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Prepublished online as a Blood First Edition Paper on June 21, 2002; DOI 10.1182/blood-2002-03-0723.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Centre for Cardiovascular Biology and Medicine,
King's College London, New Hunt's House, Guy's Campus, London; the
Neurobiotechnology Centre, Ohio State University, Columbus; and the
Institut für Pharmakologie und Toxikologie der Universität
des Saarlandes, Homburg, Germany.
Store-operated Ca++ entry (SOCE) is thought to comprise
the major pathway for Ca++ entry in platelets. Recently, a
number of transient receptor potential (TRP) proteins, which have been
divided into 3 groups (TRPC, TRPM, and TRPV), have been suggested as
SOCE channels. We report the expression and function of TRPC proteins
in human platelets. TRPC6 is found at high levels and TRPC1 at low
levels. Using purified plasma (PM) and intracellular membranes (IM),
TRPC6 is found in the PM, but TRPC1 is localized to the IM. Using
Fura-2-loaded platelets, we report that, in line with TRPC6
expression, 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulated the entry of
Ca++ and Ba2+ independently of protein kinase
C. Thrombin also induced the entry of Ca++ and
Ba2+, but thapsigargin, which depletes the stores, induced
the entry of only Ca++. Thus, thrombin activated TRPC6 via
a SOCE-independent mechanism. In phosphorylation studies, we report
that neither TRPC6 nor TRPC1 was a substrate for tyrosine kinases.
TRPC6 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK) and
associated with other cAMP-PK substrates. TRPC1 was not phosphorylated
by cAMP-PK but also associated with other substrates. Activation of
cAMP-PK inhibited Ca++ but not Ba2+ entry
induced by thrombin and neither Ca++ nor Ba2+
entry stimulated by OAG. These results suggest that TRPC6 is a
SOCE-independent, nonselective cation entry channel stimulated by
thrombin and OAG. TRPC6 is a substrate for cAMP-PK, although phosphorylation appears to not affect cation permeation. TRPC1 is
located in IM, suggesting a role at the level of the stores.
(Blood. 2002;100:2801-2811) Platelet activation forms an integral component of
hemostasis and contributes to the events leading to thrombosis.
Complete activation of platelets by all stimulatory agents leads to an increase of cytosolic Ca++ levels, which triggers many
intracellular signaling processes important for the expression of
functional responses.1 Conversely, the
vasodilators prostacyclin (PGI2) and nitric oxide
(NO) inhibit platelet function, with inhibition of Ca++
elevation an identified mechanism.2 Cytosolic
Ca++ elevation occurs as a consequence of release of the
cation from intracellular stores and influx from the outside medium.
Whilst the mechanism for Ca++ release from the stores in
nonexcitable cells is well accepted, Ca++ entry mechanisms
are less understood. The key elements involved in Ca++
signaling include activated surface receptors that lead to the stimulation of phospholipase C (PLC), resulting in the hydrolysis of
the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to release inositol 1,4,5-trisphosphate
(IP3) and diacylglycerol (DAG). IP3 binds to
the IP3 receptor (IP3R) on intracellular
stores, releasing Ca++, and DAG is a potent activator of
protein kinase C (PKC). Ca++ entry is thought to occur
predominantly as a consequence of store depletion and has been referred
to as store-operated Ca++ entry (SOCE) or capacitative
Ca++ entry (CCE).3 However, the details of the
SOCE pathway or the identity of the SOCE channel in the plasma membrane
(PM) has remained elusive. Many models have been proposed,
which include a conformational coupling mechanism involving the
IP3R at the stores signaling to the PM
channel,4 release of a soluble Ca++ influx
factor (CIF) from the stores that may trigger the opening of the cation
channel in the PM,5 involvement of tyrosine
kinases,6,7 and modifications of the conformational
coupling mechanism that include a secretionlike mechanism involving
rearrangements of the cytoskeleton8 and possible channel
insertion.9 Importantly, there is as yet no consensus
regarding the identity of the SOCE channel, and until such
identification is established, the mechanisms for its regulation will
remain contentious. There is also variability in the cation selectivity
of the SOCE pathway. A Ca++ release-activated
Ca++ current (ICRAC) that is highly
selective for Ca++ has been described in hematopoietic
cells.10 Endothelial cells have been reported to express
SOCE activities11 that have moderate selectivity for
Ca++, while some smooth muscle cell types express
relatively nonselective SOCE pathways.12 Thus, the
identities of the SOCE channels themselves may vary between cells.
At the molecular level, the transient receptor potential (TRP) proteins
have been proposed as candidates for SOCE channels. TRP was first
described as a Drosophila mutant that had an impaired visual
transduction response (hence, transient receptor
potential).13 Subsequent to cloning,14 the
Drosophila (d)TRP protein was shown in vitro to be operated
by a SOCE mechanism, although in vivo its gating mechanism remains to
be established.15 There are currently known to be 3 Drosophila TRP genes (trp, trpl, and
trp In platelets and megakaryocytic cells, there is currently information
on the expression of TRPC proteins. Using reverse
transcriptase-polymerase chain reaction (RT-PCR), we first
reported that megakaryocytes (using the cell lines MEG01, DAMI, and
HEL) expressed TRPC1, 2, and 3.21 Our studies have been
recently confirmed and extended to include TRPC6 and TRPC4 mRNA in
megakaryocytic cells and also in platelets, if very large numbers of
cells are used to extract the mRNA.22 Recently, Rosado and
Sage23,24 have reported the detection of TRPC1 protein in
platelets and suggested its coupling to the type II IP3R
upon depletion of intracellular stores. In this study, we sought to
further examine the expression, role, and phosphorylation of TRPC
proteins in human platelets. Using a range of highly specific
antibodies, we report the high expression of TRPC6 and low expression
of TRPC1. Further, we report that TRPC6 is activated by thrombin and
diacylglycerol by a SOCE-independent mechanism and that it is a
substrate for cAMP-dependent protein kinase (cAMP-PK), making it a
target for physiologically important vasodilators. We also report that,
surprisingly, TRPC1 is located in platelet intracellular membranes,
suggesting a primary role at the level of the Ca++ stores.
Materials
Antibodies
Cell culture and transfection of TRPC constructs cDNA for hTRPC1, mTRPC2, and hTRPC3 were subcloned into the vector pcDNA3.30 hTRPC1 contained an HA epitope at the N-terminus and hTRPC3 at the C-terminus. cDNA for mTRPC4 , mTRPC4 ,
mTRPC5, and mTRPC6 were subcloned into the pIRESneo vector.
Amplification of the plasmids was carried out using Escherichia
coli JM109 and maxipreps prepared using a Qiagen Maxi preparation
kit as described by the manufacturer (Qiagen, Crawley, West Sussex,
United Kingdom). DNA purity of the constructs was checked using
restriction enzyme digestion.
Transfections were carried out using QBI-293A cells (Qbiogene,
Livingstone, United Kingdom), which are a subclone of HEK-293 cells
selected for improved transfection properties. The QBI-293A cells were
grown in Dulbecco modified Eagle medium (DMEM) supplemented with 110 mg/L sodium pyruvate, 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were maintained in T175cm2 flasks until they were approximately 70% confluent and were transfected using a standard calcium phosphate precipitation protocol with TRPC constructs (30-60 µg plasmid DNA in
900 µL, 1 mL of 2 X BES buffer, 100 µL of 2.5 M CaCl2
per T175cm2 flask). The medium was replaced 24 hours later,
and the cells were allowed to grow for a further 24 hours, after which they were harvested and frozen at Preparation of Fura-2-labeled platelets Blood was taken from donors, who denied taking any medication for the last 9 days, into 0.1 volume 3.2% trisodium citrate and platelet-rich plasma (PRP) was isolated after centrifugation at 200g for 15 minutes. Fura-2-labeling was carried out by incubating PRP (acidified to pH 6.5 using 0.3 M citric acid) at 37°C with 3 µM Fura-2/AM (acetoxymethyl ester) for 1 hour. The PRP was then allowed to cool to room temperature and centrifuged at 1200g for 15 minutes. The platelet pellet was resuspended at 1-1.5 × 108 cells/mL in a medium consisting of 10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM glucose, 3 µM indomethacin, and 1 U/mL apyrase (grade III; Sigma Chemical, United Kingdom). Cation elevation studies were carried out using 2-mL suspensions at 37°C with sample mixing carried out by cuvette inversion. Fluorescence measurements were made using a rotating wheel spectrofluorimeter (Cairns Research, Faversham, Kent, United Kingdom) with excitation at 340 and 380 nm and emission at 510 nm. [Ca++]i or [Ba2+]i is reported as the 340 nm:380 nm ratio (R340/380). Additions of agents were made as stated in the "Results" section or legends to the relevant Figures. Autofluorescence was estimated by addition of 2 mM MnCl2 in the presence of 10 µM ionomycin. Where shown, calculation of [Ca++]i was carried out as described by Sage.31Preparation of highly purified platelet plasma and intracellular membranes using high-voltage free flow electrophoresis Platelet plasma membranes (PMs) and intracellular membranes (IMs) were prepared as described in considerable detail in previous publications.32,33 Briefly, platelets were separated from human blood and treated with neuraminidase (type X, 0.05 U/mL) for 20 minutes at 37°C. After washing, platelets were sonicated at 4°C (with 2 × 10-second bursts, amplitude setting at 50) in sonication medium and centrifuged at 42 000g for 90 minutes on a linear (1-3.5 M) sorbitol density gradient to obtain a mixed membrane (MM) fraction (free of granular contamination). MMs were concentrated by centrifugation (100 000g, 60 minutes), resuspended in 0.4 M sorbitol, 10 mM triethanolamine pH 7.2 with a conductivity of 750 µS/cm, and separated into PM and IM by free flow electrophoresis (FFE) using an Octopus FFE apparatus (Dr Weber, Gmbh, Germany) running at 750 V, 100 mA. Two peaks comprising of PM (less electronegative) and IM (more electronegative) were obtained. Tops of peaks were pooled, centrifuged (100 000g, 60 minutes), and resuspended in 0.4 M sorbitol, 10 mM triethanolamine pH 7.2.Labeling of intact platelets with [32P]Pi and immunoprecipitation Freshly prepared platelets were resuspended in a wash buffer consisting of 36 mM citric acid, 103 mM NaCl, 5 mM KCl, 5 mM glucose, 1mM EDTA (ethylenediaminetetraacetic acid) pH6.5 with 60 nM prostacyclin (PGI2). They were then incubated for 90 minutes at 37°C with 0.25 mCi (9.3 MBq) carrier-free [32P]Pi/mL and washed twice with fresh wash buffer before resuspension in a HEPES tyrode medium containing 10 mM HEPES, 145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 5 mM glucose pH 7.4 at 2 × 109 cells/mL. Incubations were carried out in aggregation cuvettes using a Peyton dual channel aggregometer (ISMS; Dorking, Surrey, United Kingdom) with 500 µL suspensions at 37°C with mixing. Additions of stimulatory or inhibitory agonists were for the times stated in the "Results" section and reactions stopped by addition of equal volume of Triton X-100 solubilization buffer with composition 1% Triton, 10 mM EGTA (ethyleneglycoltetraacetic acid), 150 mM NaCl, 40 mM Tris pH 7.8, 2 mM PMSF, 2 mM sodium vanadate, 0.2 mM leupeptin, 10 µg/mL pepstatin A, 0.4 U/mL aprotinin, 0.2 mg/mL soybean trypsin inhibitor, and 10 µM E64d. After 30 minutes' rotating at 4°C, the mixtures were centrifuged at 17 900g for 5 minutes at 4°C and the supernatants used for immunoprecipitation. Supernatants were first precleared with 0.1 volume 20% protein-G sepharose (PGS) for 30 minutes. After centrifugation at 420g, 5 minutes at 4°C (in an eppendorf centrifuge), the respective primary antibody was added to the supernatant and incubated overnight rotating at 4°C. The following morning, 50 µL 20% PGS was added and incubated for at least 4 hours at 4°C. The immunoadsorbant was spun down, washed 4 times with washing buffer (0.2% Triton X100, 0.1% BSA, 0.01% sodium azide in PBS), with the last wash for at least 4 hours or overnight and then once with buffer lacking BSA. Sodium dodecyl sulfate (SDS)-Laemmli sample buffer was added to the pellet and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting and autoradiography.Western blotting Samples were applied to SDS-PAGE gradient gels (5%-15%) following the method of Laemmli34 and subjected to electrophoresis. Separated proteins were transferred onto nitrocellulose membrane by semidry blotting using a current density of 0.8 mA/cm2 for 1.5 hours, and the nitrocellulose was then blocked for 3 hours (or overnight) in blocking medium (5% dried milk, 1% normal goat serum [NGS] in TBS-Tween [20 mM Tris pH 7.4, 500 mM NaCl, 0.2% Tween]). Filters were washed 3 times in TBS-Tween, followed by incubation with the corresponding primary antibody (Ank, anti-TRPC6, etc) in TBS-Tween + 20 mg/mL BSA for at least 1 hour or overnight. After washing, the membranes were incubated with an appropriate second antibody (eg, goat antirabbit) conjugated to horseradish peroxidase in TBS-Tween + 2 mg/mL BSA for 1 hour, followed by detection using enhanced chemiluminescence (ECL) reagents.Phosphorylation of QBI-293A microsomes by catalytic subunit of cAMP-PK Phosphorylation of overexpressed hTRPC1 or mTRPC6 containing microsomes was carried out using a procedure used to phosphorylate platelet membranes.35 The reaction mixture consisted of 50 mM HEPES buffer (pH 7.4), 10 mM MgCl2, 1 mM dithiothreitol (DTT), 0.2 mM EGTA, 500 µg membrane protein, 50 µM ATP containing 10 µCi (0.37 MBq) -[32P] ATP,
and, where indicated, 350 U catalytic subunit (CAT) or 1 µM cAMP-PK
inhibitor peptide. The reaction was initiated by adding ATP and CAT,
mixing and incubation at 30°C for the time stated. The
reaction was terminated by the addition of cold solubilization buffer
(at final concentration of 0.5% Triton X-100, 20 mM Tris pH 7.8, 5 mM
EDTA, 75 mM NaCl, 1 mM PMSF, 1 mM sodium vanadate, 0.1 mM leupeptin, 5 µg/mL pepstatin, 0.2 U/mL aprotinin, 0.1 mg/mL soybean trypsin
inhibitor, and 5 µM E64d). After 30 minutes' rotating at 4°C, the
samples were centrifuged at 17 900g for 5 minutes. The
supernatant was precleared and immunoprecipitated as described above.
All Western blots and autoradiographs shown represent typical results obtained with at least 3 different platelet or membrane preparations, and analysis of statistical significance (Student t test using Microsoft Excel, Seattle, WA), where appropriate, is reported in figure legends.
Antibodies and the expression of TRPC proteins in human platelets Figure 1 presents the characterization of an affinity-purified polyclonal antibody (Ank) that was raised to a peptide sequence in the first ankyrin domain of hTRPC1. Ank recognized hTRPC1 overexpressed in QBI-293A cells with the protein migrating at approximately 80 kDa (Figure 1A). The overexpressed protein also contained an HA epitope at the N terminus, allowing its detection with an anti-HA epitope antibody (results not shown). Ank also was effective at immunoprecipitating overexpressed hTRPC1, as determined by probing the immunoprecipitates with the HA-epitope antibody in Western blots. This confirmed the size of the overexpressed protein as approximately 80 kDa (Figure 1B). Ank is specific for hTRPC1, as it did not recognize mTRPC2, hTRPC3, mTRPC4, mTRPC5, or mTRPC6, all overexpressed in
QBI-293A cells (results not shown).
Ank was then used to determine the expression of TRPC1 in human
platelets. Figure 1C shows that in Western blots of platelet lysates,
Ank recognized 3 bands between 65 and 105 kDa migration positions,
namely at 100, 80, and 70 kDa. These bands may reflect different
glycosylation states or alternatively spliced forms of TRPC1 in
platelets. Although the migration size of overexpressed hTRPC1 is 80 kDa, recent studies in the literature have reported endogenous
mammalian TRPC1 to be 100, 92, 80, and 65 kDa,36,23,26 suggesting that more than one detectable form may be present (hTRPC1 Platelets also were tested for the presence of other TRPC proteins,
using a range of polyclonal antibodies, each specifically recognizing
its corresponding protein. An anti-TRPC6 antibody (raised to an
N-terminal sequence of rTRPC6) recognized mTRPC6 overexpressed in
QBI-293A cells as a doublet protein band migrating at 100 and 110 kDa,
and also recognized well a 110-kDa band in platelet mixed membranes,
suggesting a good expression of this cation channel in platelets
(Figure 1D). We believe that the doublet band of overexpressed mTRPC6
is due to full and partial glycosylation of the protein, as treatment
of solubilized membranes with N-glycosidase F (Boehringer Mannheim,
Mannheim, Germany; to remove attached carbohydrate residues) resulted
in the detection of only the 100-kDa band (results not shown). An
antibody raised to the C-terminal sequence of bovine (b)TRPC4
recognized overexpressed mTRPC4 Our laboratory has, for a number of years, used the technique of FFE to
separate PM and IM from human platelets. Full characterization of these
membrane fractions has been described in many previous publications.32,39 Using this technique, we investigated
the localization of TRPC1 and TRPC6. Figure
2B shows that detection of the 110-kDa TRPC6
protein band was observed in mixed membranes (MM) prior to separation
by FFE, and in PM after electrophoresis, but there was
little or no detection of the protein in IM. Surprisingly, TRPC1 (100 kDa) with, again, weak detection, was located in MM and IM, suggesting
that its predominant location was not in the PM and
therefore may not play a direct role in cation entry in platelets.
Cation influx studies using Fura-2-loaded platelets The above results indicated that human platelets express TRPC6 in the platelet PM. Recent studies suggest that in heterologous systems, members of the TRPC3, 6, and 7 subfamilies can be activated by the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) to stimulate cation entry.18,40-43 We therefore carried out studies on Fura-2-labeled platelets to determine if OAG stimulated endogenous TRPC6 to allow calcium entry. Figure 3 shows the results. In the presence of 1 mM extracellular Ca++, addition of OAG stimulated a dose-related increase of (340/380 ratio) fluorescence of Fura-2-loaded platelets, signifying an increase of cytosolic [Ca++] (Figure 3A). Minimal increase was observed if no Ca++ was added to the incubation medium, suggesting that the major component of Ca++ increase was due to entry of the cation (results not shown). Ca++ increase induced by OAG was steady and near linear over time, whereas that by thrombin was rapid, reaching a peak followed by a slow decline (Figure 3B). The mechanism of action of OAG on the TRPC3, 6, and 7 subfamilies is not known but has been suggested to act via a membrane-delimiting manner and independent of PKC activity.18,40 The latter has been reported to be activated maximally by 75 µM OAG.44 In our experiments, incubation of platelets with a PKC inhibitor bisindolylmaleimide I (Bis I, 0.5 µM) 5 minutes prior to addition of OAG did not significantly affect the rate of Ca++ entry induced by 60 µM OAG and confirmed that this cation entry was independent of PKC (increase of ratio units above basal 4 minutes after OAG addition was 0.44 ± 0.09 [n = 4] and 0.57 ± 0.04 [n = 3] in the presence and absence of Bis I respectively, P = .30). However, 60 µM OAG-induced platelet aggregation (which is known to be dependent on PKC) was totally inhibited by 0.5 µM Bis I (results not shown).
We further investigated if OAG led to the activation of entry of other
cations, particularly Ba2+, as this has been used to
monitor the activity of heterologously expressed
TRPC3.41,43 Figure 4A shows that
60 µM OAG was effective at inducing the entry of Ba2+
into platelets at a rate similar to that of Ca++, further
correlating the expression of TRPC6 in the platelet PM. We
then went on to determine the contribution of TRPC6 to SOCE and
receptor-activated cation entry pathways. For these studies, the cells
were either stimulated with thapsigargin, which inhibits SERCA pumps
and depletes intracellular stores, or with thrombin, which acts on its
receptor and induces G protein-dependent phospholipase C signaling.
Experiments were carried out with the agonist added in the absence of
extracellular Ca++ to monitor release from stores, followed
by addition of the respective cation to determine entry (Figure 4B-C).
Addition of thapsigargin (3 µM) caused a slow release of
Ca++ from stores and a rapid entry of Ca++ upon
addition of extracellular Ca++, but was ineffective at
inducing sufficient entry of Ba2+ (Figure 4B). This
demonstrated that in platelets, SOCE is selective for Ca++
over Ba2+. In similar experiments, addition of 1 U/mL
thrombin showed rapid release of Ca++ from intracellular
stores and the rapid entry of either Ca++ or
Ba2+ (Ca++ > Ba2+) upon
either cation addition (Figure 4C). The kinetics of Ba2+
entry (ie, increase to a plateau) suggested that unlike
Ca++, Ba2+ was a poor substrate for platelet
Ca++ pumps. These results indicated that thrombin signaling
to PLC and the resultant IP3 and DAG formation activated
not only SOCE channels by IP3-induced depletion of the
stores but also non-SOCE channels such as TRPC6 that could be monitored
by the DAG (OAG)-induced entry of Ba2+.
Phosphorylation status of TRPC proteins in human platelets Our above findings suggested that platelet TRPC6 was activated upon thrombin addition. We next sought to examine if the phosphorylation status of TRPC proteins changed when platelets were treated with stimulatory or inhibitory agonists. Analysis of the primary sequence of hTRPC1 and hTRPC6 suggest that both proteins contain consensus sequences for cAMP-PK, cGMP-PK, PKC, casein kinase II, and tyrosine kinases (Prosite, ExPasy Molecular Biology Server, Geneva, Switzerland, http://www.expasy.ch/). We first examined tyrosine phosphorylation of TRPC proteins, as a number of studies have suggested that tyrosine kinases play a role in Ca++ entry when platelets undergo activation.6,7 Studies were carried out on freshly isolated platelets in aggregation tubes and, after stimulation with appropriate agents, lysates were prepared and immunoprecipitation carried out using the Ank and anti-TRPC6 antibodies (to extract TRPC1 and TRPC6, respectively). Western blots were probed with the antiphosphotyrosine antibody 4G10. Analysis of platelet lysates after cell stimulation with thrombin and collagen revealed that there was an increase of tyrosine-phosphorylated proteins, particularly of molecular sizes 140, 105, and 80 kDa, as observed by many investigators previously (Figure 5A). However, immunoprecipitates of TRPC1 using Ank suggested that TRPC1 (at 100 kDa) did not undergo tyrosine phosphorylation when platelets were stimulated with 2 U/mL thrombin or 30 µg/mL collagen, even though significant extraction of the 100-kDa protein was evident. TRPC6 also did not undergo tyrosine phosphorylation when platelets were stimulated with thrombin or collagen (Figure 5A-B). This suggested that direct tyrosine phosphorylation of TRPC1 or TRPC6 was not a mechanism for triggering cation entry into platelets. In experiments involving TRPC6, we have noticed a decrease in the level of the TRPC6 protein in Triton X-100 soluble lysates, its extraction by the antibody, and an increase of the protein in the insoluble pellet after stimulation of platelets with thrombin but not with collagen (results not shown). This may reflect an increased association with the cytoskeleton upon thrombin stimulation, and currently it is not known whether the TRPC6 protein associated with the Triton insoluble pellet undergoes tyrosine phosphorylation. However, this issue was not further investigated in this study.
Studies were then carried out to determine if the TRPC proteins
underwent changes in serine or threonine phosphorylation upon platelet
activation or inhibition using [32P]Pi-labeled cells.
Activation was achieved using 2 U/mL thrombin or 3 µM thapsigargin;
inhibition using 500 µM BIMPS, 500 µM 8PCPT, or 10 µM
PGE1. As with the tyrosine phosphorylation experiments, incubations were carried out in aggregation tubes, followed by lysate
preparation and extraction of TRPC proteins using the Ank and
antiTRPC6 antibodies. Detection of phosphorylation was carried out by autoradiography of the dried-down gels or Western blots. Figure
6A shows that under conditions where thrombin
caused complete aggregation of platelets, examination of lysates by
autoradiography showed the phosphorylation of pleckstrin at 45 kDa, a
well-established substrate of protein kinase C (lane T, Figure 6A).
Thapsigargin stimulation also caused aggregation of platelets and
phosphorylation of pleckstrin (results not shown). BIMPS and 8pCPT did
not cause aggregation of platelets or increase phosphorylation of
pleckstrin but did stimulate the phosphorylation of the 50-kDa protein
VASP, which is a well-established substrate of cAMP-PK and cGMP-PK
(lanes B and 8P, Figure 6A).29 BIMPS- and 8PCPT-induced
VASP phosphorylation also was detected using a polyclonal antibody that
recognized both the 50-kDa (phosphorylated) and 46-kDa
(nonphosphorylated) forms, confirming that cAMP-PK and cGMP-PK were
activated with these 2 agents (results not shown). Autoradiographic
analysis of immunoprecipitates of TRPC1 from resting platelets showed
phosphoproteins of molecular size 250 and 120 kDa but not at 100 kDa
(the latter being the migration size of extracted TRPC1, Figure 6B).
Upon stimulation of the cells with thrombin, these bands were
completely dephosphorylated. In the presence of BIMPS or 8pCPT, the
TRPC1 antibody Ank extracted phosphoproteins at molecular sizes 250, 120, 85, and 70 kDa. Occasionally, a phosphoprotein of a size greater
than 250 kDa was detected and other minor bands at 55 and 30 kDa
(Figure 6B). Although the 85-kDa phosphoprotein migrated at the size of
overexpressed hTRPC1, this identity from platelet extracts was not
confirmed, as probing the Western blots with Ank (or XTRP-1 antibody)
revealed only detection of the 100-kDa immunoprecipitated band. This
suggested that in platelets, TRPC1 was not phosphorylated by PKC,
cAMP-PK, or cGMP-PK, but associated with a number of substrates of
cAMP-PK and cGMP-PK. The identity of these substrates was not further
examined in this study. In separate experiments, the incubation of
[32P]Pi-labeled platelets with 100 µM sodium
nitroprusside (which acts as a NO donor to elevate cGMP in platelets,
resulting in activation of cGMP-PK) also resulted in the extraction by
Ank of phosphoproteins of molecular size 250, 120, 85, 70, and 55 kDa
(results not shown). Further, the inclusion of a partially permeable
specific inhibitor of cGMP-PK (Rp-8-pCPT-cGMPS) inhibited the
phosphorylation of these bands. This suggested that the phosphorylation of these substrates was physiologically relevant and, because of their association, they may be involved in the regulation or modulation of TRPC1 function. Figure 6C shows that the TRPC6 antibody immunoprecipitated weakly phosphorylated bands at 250, 120, 110, and 85 kDa from resting platelets. In the presence of BIMPS or 8PCPT, these
proteins underwent further phosphorylation, while if platelets were
stimulated with thrombin, they were totally dephosphorylated. The
110-kDa band was immunologically identified as TRPC6 by Western
blotting, suggesting that unlike TRPC1, it is a substrate for cAMP-PK
and cGMP-PK.
Studies were then carried out to determine if overexpressed hTRPC1 or
mTRPC6 could be phosphorylated by exogenous addition of the CAT of
cAMP-PK. Microsomal membranes from QBI-293A cells overexpressing hTRPC1
or mTRPC6 were incubated with
Studies were then designed to test if phosphorylation of TRPC6 by
cAMP-PK resulted in any effect on cation entry into Fura-2-loaded platelets. Many previous studies have demonstrated an action
of cAMP-PK on both a reduction of agonist-stimulated IP3
formation, an inhibition of Ca++ release from intracellular
stores, and the promotion of resequestration and extrusion (for a
review, see Authi1). As this study focused on entry
channels, experiments were carried out adding 250 µM BIMPS to
platelet suspensions after stores had been depleted but before the
addition of extracellular Ca++. Figure
8A shows that activation of cAMP-PK 2 minutes
prior to addition of extracellular Ca++ resulted in a 54%
reduction of thrombin-stimulated Ca++ entry
(P < .001), even though depletion of stores was similar to vehicle-treated control platelets. This clearly suggested that cAMP-PK inhibited calcium entry at a step downstream of store depletion
and possibly at the level of the entry channel. Similar experiments,
adding extracellular Ba2+ after store depletion, showed
that BIMPS had little or no effect on thrombin-stimulated
Ba2+ entry (Figure 8B). This result was surprising, as
TRPC6 was phosphorylated by cAMP-PK in both platelets and
overexpression systems, and suggested that phosphorylation by cAMP-PK
did not affect channel function. However, addition of BIMPS 2 minutes
prior to thrombin did inhibit both Ca++ release from stores
(via an inhibition of PLC and stimulation of resequestration) and entry
of Ba2+ (Figure 8C). Further, addition of the PLC inhibitor
U73122 (10 µM) 2 minutes prior to thrombin drastically inhibited
Ca++ release from stores and entry of Ba2+
(increase of ratio units 4 minutes after Ba2+ addition was
1.26 ± 0.02 [n = 3] and 0.69 ± 0.02 [n = 3] in the absence and presence of U73122 [P < .001]), suggesting
that inhibition of thrombin-stimulated PLC reduced channel activation.
BIMPS also was used to determine if cAMP-PK affected OAG-induced cation
entry. No effect was observed on Ba2+ entry (Figure 8D) or
Ca++ entry (results not shown), confirming that
phosphorylation of TRPC6 by cAMP-PK did not affect cation permeability
through the channel.
In this study we have shown for the first time expression of TRPC6 in the human platelet PM, its involvement in cation entry by the surface receptor-activating agent thrombin, in addition to direct activation by OAG, and we have also shown that it is a substrate for cAMP-PK. Compared with Ca++ release from intracellular stores, Ca++ entry is still little understood. SOCE has been suggested as the major mechanism responsible in nonexcitable cells, but neither the identity of the entry channels nor the mechanism for their gating are established. Members of the TRP family of ion channels represent the first potential candidates for SOCE channels. However, information accumulated from overexpression systems suggest that most members of the TRP family form receptor-activated channels acting independently from store regulation.16,17 Most cells are postulated to contain more than one member of the TRP family contributing either part or all of the entry channels in the PM. Analysis at the protein level, however, is incomplete and mostly to be determined. Here we show that a low level of TRPC1 is present in platelet IM and a higher expression of TRPC6 in the PM. TRPC4 and 5 were undetectable using an antibody that detected both overexpressed TRPC4 and TRPC5, and TRPC3 was also undetectable using a similarly high-affinity antibody. Currently we do not have an antibody that recognizes TRPC7. Recently, Rosado and Sage23,24 reported the presence of TRPC1 protein in platelets and suggested that it mediated Ca++ entry via a store depletion-dependent coupling to the type II IP3R. They presented their findings as evidence for a secretionlike conformational coupling mechanism with the type II IP3R at the stores linking with TRPC1 (presumed to be at the PM) only when stores were depleted. Further, they reported that Jasplakinolide, which induced cortical actin assembly and inhibited Ca++ entry, also disrupted the coupling of TRPC1 with the type II IP3R.24 TRPC1 has been suggested to play a role in SOCE in a number of other cell types, including salivary gland cells,45 A549 and pulmonary artery cells,46 and vascular smooth muscle cells,47 although the mechanism of gating in these cells has not been elucidated. Our studies present new evidence that supports an indirect role for TRPC1 in cation entry in platelets. We propose a function for TRPC1 in calcium signaling at the level of the stores that we and others have shown to contain the type I and type II IP3R.32,48 The possibility that it may become inserted into the PM upon cell activation, as has been described in Xenopus oocytes,9 cannot be ruled out. Further, a recent study has described that in a HEK-293 cell line expressing hTRPC3, Jasplakinolide (and calyculin-A) inhibited Ca++ entry by the internalization of TRPC3 constitutively associated with the type III IP3R and other Ca++ regulatory proteins without disruption of the multimolecular complex.49 Thus, membrane fusion to and internalization from the PM could possibly provide a mechanism for regulation of Ca++ entry. However, our studies suggest that SOCE in platelets is selective for Ca++ over Ba2+ entry and thus should be mediated by an ion channel bearing these properties. TRPC1, however, is known to be a nonselective channel with similar permeabilities37,50 for Ca++, Na+, Cs+, and Ba2+ and thus on its own is unlikely to account for the Ca++ selectivity of SOCE function at the PM. The reported properties of CaT1 and CaT2 (TRPV6 and TRPV5, respectively) and the suggestion that CaT1 forms a part or all of ICRAC51,52 should necessitate a study of the expression of these proteins in platelets. TRPC6 is well expressed in platelets and located in the PM.
In heterologous systems, TRPC6 has been shown to be a nonselective cation channel (though favoring Ca++ over Na+
by 5 to 1) that is activated by receptors linked to phospholipase C
signaling18 but independent of store depletion. TRPC6 is
closely related to TRPC3 and TRPC7, with which it shares approximately 75% sequence identity and, along with these members, it can be directly activated by DAG analogs. Indeed, we have shown that in line
with the expression of TRPC6, OAG activates both Ca++ and
Ba2+ entry into platelets. The ability of TRPC6, 3, and 7 to allow Ba2+ passage has proved useful in examining the
gating properties of these channels, and our finding that thrombin
induces Ba2+ entry is highly indicative of the
activation of TRPC6 by surface receptor-activating stimuli. This
therefore represents (as far as we are aware) the first demonstration
of the activation of an identified endogenous cation channel in the
PM of platelets. TRPC6 has been shown to be important for
the The finding that TRPC6 and (to a lesser extent) TRPC1 were present in human platelets prompted us to examine the phosphorylation status of these proteins during activation and inhibition of platelet function. Both TRPC6 and TRPC1 contain consensus sequences for a number of protein kinases including tyrosine kinases, PKC, casein kinase II, cAMP-PKs, and cGMP-PKs. Although controversial and little understood, tyrosine kinases have been suggested to play a role in SOCE-mediated Ca++ entry,7,57 with a recent study that suggests an action by tyrosine kinases on the cytoskeleton and further, that tyrosine kinase inhibitors prevented actin polymerization.58 Our experiments revealed little evidence of tyrosine phosphorylation of either TRPC6 or TRPC1 under conditions where thrombin and collagen had induced the widespread tyrosine phosphorylation of platelet proteins, suggesting that the channels themselves were not direct targets of tyrosine kinases. In [32P]Pi-labeled platelets, TRPC6 was found to be a substrate of cAMP-PK and cGMP-PK and associated with 250-, 120-, and 85-kDa phosphoproteins. TRPC1 was not a good substrate for this kinase but was similarly associated with a number of cAMP-PK and cGMP-PK substrates. These findings are significant, as the vasodilators NO and PGI2 inhibit platelet function predominantly via cGMP-PK and cAMP-PK, respectively. TRPC6 contains 2 sites for cyclic nucleotide kinase action at RRQT70 and KKLS,322 and currently it is not known whether both sites are phosphorylated or what their relationship is to the regulation of TRPC6 function. Our studies using Fura-2-labeled cells show that under conditions where store depletion had occurred, activation of cAMP-PK resulted in inhibition of Ca++ entry induced by thrombin but not of Ba2+ entry. As we have shown that Ba2+ entry occurred by a SOCE-independent mechanism, most probably mediated by DAG, this suggested that cAMP-PK acted on the SOCE pathway, at a site downstream of store depletion, either on the gating mechanism or on the SOCE channel itself. Targets for cAMP-PK previously identified include PLC activation,59 the IP3R itself,32 the Ca++ pumps,60 and many more (for a review, see Koesling et al61). Inhibition of Ba2+ entry was seen if BIMPS was added 2 minutes prior to thrombin addition, reflecting phospholipase C inhibition. Rosado et al62 have recently reported an action of cAMP on tyrosine phosphatases that may be involved with the SOCE mechanism and suggested that it was independent of cAMP-PK. However, there is currently controversy regarding the action and efficacy of the KT series of cyclic nucleotide kinase inhibitors in intact platelets,63 and therefore further studies are required to clarify these suggestions. Interestingly, direct activation of TRPC6 with OAG also was not affected by cAMP-PK activation, further confirming our suggestion that thrombin acts on TRPC6 via the formation of DAG. Our results raise the question, "Why is TRPC6 a substrate for cAMP-PK if after phosphorylation there is no effect on channel activity?" Currently, the answer to this question is not known, but cAMP-PK may affect other functions of TRPC6 not tested in this study, such as its association to other proteins and particularly to components of the cytoskeleton. Alternatively, the possibility that Ba2+ entry resulted from the presence of another Ca++/Ba2+ channel cannot be ruled out. In this study, we have not addressed the identities of the phosphoproteins that associate with either TRPC6 or TRPC1. Clearly, suitable candidates for the 250-kDa protein are the IP3Rs and the actin-binding protein filamin. The possibility that the 120-kDa phosphoprotein could be the newly described IP3R-associated G-kinase substrate (IRAG)64 and known to be involved with Ca++ regulation is equally fascinating. Our future studies will investigate the relationship of components of this multimolecular complex with the cAMP-PKs and cGMP-PKs. In conclusion, our studies report the presence of high levels of TRPC6 and low levels of TRPC1 in platelet membranes. The presence of TRPC6 in the plasma membrane, its activation by thrombin, and its demonstration as a substrate of cAMP-PK represent the first identification of a non-SOCE-regulated cation channel in human platelets. A similar search for other members of the TRP family may reveal the molecular identity of the SOCE channel and a better understanding of how information from the stores leads to their gating. Our studies also suggest a role for TRPC1 at the level of the intracellular Ca++ stores. During the review of this manuscript, Mori et al65 reported that TRP1 knock-out in the DT40 cell line resulted in a reduction of agonist-mediated release of Ca++ from the stores and a reduction of IP3-mediated Ca++ release from membranes. This further supports a role for TRPC1 at the level of the intracellular stores.
We thank Professor G. Barritt (Adelaide, Australia) for the
generous gift of anti-XTRP-1 antibody, and Professor C. Montell (Baltimore, MD) for the generous gift of anti-
Submitted March 7, 2002; accepted May 22, 2002.
Prepublished online as Blood First Edition Paper, June 21, 2002; DOI 10.1182/blood-2002-03-0723.
Supported by grants from the British Heart Foundation and from the National Institutes of Health (GM54235, M.X.Z.).
Correspondence: Kalwant S. Authi, Centre for Cardiovascular Biology and Medicine, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL; e-mail: kalwant.authi{at}kcl.ac.uk.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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G. Vazquez, B. J. Wedel, B. T. Kawasaki, G. St. J. Bird, and J. W. Putney Jr. Obligatory Role of Src Kinase in the Signaling Mechanism for TRPC3 Cation Channels J. Biol. Chem., September 24, 2004; 279(39): 40521 - 40528. [Abstract] [Full Text] [PDF]
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C. Hisatsune, Y. Kuroda, K. Nakamura, T. Inoue, T. Nakamura, T. Michikawa, A. Mizutani, and K. Mikoshiba Regulation of TRPC6 Channel Activity by Tyrosine Phosphorylation J. Biol. Chem., April 30, 2004; 279(18): 18887 - 18894. [Abstract] [Full Text] [PDF]
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A. Dietrich, M. Mederos y Schnitzler, J. Emmel, H. Kalwa, T. Hofmann, and T. Gudermann N-Linked Protein Glycosylation Is a Major Determinant for Basal TRPC3 and TRPC6 Channel Activity J. Biol. Chem., November 28, 2003; 278(48): 47842 - 47852. [Abstract] [Full Text] [PDF]
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S. O. Sage, S. L. Brownlow, J. A. Rosado, K. S. Authi, S. Hassock, M. X. Zhu, V. Flockerzi, and C. Trost TRP channels and calcium entry in human platelets Blood, December 1, 2002; 100(12): 4245 - 4246. [Full Text] [PDF]
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Top
Abstract
Introduction
-aminohexyl agarose column using
N-
80°C until required. The cell pellets were thawed on ice and resuspended in sonication medium (0.34 M
sorbitol, 10 mM HEPES
[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] pH 7.2 with a cocktail of inhibitors including aprotinin [0.2
U/mL], pepstatin A [10 µg/mL], PMSF [phenylmethylsulfonyl fluoride] [0.1 mM], and soybean trypsin inhibitor [0.2
mg/mL]). Homogenization was carried out on ice using a Vibra Cell
ultrasonicator (Jencons Scientific, Leighton Buzzard, United Kingdom),
with 2 × 10-second bursts at an amplitude setting of 30. The
homogenates were centrifuged at 100 000g for 90 minutes to
generate a microsomal pellet that was resuspended in sonication medium
plus 5% glycerol at approximately 2 mg/mL. These were used for all
subsequent analyses, including Western blotting and phosphorylation by
exogenous kinases.
a possible mechanism.
FEBS Lett.
1990;263:5-9
/