblotted again by using an anti-HA anti-
body (Lower). Proteins detected in total cell lysates
(lane 1, Input), immunocomplexes (lane 2, IP-HA-C3)
and lysates precipitated only with beads (lane 3, Ctrl.).
(C) Coimmunoprecipitations of cell homogenates using
antibodies against the HA-tag and calcineurin, and
immunoblotted against calcineurin. Proteins detected
in total cell lysates (lane 1, Input), immunocomplexes
(lane 2, IP-HA-C3; lane 3, IP-CN), and lysates pre-
cipitated only with beads (lane 4, Ctrl.).
ABCD
Fig. 5. Phosphorylation of threonine 573 of the TRPC3 channel protein is essential for the activation of the calcineurin/NFAT pathway in HL-1 atrial myocytes. (A)
Representative traces of fura-2 Ca
2+
-imaging experiments in cells transfected with either TRPC3-WT or TRPC3-T573A and TRPC3-WT (DNA ratio 3:1, MWK/TRPC3-
WT), stimulated by 100 nM endothelin (arrow) and in the absence or presence of 3 μM nifedipine (+ Nif) or 2 μM GF109203X (+ GFX), as indicated. (B) Mean Δratio
values (±SEM, n>30) of fura-2 Ca
2+
-imaging experiments. Asterisks indicate statistically significant nifedipine-induced inhibition. (C) Mean nucleus/cytosol
fluorescence intensity ratio (±SEM, n>9) in HL-1 cells expressing GFP-NFAT and the respective (mutant) channel protein after stimulation and application of the
same protocol as used in fura-2 experiments. (Band C) Asterisks indicate statistically significant difference to TRPC3-WT–expressing cells in the absence of
inhibitors (white bar). (D) Representative fluorescence images recorded in GFP-NFAT translocation experiments. Individual nucleus/cytosol fluorescence ratios
are given, and positions of nuclei are indicated by arrows.
10560
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www.pnas.org/cgi/doi/10.1073/pnas.1106183108 Poteser et al.
DNA and Mutagenesis. Site-directed mutagenesis was performed with stan-
dard protocols. Details on cDNA constructs and cloning procedures are
provided in SI Materials and Methods.
Cell Culture and Transfection. Cell lines were cultured at 37 °C and 5% CO
2
.For
HEK293 cells DMEM (Invitrogen) supplied with 10% FCS and for HL-1 atrial
myocytes Claycomb medium (Sigma) supplied with 100 μM norepinephrin,
4mML-glutamin and 10% FCS were used. Lipofection was used for genetrans-
fer; for details on DNA amounts and reagents, see SI Materials and Methods.
Electrophysiology. Standard patch clamp protocols were used (SI Materials
and Methods). Standard bath solutions contained 140 or 0 mM NaCl, 0 or
140 mM NMDG, 2 mM MgCl
2
, 10 mM glucose, 10 mM Hepes, 2 or 0 mM
CaCl
2
, and 0 or 2 mM BaCl
2
at pH adjusted to 7.4 with NaOH or NMDG. Pi-
pette solution contained 120 mM cesium methanesulfonate, 20 mM CsCl,
15 mM Hepes, 5 mM MgCl
2
, and 3 mM EGTA, at pH adjusted to pH 7.3 with
CsOH. For delineation of Ca
2+
permeability of TRPC3 mutants, a bath solu-
tion containing 132 mM NMDG, 2 mM MgCl
2
, 10 mM Glucose, 10 mM Hepes,
3 mM CaCl
2
, 7 mM Ca-Gluconate, at pH adjusted to 7.4 with methanesulfonic
acid and a pipette solution composed of 140 mM cesium methanesulfonate,
15 mM Hepes, 5 mM MgCl
2
, and 10 mM BAPTA at pH 7.3 was used.
Measurement of NFAT-Translocation. Cells were transfected to express an N-
terminally GFP-tagged NFATc1 fusion (15) and plated on coverslips. For
buffers and solutions see SI Materials and Methods. Agonists as well as
inhibitors (Pyr3, GFX109203, nifedipine, or KB-R7943) remained present
continuously after administration. GFP-NFAT translocation was monitored
(488 nm excitation) with standard fluorescence microscopy (Zeiss Axiovert
equipped with Coolsnap HQ). GFP-NFAT and YFP-TRPC3-WT/-mutant fluo-
rescence were discriminated by specific cellular localization. Nuclear/cytosol
fluorescence intensity ratios of cells were calculated with ImageJ software.
Measurement of Intracellular Ca
2+
Signaling. For details on fura-2 calcium
imaging experiments see SI Materials and Methods.
Immunoprecipation. In short, protein-A- or protein-G-bead-precleared
supernatants of lysates from stimulated HEK293 cells were incubated with
precipitating antibody overnight. After the addition of respective protein-A-
or protein-G-beads, washing, and denaturation in Lämmli buffer, the im-
munocomplexes were separated by SDS/PAGE and subjected to Western
blotting. See SI Materials and Methods for details.
Reagents. Chemicals, reagents, and antibodies were purchased from Sigma
Aldrich. KB-R7943 and GF109203X were from Tocris Biosciences. The TRPC3
pore blocker Pyr3 was synthesized as published (16).
Statistics. Data are presented as mean values ±SEM and was tested for
statistical significance by using the Student ttest (*P<0.05).
ACKNOWLEDGMENTS. We thank Dr. R. Kehlenbach for providing the GFP-
NFAT construct and Mrs. R. Schmidt for excellent technical assistance. This
work was supported by FWF (Austrian Science Fund) Grant P21925-B19
(to K.G.), P22565 (to C.R.), and DK+Metabolic and Cardovascular Disease
Grant W2126-B18.
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Fig. 6. Phosporylation of TRPC3 at position 573 (via PLC) enables Ca
2+
/
calmodulin/calcineurin (Ca
2+
, Cm, CaN)-dependent activation upon receptor-
activated Ca
2+
entry through TRPC3. Dephosporylation of position 573 turns
TRPC transcriptionally silentwhile enhancing itsgeneral activity.L-type channels
are controlled by TRPC3-induced changes in membrane potential, providing
only negligible effects on Ca
2+
-induced NFAT activation in HL-1 atrial myocytes.
Poteser et al. PNAS
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no. 26
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10561
CELL BIOLOGY
Correction
CELL BIOLOGY
Correction for “PKC-dependent coupling of calcium permeation
through transient receptor potential canonical 3 (TRPC3) to
calcineurin signaling in HL-1 myocytes,”by Michael Poteser,
Hannes Schleifer, Michaela Lichtenegger, Michaela Schern-
thaner, Thomas Stockner, C. Oliver Kappe, Toma N. Glasnov,
Christoph Romanin, and Klaus Groschner, which appeared in
issue 26, June 28, 2011, of Proc Natl Acad Sci USA (108:10556–
10561; first published June 8, 2011; 10.1073/pnas.1106183108).
The authors note that Figs. 2, 3, and 5 appeared incorrectly.
The corrected figures and their corresponding legends appear
below.
ABCD
Fig. 2. Receptor-stimulated Ca
2+
influx as well as NFAT translocation are impaired in HEK293 cells expressing the Ca
2+
impermeable TRPC3-E630Q or the
impermeant TRPC3-E630K, compared with cells expressing wild-type TRPC3. (A) Representative traces of fura-2 Ca
2+
-imaging experiments. Cells were stimu-
lated by 100 μM carbachol (arrow). (B) Mean Δratio values (±SEM, n>40) derived from fura-2 Ca
2+
-imaging. Black bars indicate the basal (unstimulated) Ca
2+
entry at indicated transfections. (C) Mean nuclear/cytosol fluorescence intensity ratio (±SEM, n>11) of HEK293 cells expressing GFP-NFAT and the respective
channel protein after stimulation and application of the same protocol as used in fura-2 experiments. Black bar (basal) represents mean nuclear/cytosol
fluorescence intensity ratio in HEK293 cells transfected with GFP-NFAT only. (Band C) Asterisks indicate statistically significance of difference to TRPC3-WT–
expressing cells. (D) Representative fluorescence images recorded in GFP-NFAT translocation experiments shown at Left. Positions of nuclei are indicated by
arrowheads. Nucleus/cytosol fluorescence ratios of example images are indicated.
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no. 33 www.pnas.org
A
B
C
D
Fig. 3. Ca
2+
entry through TRPC3 is critical for activation of the calcineurin/NFAT pathway in HL-1 atrial myocytes. HL-1 cells were transfected with vector
control (A), TRPC3-WT (B), or the indicated pore mutant (Cand D). (Left) Representative traces of fura-2 imaging in cells at basal conditions (unstimulated +
Ca
2+
readdition) and stimulated with 100 nM endothelin (+ ET-1, arrow) in the absence or presence of 3 μM nifedipine (+ Nif). (Center Left) Mean fura-2 Δ
ratio values (±SEM, n>20). (Center Right) Mean nuclear/cytosolic NFAT-GFP fluorescence ratio (±SEM, n>8) in unstimulated HL-1 cells (basal), HL-1 cells
stimulated with 100 nM endothelin (+ ET-1) in the absence and presence of 3 μM nifedipine (+ Nif) and application of the same protocol as used in fura-2
experiments. Asterisks indicate statistically significant inhibition by nifedipine. (Right) Representative images of NFAT-localization before stimulation and Ca
2+
readdition (control), after Ca
2+
readdition (basal), and after stimulation by endothelin and subsequent Ca
2+
readdition (+ ET-1) in the absence and presence of
3μM nifedipine (+ Nif). Positions of nuclei are indicated by arrowheads. Nucleus/cytosol fluorescence ratios of example images are indicated.
PNAS
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CORRECTION
www.pnas.org/cgi/doi/10.1073/pnas.1111388108
ABCD
Fig. 5. Phosphorylation of threonine 573 of the TRPC3 channel protein is essential for the activation of the calcineurin/NFAT pathway in HL-1 atrial myocytes.
(A) Representative traces of fura-2 Ca
2+
-imaging experiments in cells transfected with either TRPC3-WT or TRPC3-T573A and TRPC3-WT (DNA ratio 3:1, MWK/
TRPC3-WT), stimulated by 100 nM endothelin (arrow) and in the absence or presence of 3 μM nifedipine (+ Nif) or 2 μM GF109203X (+ GFX), as indicated. (B)
Mean Δratio values (±SEM, n>30) of fura-2 Ca
2+
-imaging experiments. Asterisks indicate statistically significant nifedipine-induced inhibition. (C) Mean
nucleus/cytosol fluorescence intensity ratio (±SEM, n>9) in HL-1 cells expressing GFP-NFAT and the respective (mutant) channel protein after stimulation and
application of the same protocol as used in fura-2 experiments. (Band C) Asterisks indicate statistically significant difference to TRPC3-WT–expressing cells in
the absence of inhibitors (white bar). (D) Representative fluorescence images recorded in GFP-NFAT translocation experiments. Individual nucleus/cytosol
fluorescence ratios are given, and positions of nuclei are indicated by arrowheads.
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