Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets

Available online at www.sciencedirect.com

ta 1783 (2008) 84–97www.elsevier.com/locate/bbamcr

Biochimica et Biophysica Ac

Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entrythrough hTRPC6 channel in human platelets

Isaac Jardín a, Pedro C. Redondo b, Ginés M. Salido a, Juan A. Rosado a,⁎

a Department of Physiology (Cell Physiology Research Group), University of Extremadura, 10071 Cáceres, Spainb Department of Physiology, Development and Neuroscience, University of Cambridge, CB2 3EG Cambridge, UK

Received 3 May 2007; received in revised form 29 June 2007; accepted 19 July 2007Available online 29 July 2007

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a versatile regulator of TRP channels.We report that inclusion of a PIP2 analogue, PIP2 1,2-dioctanoyl,does not induce non-capacitative Ca2+ entry per se but enhanced Ca2+ entry stimulated either by thrombin or by selective depletion of the Ca2+ stores inplatelets, the dense tubular system, using 10 nMTG, and the acidic stores, using 20 μM2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Reduction of PIP2levels by blocking PIP2 resynthesiswithLi

+ or introducing amonoclonal anti-PIP2 antibody, or sequestering PIP2 using poly-lysine, attenuatedCa2+ entry

induced by thrombin, TG and TBHQ, and reduced thrombin-evoked, but not TG- or TBHQ-induced, Ca2+ release from the stores. Incubation with theanti-hTRPC1 antibody did not alter the stimulation of Ca2+ entry by PIP2, whilst introduction of anti-hTRPC6 antibody directed towards the C-terminusof hTRPC6 reduced Ca2+ and Mn2+ entry induced by thrombin, TG or TBHQ, and abolished the stimulation of Ca2+ entry by PIP2. The anti-hTRPC6antibody, but not the anti-hTRPC1 antibody or PIP2, reduced non-capacitative Ca2+ entry by the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol. Insummary, hTRPC6 plays a role both in store-operated and in non-capacitative Ca2+ entry. PIP2 enhances store-operated Ca

2+ entry in human platelets,most probably by stimulation of hTRPC6 channels.© 2007 Elsevier B.V. All rights reserved.

Keywords: Ca2+ influx; PIP2; Platelet; Thrombin; TG; TBHQ; hTRPC6; hTRPC1

1. Introduction

The transient receptor potential (TRP) proteins contain sixtransmembrane domains and form cation-selective ion channels.At present 29 vertebrate TRP isoforms have been recognized,distributed among seven subfamilies (TRPC, TRPV, TRPM,TRPML, TRPP, TRPA and TRPN) [1]. TRP channel subunitshave been shown to assemble into homo- or heterotetramericchannel complexes. The subunit composition presumably influ-ences the biophysical and regulatory properties of the resulting

Abbreviations: BSA, bovine serum albumin; [Ca2+]c, cytosolic free calciumconcentration; DTS, dense tubular system; HBS, HEPES-buffered saline;hTRPC1, human canonical transient receptor potential 1; hTRPC6, humancanonical transient receptor potential 6; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3receptor; PIP2, Phosphatidylinositol 4,5-bisphosphate; PMCA, plasma membraneCa2+ ATPase; TBHQ, 2,5-di-(tert-butyl)-1,4-hydroquinone; TG, thapsigargin⁎ Corresponding author. Tel.: +34 927257154; fax: +34 927257110.E-mail address: [email protected] (J.A. Rosado).

0167-4889/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.bbamcr.2007.07.007

channel [2]. The canonical transient receptor potential (TRPC)cation channels are mammalian homologues of the photoreceptorTRP channel inDrosophila melanogaster [3]. A number of recentstudies have provided evidence for the role of TRPC channels inCa2+ entry pathways, as well as for the different mechanisms ofregulation of channel gating [4–8]. TRPC channels has beensuggested to function as integrators of a number of intracellularsignals resulting from receptor-induced PLC activation, whichcatalyzes the synthesis of inositol 1,4,5-trisphosphate (IP3) anddiacylglycerol (DAG) through the hydrolysis of phosphatidyli-nositol 4,5-bisphosphate (PIP2) [9]. But, in addition to the role ofDAG on TRP channel function [10], its precursor, PIP2, has beenpresented as a versatile regulator of TRP channels, which ofteninvolves the interaction of the TRP subunits with the lipid itself,independently of its hydrolysis products [11]. A number of TRPchannels are regulated by PIP2, including TRPV1, which has beenreported to be inhibited by PIP2 by interaction with a PIP2-bindingdomain in the C-terminus of TRPV1, characterised by clusters of

mailto:[email protected]

http://dx.doi.org/10.1016/j.bbamcr.2007.07.007

85I. Jardín et al. / Biochimica et Biophysica Acta 1783 (2008) 84–97

positively charged residues interspersed with hydrophobic aminoacids [12]. In contrast, several members of the TRPM family,including TRPM4, 5, 7 and 8 are positively regulated by PIP2 andunder experimental conditions that prevent PIP2 resynthesis (byphosphatidylinositol (PI) kinase inhibitors) or decrease PIP2 avail-able (using PIP2 antibodies) these channels rundown or desensitise[1,13,14]. In TRPM4 this regulation has been suggested to bemediated by interaction with a putative PIP2-binding pleckstrinhomology (PH) domain located in the C-terminus [14].

Growing evidence suggests that TRPC channels are alsoregulated by PIP2. TRPC3 complexed with PLCγ forms anintermolecular PH domain, which shows a high affinity for PIP2[9]. In addition, TRPC6 has been shown to be activated byhydrolysis of PIP2 in neurones [15]. From the seven identifiedmembers of the TRPC family (TRPC1 through TRPC7),platelets have been shown to express TRPC1, TRPC3,TRPC4, TRPC5 and TRPC6 [16–18]. TRPC1, TRPC4 andTRPC5 have been shown to form a heteromultimer associatedwith platelet lipid raft domains, whereas TRPC3 and TRPC6also associate but, in this case, independently of lipid rafts [18].TRPC1 and TRPC6 are putative store-operated Ca2+ channels[5,6,19,20]. In addition TRPC6 has been suggested to beinvolved in receptor-activated, diacylglycerol-mediated cationentry [1,21]. We have previously shown that impairment of PIP2resynthesis in platelets by inhibition of PI4 kinase results ininhibition of store-operated Ca2+ entry (SOCE) [22]. Hence, wehave investigated the role of PIP2 in the regulation of TRPC1and TRPC6 channel function in human platelets.

2. Materials and methods

2.1. Materials

Fura-2 acetoxymethyl ester (fura-2/AM) and calcein-AM were fromMolecular Probes (Leiden, The Netherlands). Apyrase (grade VII), aspirin,thapsigargin (TG), thrombin, lithium chloride, poly-lysine and bovine serumalbumin (BSA) were from Sigma (Madrid, Spain). Ionomycin and 1-oleoyl-2-acetyl-sn-glycerol (OAG) were from Calbiochem (Nottingham, UK). 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ) was from Alexis (Nottingham, UK).N-(p-amylcinnamoyl)anthranilic acid was from BIOMOL Research Labora-tories (Plymouth Meeting, PA, USA). Phosphatidylinositol 4,5-bisphosphate(PIP2) (1,2-dioctanoyl) was from Scharlab (Barcelona, Spain). Monoclonalanti-phosphatidylinositol 4,5-bisphosphate antibody (anti-PIP2) was fromAssay Design Inc (Ann Arbor, MI, USA). Anti-hTRPC1 polyclonal antibodywas from Alomone Laboratories (Jerusalem, Israel). Anti-hTRPC6 polyclonalantibody and control antigen peptide were from Santa Cruz (Santa Cruz, CA,USA). All other reagents were of analytical grade.

2.2. Platelet preparation

Fura-2-loaded platelets were prepared as previously described [23] asapproved by Local Ethical Committees and in accordance with the Declarationof Helsinki. Briefly, blood was obtained from healthy drug-free volunteers andmixed with one-sixth volume of acid/citrate dextrose anticoagulant containing(in mM): 85 sodium citrate, 78 citric acid and 111 D-glucose. Platelet-richplasma (PRP) was then prepared by centrifugation for 5 min at 700×g andaspirin (100 μM) and apyrase (40 μg/mL) were added. Platelet-rich plasma wasincubated at 37 °C with 2 μM fura-2 acetoxymethyl ester for 45 min. Cells werethen collected by centrifugation at 350×g for 20 min and resuspended inHEPES-buffered saline (HBS), pH 7.45, containing (in mM): 145 NaCl, 10HEPES, 10 D-glucose, 5 KCl, 1 MgSO4 and supplemented with 0.1% BSA and40 μg/mL apyrase.

2.3. Cell viability

Cell viability was assessed using calcein and trypan blue. For calcein loading,cells were incubated for 30 min with 5 μM calcein-AM at 37 °C, centrifuged andthe pellet was resuspended in fresh HBS. Fluorescence was recorded from 2 mLaliquots using a Cary Eclipse Spectrophotometer (Varian Ltd., Madrid, Spain).Samples were excited at 494 nm and the resulting fluorescence was measured at535 nm. The results obtained with calcein were confirmed using the trypan blueexclusion technique. 95% of cells were viable in our platelet preparations, at leastduring the performance of the experiments.

2.4. Measurement of cytosolic free calcium concentration ([Ca2+]c )

Fluorescence was recorded from 2 mL aliquots of magnetically stirred plateletsuspension (2×108 cells/mL) at 37 °Cusing a fluorescence spectrophotometerwithexcitation wavelengths of 340 and 380 nm and emission at 505 nm. Changes in[Ca2+]c were monitored using the fura-2 340/380 fluorescence ratio and calibratedaccording to the method of Grynkiewicz and coworkers [24]. Mn2+ influx wasmonitored as a quenching of fura-2 fluorescence at the isoemissive wavelength of360 nm, which is presented on an arbitrary linear scale [25].

Ca2+ entry was estimated using the integral of the rise in [Ca2+]c for 2.5min afteraddition of CaCl2 [26]. Control experiments were performed for all experimentalprocedures in order to correct Ca2+ entry by subtraction of the [Ca2+]c elevation dueto leakage of the indicator. Ca2+ release was estimated using the integral of the rise in[Ca2+]c for 3 min after the addition of thrombin, TG or TBHQ. Both Ca2+ entry andrelease are expressed as nM.s, as previously described [27,28]. To calculate the initialrate of Ca2+ elevation after the addition of Ca2+ to the medium, the traces were fittedto the equation y=A×(1−e−KX) and to estimate the initial rate of fura-2 fluorescencequenching after the addition of Mn2+ to the medium, the traces were fitted to theequation y=S×e−KX+A, whereK is the slope, S is the span andA is the plateau [29].

To compare the rate of decay of [Ca2+]c to basal values after platelet stimulationwith TG plus ionomycin in the absence or presence of PIP2 we used the constant ofthe exponential decay. Traces were fitted to the equation y=S×(1−e−K1T)×e−K2T,as previously described [30], where K1 and K2 are the constants of the exponentialincrease and decay, respectively, T is time and S is the span.

2.5. Reversible electroporation procedure

The platelet suspension was transferred to an electroporation chambercontaining antibodies at a final concentration of 2 μg/mL, and the antibodieswere transjected according to published methods [31–33]. Reversible electro-permeabilization was performed at 4 kV/cm at a setting of 25-microfaradcapacitance and was achieved by 7 pulses using a Bio-Rad Gene Pulser XcellElectroporation System (Bio-Rad, Hercules, CA, USA). Following electropora-tion, cells were incubated with antibodies for an additional 60 min at 37 °C thenwere centrifuged at 350×g for 20 min and resuspended in HBS containing200 μM CaCl2. At the time of experiment 250 μM EGTA was added.

2.6. Immunofluorescence

Samples of platelet suspension (200 μM; 108 cells/mL) were transferred to200 μL of ice-cold 3% (w/v) formaldehyde in phosphate-buffered saline (PBS;containing 137 mM NaCl, 2.7 mM KCl, 5.62 mM Na2HPO4, 1.09 mMNaH2PO4, 1.47mMKH2PO4, pH 7.2, and supplementedwith 0.5% (w/v) bovineserum albumin) for 10 min and then incubated for 1 h with 1 μg/mL anti-PIP2antibody. The platelets were then collected by centrifugation and washed twice inPBS. To detect the primary antibody, samples were incubated with 0.02 μg/mLFITC-conjugated goat anti-mouse IgG antibody for 1 h andwashed twice in PBS.Fluorescence was measured using a fluorescence spectrophotometer (VarianLtd., Madrid, Spain). Samples were excited at 496 nm, and emission was at516 nm.

2.7. Statistical analysis

Analysis of statistical significance was performed using one-way analysis ofvariance. For comparison between two groups Student's t test was used. pb0.05was considered to be significant for a difference.

Fig. 2. PIP2 enhances TG-induced cation entry in human platelets. (A) Fura-2-loaded human platelets were treated in a Ca2+-free medium (100 μM EGTAwasadded) with various concentrations of PIP2 1,2-dioctanoyl (1–10 μM) or thevehicle (HBS) and 1 min later were stimulated with 10 nM TG followed byaddition of CaCl2 (final concentration 300 μM) to initiate Ca2+ entry.Modifications in [Ca2+]c were monitored using the 340/380 nm ratio and traceswere calibrated in terms of [Ca2+]c. (B) Human platelets were loaded with Fura-2

2+

86 I. Jardín et al. / Biochimica et Biophysica Acta 1783 (2008) 84–97

3. Results

3.1. PIP2 enhances store-operated divalent cation entry inhuman platelets

In the absence of extracellular Ca2+ (100 μM EGTA wasadded), treatment of fura-2-loaded human platelets in stirredcuvettes at 37 °C with 0.1 U/mL thrombin induced a transientincrease in [Ca2+]c. Subsequent addition of Ca2+ to theextracellular medium (300 μM) resulted in a rapid rise in [Ca2+]cindicative of Ca2+ entry (Fig. 1, top panel). Treatment of plateletswith 1, 3 or 10 μM of the water-soluble PIP2 analogue, PIP2 1,2-dioctanoyl, significantly increased thrombin-evokedCa2+ entry by127±6%, 137±10% and 140±5%, respectively (Fig. 1, top panel;mean±S.E.M.; one-way analysis of variance; pb0.05; n=5). Theinitial slope for the rise in [Ca2+]c after the addition of Ca2+ wassignificantly enhanced from 0.0565±0.0065 to 0.0792±0.0067 incontrol and PIP2 1,2-dioctanoyl (10μM)-treated cells, respectively(pb0.05, Student's t test). Addition of PIP2 1,2-dioctanoyl beforethrombin reduced thrombin-evoked Ca2+ release from the intra-cellular stores in a concentration-dependent manner (Ca2+ releaseestimated as the integral of the rise in [Ca2+]c for 3 min after theaddition of thrombin in a Ca2+-free mediumwas 86±3%, 79±9%and 64±9% of control at 1, 3 and 10 μM PIP2 1,2-dioctanoyl;Fig. 1, top panel; pb0.05; n=5). As shown in Fig. 1, lower panel,addition of 10 μM PIP2 1,2-dioctanoyl to the platelet suspensionhas a negligible effect on cytosolic Ca2+ mobilization either in theabsence or presence of extracellular Ca2+, which strongly supportthat PIP2 does not induce non-capacitative Ca2+ entry in thesecells.

Fig. 1. PIP2 increases thrombin-evoked Ca2+ entry in human platelets. Toppanel, Fura-2-loaded human platelets were treated in a Ca2+-free medium(100 μM EGTA was added) with various concentrations of the water-solublePIP2 analogue, PIP2 1,2-dioctanoyl (1–10 μM), or the vehicle (HBS) and 1 minlater were stimulated with 0.1 U/mL thrombin followed by addition of CaCl2(final concentration 300 μM) to initiate Ca2+ entry. Bottom panel, cells weretreated in a Ca2+-free medium with 10 μM PIP2 1,2-dioctanoyl followed by theaddition of CaCl2 as above. Modifications in [Ca2+]c were monitored using the340/380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces arerepresentative of five separate experiments.

and resuspended in a Ca -free medium. Fura-2-fluorescence was measuredwith an excitation wavelength of 360 nm, the isoemissive wavelength. Plateletswere treated in the absence or presence of 3 μM PIP2 1,2-dioctanoyl and 1 minlater cells were stimulated with 10 nM TG or DMSO (as control) followed by theaddition of MnCl2 (final concentration 300 μM). Mn2+ was added to untreatedcontrol cells (DMSO) or cells treated with TG in the absence (TG) or presence ofPIP2 (TG+PIP2). Traces are representative of six separate experiments.

We have recently identified two mechanisms for SOCE inhuman platelets regulated by two separate Ca2+ compartments:the dense tubular system (DTS) and acidic stores [34]. The DTScontains an isoform of SERCA that shows a high sensitivity to TGand is insensitive to TBHQ; in contrast, the acidic organellesexpress a TBHQ-sensitive SERCA isoform with low affinity forTG [35–38]. Since thrombin is able to mobilise Ca2+ from bothstores [39], we have investigated the effect of PIP2 on SOCEinduced by low concentrations of TG (by depletion of the DTS) orby TBHQ (to discharge the acidic stores) in platelets. Aspreviously reported [38,39], in the absence of extracellular Ca2+,addition of TG (10 nM) to fura-2-loaded human platelets in stirredcuvettes at 37 °C evoked a prolonged increase in [Ca2+]c due torelease of Ca2+ from the DTS. Subsequent addition of Ca2+

(300 μM) to the external medium induced a sustained increase in[Ca2+]c indicative of SOCE (Fig. 2A). Treatment with 1, 3 or10 μM PIP2 1,2-dioctanoyl, significantly enhanced Ca2+ influxinduced by TG by 157±20%, 212±30% and 297±17%,respectively (Fig. 2A; pb0.05; n=6). The initial slope for therise in [Ca2+]c after the addition of Ca2+ was significantly

Fig. 3. PIP2 enhances TBHQ-induced cation entry in human platelets. (A) Fura-2-loaded human platelets were treated in a Ca2+-free medium (100 μM EGTAwas added) with various concentrations of PIP2 1,2-dioctanoyl (1–10 μM) or thevehicle (HBS) and 1 min later were stimulated with 20 μM TBHQ followed byaddition of CaCl2 (final concentration 300 μM) to initiate Ca2+ entry.Modifications in [Ca2+]c were monitored using the 340/380 nm ratio and traceswere calibrated in terms of [Ca2+]c. (B) Human platelets were loaded with Fura-2and resuspended in a Ca2+-free medium. Fura-2-fluorescence was measuredwith an excitation wavelength of 360 nm, the isoemissive wavelength. Plateletswere treated in the absence or presence of 3 μM PIP2 1,2-dioctanoyl and 1 minlater cells were stimulated with 20 μM TBHQ or DMSO (as control) followedby the addition of MnCl2 (final concentration 300 μM). Mn2+ was added tountreated control cells (DMSO) or cells treated with TBHQ in the absence(TBHQ) or presence of PIP2 (TBHQ+PIP2). Traces are representative of sixseparate experiments.

Fig. 4. Effect of PIP2 on restoration of [Ca2+]c in human platelets. Fura-2-loadedhuman platelets were treated in a Ca2+-free medium (100 μM EGTAwas added)with PIP2 1,2-dioctanoyl (3 μM; PIP2) or the vehicle (Control) and 1 min laterwere stimulated with TG (1 μM) combined with ionomycin (50 nM). Elevations in[Ca2+]i were monitored using the 340/380 nm ratio and traces were calibrated interms of [Ca2+]c. Traces shown are representative of five independent experiments.


enhanced from 0.0605±0.0055 to 0.0822±0.0062 in control andPIP2 1,2-dioctanoyl (10 μM)-treated cells, respectively (pb0.05).Treatment with PIP2 1,2-dioctanoyl had no significant effects onCa2+ release from the intracellular store, indicating that accu-mulation of Ca2+ in the DTS was unaffected by addition of PIP21,2-dioctanoyl (Fig. 2A; n=6).

Mn2+ was used to evaluate the effect of PIP2 on divalent cationinflux. This cation can be used as a surrogate for Ca2+ entry givenits quenching effect on fura-2 [25]. Fura-2 was excited at theisoemissive wavelength, 360 nm, to allow monitoring of quench-ing of fluorescence by Mn2+. Addition of Mn2+ (300 μM) toplatelets treatedwith TG resulted in a sustained quenching of fura-2fluorescence (Fig. 2B, traces TG) compared with non-stimulatedcells (Fig. 2B, trace DMSO). Addition of 3 μM PIP2 1,2-dioctanoyl before TG significantly increased the rate of Mn2+

entry. The initial slope for the rate of fura-2 fluorescence quenchingafter the addition of Mn2+ were 0.0409±0.0021 and 0.0491±0.0020 for control and PIP2 1,2-dioctanoyl-treated cells. Theseresults indicate that PIP2 enhances DTS-regulated divalent cationentry.

Our studies investigating the effect of PIP2 on SOCE stim-ulated by TBHQ reported similar results. As shown in Fig. 3A,in the absence of extracellular Ca2+, TBHQ (20 μM), whichfully depletes the acidic stores [34,38,39], induced a sustainedelevation in [Ca2+]c. Subsequent addition of Ca2+ (300 μM)induced a sustained increase in [Ca2+]c indicative of SOCE.Addition of PIP2 1,2-dioctanoyl significantly enhanced TBHQ-evoked Ca2+ influx by 131±19%, 150±10% and 179±14% atthe concentrations 1, 3 and 10 μM, respectively, without havingany effect on Ca2+ release from the stores (Fig. 3A; pb0.05;n=6). The initial slope for the rise in [Ca2+]c after the additionof Ca2+ was enhanced from 0.0525±0.0035 to 0.0732±0.0047in control and PIP2 1,2-dioctanoyl (10 μM)-treated cells, re-spectively ( pb0.05). In addition, PIP2 1,2-dioctanoyl (3 μM)significantly enhanced the rate of Mn2+ entry induced byTBHQ. The initial slope for the rate of fura-2 fluorescencequenching after the addition of Mn2+ were 0.0329±0.0047 and0.0451±0.0034 for control and PIP2 1,2-dioctanoyl-treatedcells. These findings indicate that PIP2 enhances divalent cationentry induced by depletion of the acidic organelles in platelets.

3.2. PIP2 does not alter Ca2+ extrusion

Established routes for Ca2+ extrusion are the Na+/Ca2+ ex-changer and the plasma membrane Ca2+-ATPase (PMCA),although in platelets the main mechanism for Ca2+ extrusion atlow [Ca2+]c has been reported to be the PMCA [30,40]. Using apreviously established procedure we have estimated Ca2+ extru-sion in platelets as the rate of decay of [Ca2+]c to basal levels afteractivation with 1 μM TG combined with a low concentration ofionomycin (50 nM) to achieve extensive depletion of the internalCa2+ stores [30]. Treatment of platelets at 37 °C with 3 μM PIP21,2-dioctanoyl did not modify Ca2+ release from the intracellularstores induced by TG plus ionomycin. In addition, PIP2 1,2-dioctanoyl has a negligible effect on the rate of decay of [Ca2+]c tobasal levels. The decay constants were 0.0071±0.0005 and0.0073±0.0006 in control and PIP2-treated platelets (Fig. 4; n=5).

Fig. 5. LiCl attenuatesCa2+ entry in human platelets. (A andB)Human platelets in PRPwere preincubated in the absence (A) or presence of 10mMLiCl (B) for 12 h, loadedwith fura-2 and resuspended in a Ca2+-free HBS. Cells were then stimulated with 0.1 U/mL thrombin followed by the addition of CaCl2 (final concentration 300 μM) toinitiate Ca2+ entry. (C and D) Platelets were preincubated with 10mMLiCl for 12 h, loaded with fura-2 and resuspended in a Ca2+-free HBS. Cells were treated with 20 μMTBHQ followed by addition of 0.1 U/mL thrombin. 3 min later CaCl2 (final concentration 300 μM; C) or TG (1 μM) combined with ionomycin (50 nM; D) were added.(E and F) Platelets were preincubated in the absence (Control) or presence of 10mMLiCl (Li+) for 12 h, as indicated, loadedwith fura-2 and resuspended in a Ca2+-freeHBS.Cells were treated with 10 nM TG (E) or 20 μMTBHQ (F) followed by addition of CaCl2 (final concentration 300 μM) to initiate Ca2+ entry. G, Platelets were incubated inthe absence and presence of poly-lysine (10 μg/mL, molecular weight ∼1000) for 1 h, as indicated, and then stimulated with 0.1 U/mL thrombin in a Ca2+-free medium(100 μMEGTAwas added) followed by the addition of CaCl2 (final concentration 300 μM) to initiate Ca2+ entry. Modifications in [Ca2+]c were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces are representative of six separate experiments.


Fig. 6. Electrotransjection of anti-PIP2 antibody attenuates Ca2+ entry. (A) Fixed human plateletswere incubatedwith either 1μg/mL anti-PIP2 antibody for 1 h (b), 0.02μg/mL

FITC-conjugated goat anti-mouse IgG antibody for 1 h (c) for 1 h with 1 μg/mL anti-PIP2 antibody followed by incubation with the FITC-conjugated anti rabbit IgG for afurther 1 h (d) or left untreated (a). In (e) cellswere treatedwith 10mMLiCl for 12 h and then incubated for 1 hwith 1μg/mLanti-PIP2 antibody followed by incubationwith theFITC-conjugated anti rabbit IgG for a further 1 h. Histograms indicate the percentage of immunofluorescence under different experimental conditions relative to their control(cells not treated with LiCl and incubated with both antibodies).Values are mean±S.E.M. of 4 independent experiments. (B–G) Human platelets (109 cells/mL) wereelectropermeabilized in aGene Pulser as described inMaterials andmethods. Following electroporation, cells were incubated in the absence (B, D and F) or presence (C, E andG) of 1μg/mL anti-PIP2 antibody for an additional 60min at 37 °C, loadedwith fura-2, centrifuged at 350×g for 20min and resuspended in HBS containing 200 μMCaCl2. Atthe time of experiment 250 μMEGTAwas added. Fura-2-loaded human platelets were stimulated either with 0.1 U/mL thrombin (B and C), 10 nM TG (D and E) or 20 μMTBHQ (F andG) and 4min later CaCl2, final concentration 300 μM,was added to themedium.Changes in fura-2 fluorescenceweremonitored using the 340-nm/380-nm ratioand calibrated in terms of [Ca2+]c. Traces are representative of six independent experiments.


Fig. 7. Effect of incubation with anti-hTRPC1 antibody on PIP2-induced increase in Ca2+ entry. Fura-2-loaded human platelets were incubated for 30 min with15 μg/mL of anti-hTRPC1 antibody (C and D) or the vehicle (A and B), as indicated. Cells were treated in a Ca2+-free medium with PIP2 1,2-dioctanoyl (3 μM;B and D) or the vehicle (HBS; A and C) and then stimulated with thrombin (0.1 U/mL) followed by addition of CaCl2 (final concentration 300 μM) to initiate Ca2+

entry. Modifications in [Ca2+]c were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces are representative of five separateexperiments.


3.3. Inhibition of PIP2 resynthesis by lithium reduces SOCE

The role of PIP2 on SOCE in platelets was further inves-tigated by blocking its resynthesis by incubating the PRP withLiCl (10 mM) for 12 h. In cells preincubated with Li+, thrombin-evoked Ca2+ release was significantly reduced by 78±6%(Fig. 5B vs. A; n=6; pb0.001), which is likely due to bothinhibition of PIP2 resynthesis and IP3 production. We havepreviously shown that thrombin induces Ca2+ release from theDTS and the acidic stores by synthesis of the intracellularmessengers IP3 and NAADP, respectively [39]; therefore, Ca2+

accumulated in the acidic stores is expected to be releasable bythrombin independently of IP3 generation. To test thispossibility, the acidic stores in Li+-treated cells were depletedby treatment with 20 μM TBHQ prior to the stimulation withthrombin. As shown in Fig. 5C, treatment of Li+-pretreated cellswith TBHQ abolished thrombin-induced Ca2+ release, althoughthe subsequent addition of 1 μMTG plus 50 nM ionomycin wasable to release Ca2+ from the DTS (Fig. 5D). These findingsconfirm that Li+ treatment was essentially inhibiting PIP2 re-synthesis, inducing PIP2 depletion whilst leaving the Ca

2+ storesintact.

Pretreatment of platelets with 10mMLi+ significantly reducedthrombin-induced Ca2+ entry by 60±8% (Fig. 5B vs. 5A;pb0.05; n=6). Under these conditions SOCE induced by 10 nM

TG or 20 μM TBHQ was reduced by 67±6% and 61±5%,respectively (Fig. 5E vs. 5F; pb0.05; n=6). Pretreatment withLi+ has no significant effect onCa2+ release byTGor TBHQ fromthe intracellular stores, which further supports that PIP2 is notrequired for Ca2+ storage. Our results indicate that treatment ofplatelets with 3 μM PIP2 1,2-dioctanoyl overcame the effect ofLi+ on thrombin-induced Ca2+ entry (in the presence of PIP2 1,2-dioctanoyl thrombin-evoked Ca2+ entry in Li+-treated cells was116% of control; data not shown).

Poly-lysine, which contains positively charged residues, hasalso been used to inhibit the activity of PIP2-activated ionchannels [41,42]. As shown in Fig. 5G, incubation of plateletswith 10μg/mL poly-lysine significantly reduced thrombin-evokedCa2+ release and entry by 60% and 50%, respectively (pb0.05;n=5).

3.4. Electrotransjection of monoclonal anti-PIP2 antibodyattenuates SOCE

In order to decrease the amount of PIP2 available platelets werereversibly electroporated and incubated with monoclonal anti-PIP2 antibody following a previously published procedure [33].As shown in Fig. 6A, incubation of fixed, non-permeabilizedplatelets in suspension with 1 μg/mL anti-PIP2 antibody followedby detection using a FITC-conjugated secondary antibody

Fig. 8. Electrotransjection of anti-hTRPC6 antibody impairs PIP2-induced increase in Ca2+ entry. (A) Human platelets were lysed and whole-cell lysate was analysed

by Western blotting using either anti-hTRPC6 antibody (lane 1) or anti-hTRPC6 antibody preincubated with the control antigen peptide (CAP; lane 2) as described inMaterials and methods. The position of hTRPC6 is indicated on the left. Positions of molecular-mass markers are shown on the right. These results are representative offour independent experiments. (B–E) Human platelets (109 cells/mL) were electropermeabilized in a Gene Pulser as described in Materials and methods. Followingelectroporation, cells were incubated in the absence (B and C) or presence of 1 μg/mL anti-hTRPC6 antibody (D and E) for an additional 60 min at 37 °C, loaded withfura-2, centrifuged at 350×g for 20 min and resuspended in HBS containing 200 μMCaCl2. At the time of experiment 250 μMEGTAwas added. Platelets were treatedwith PIP2 1,2-dioctanoyl (3 μM; C and E) or the vehicle (HBS; B and D). Cells were then stimulated with thrombin (0.1 U/mL) followed by addition of CaCl2 (finalconcentration 300 μM) to initiate Ca2+ entry. (F and G) Cells were preincubated for 30 min with 10 μMN-(p-amylcinnamoyl)anthranilic acid (ACA) and then treatedin the absence (F) of presence of 3 μM PIP2 1,2-dioctanoyl (G). Cells were stimulated with thrombin (0.1 U/mL) followed by addition of CaCl2 (final concentration300 μM) to initiate Ca2+ entry. Modifications in [Ca2+]c were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces arerepresentative of five separate experiments.



revealed the interaction of the anti-PIP2 antibody with PIP2 in theplasma membrane (column d; n=4). The fluorescence observedwas not due to non-specific binding of the secondary antibody, asdemonstrated by the lower fluorescence detected in samplesincubated with this antibody alone (column c; n=4). The increasein fluorescence detected after incubation with the anti-PIP2antibody was almost completely prevented when cells werepreincubated with lithium to reduce PIP2 recycling, which con-firms specificity of the antibody.

Electrotransjection of cells with 1 μg/mL anti-PIP2 antibodysignificantly reduced thrombin-evoked Ca2+ release by 41±7%,which suggest that the antibody was effectively reducing theavailability of PIP2 to thrombin-activated PLC. In addition,electrotransjection of the anti-PIP2 antibody attenuated Ca

2+ entryevoked by the physiological agonist thrombin by 26±6%, as wellas SOCE-induced by selective depletion of the DTS or the acidicstores by using 10 nM TG or 20 μM TBHQ, respectively (in thepresence of the antibody SOCE-induced by TG or TBHQ was


reduced by 83±3% and 72±5%, respectively; Fig. 6B–G;pb0.05; n=6). Incubation of intact (non electroporated) plateletswith 1 μg/mL anti-PIP2 antibody was unable to reduce thrombin-evoked Ca2+ entry (data not shown), which indicates that theelectroporation procedure was effective introducing the antibodyinto the cells. As for Li+, electrotransjection of the anti-PIP2antibody did not significantly modify Ca2+ release induced by TGorTBHQ,which further confirms that PIP2 is not required forCa

2+

accumulation into the stores.

3.5. Incubation with anti-hTRPC1 antibody inhibits thrombin-induced Ca2+ influx but not the effect of PIP2

We have previously reported that preincubation of humanplatelets with an antibody directed towards the sequence 557–571located in the pore-forming region results in a concentration-dependent inhibition of Ca2+ and Mn2+ influx induced by TG(200 nM), as well as reduced thrombin-evoked Ca2+ entry [5].Here, we have investigated whether the effect of PIP2 could bemediated through the hTRPC1 protein by testing the effect of PIP21,2-dioctanoyl on Ca2+ entry in cells preincubated with the anti-hTRPC1 antibody. If this were the case, the enhancing effect ofPIP2 on thrombin-induced Ca2+ influx might be expected to beaffected by blockade of hTRPC1 channel function with theantibody. To assess this, human platelet suspensions wereincubated for 30 min with 15 μg/mL of anti-hTRPC1 antibodyfollowed by stimulation with 0.1 U/mL thrombin to activate Ca2+

entry. As shown in Fig. 7, incubation with the anti-hTRPC1antibody significantly reduced thrombin-evoked Ca2+ entry by47±7% (pb0.01; n=5). This effect was prevented when theantibody was incubated previously for 1 h in the presence of thecontrol antigen peptide (15 μg/mL; data not shown). Incubationwith the anti-hTRPC1 antibody did not significantly modify theability of thrombin to release Ca2+ from the intracellular stores(Fig. 7C vs. Fig. 7A). We have previously shown that incubationwith 15 μg/mL of a non-specific IgG was unable to block TG-induced SOCE [5], which shows the specificity of the assay.

In cells preincubated with the anti-hTRPC1 antibody, additionof 3 μM PIP2 1,2-dioctanoyl prior to thrombin stimulationenhanced Ca2+ entry relative to its control in the presence of theantibody by 132±7%. This effect was not found to be statisticallydifferent from the increase induced by 3 μM PIP2 1,2-dioctanoylin cells not treated with the anti-hTRPC1 antibody (137±10% asdescribed in the first section of results). These findings indicates

Fig. 9. Electrotransjection of anti-hTRPC6 antibody attenuates TG- and TBHQ-evoked catPulser as described in Materials and methods. Following electroporation, cells were incub60min at 37 °C, as indicated, loadedwith fura-2, centrifuged at 350×g for 20min and resuwas added. Plateletswere then stimulatedwith 10 nMTG(AandC) or 20μMTBHQ(Ban300 μM) 4 min later to initiate Ca2+ or Mn2+ entry, respectively. For Ca2+ mobilisation, mcalibrated in terms of [Ca2+]c. Mn2+ was added to untreated control cells (DMSO) or cells (Mn2+ entry, fura-2-fluorescence was measured with an excitation wavelength of 360electropermeabilized in aGene Pulser as described inMaterials andmethods. Following eleantibody for an additional 60min at 37 °C, as indicated, loadedwith fura-2, centrifuged at 3incubated for 30 min in the absence or presence of 15 μg/mL of anti-hTRPC1 antibody, astimulatedwith 10 nMTG (E), 20μMTBHQ(F) or 1μMTG+50 nM ionomycin (G) folloin [Ca2+]c were monitored as described above. Traces are representative of five separate e

that blockade of the hTRPC1 protein was without effect on PIP2-induced increase in Ca2+ entry.

3.6. Electrotransjection of anti-hTRPC6 antibody inhibitsthrombin-induced Ca2+ entry and the enhancing effect of PIP2

We tested for the presence of hTRPC6 in human platelet lysatesby SDS/PAGE and Western blotting using an anti-hTRPC6antibody, specific for an epitope located in the C-terminus ofhTRPC6. Immunoblotting of platelet whole-cell lysates with theanti-hTRPC6 antibody revealed the presence of hTRPC6 in thesecells (Fig. 8A, lane 1; n=4). Detection of hTRPC6 was abolishedwhen the anti-hTRPC6 antibody was previously incubated for 1 hwith a control antigen peptide, confirming the specificity of theantibody. These data support the reported presence of hTRPC6 inhuman platelets [17,43], as well as in the immature megakaryo-cytic Dami cell line and mature megakaryocytes [17]. In platelets,hTRPC6 has been shown to form heteromultimeric complexeswith hTRPC3 [18].

To investigate the regulation of hTRPC6 by PIP2 in plateletswe have induced functional knock down of hTRPC6 byelectrotransjection of cells with anti-hTRPC6 antibody, directedtowards an intracellular C-terminal sequence, following apreviously described procedure [31–33]. Here we show for thefirst time that electrotransjection with 1 μg/mL hTRPC6significantly reduced thrombin-evoked Ca2+ entry by 33±5%(Fig. 8D vs. Fig. 8B; pb0.05; n=5), without significantly alterCa2+ release (the integral of the rise in [Ca2+]c for 3 min after theaddition of thrombin in the presence of the anti-hTRPC6antibody was 106±7% of control; Fig. 8D vs. Fig. 8B).Electrotransjection with 1 μg/mL non-specific IgG following theprotocol used for the anti-hTRPC6 antibody was without effecton thrombin-induced Ca2+ mobilization, which shows thespecificity of the assay (data not shown). To assess whetherthe effect of PIP2 is mediated by regulation of the hTRPC6protein we explored the effect of PIP2 1,2-dioctanoyl on Ca2+

entry in cells electrotransjected with the anti-hTRPC6 antibody.As shown in Fig. 8, functional knock down of hTRPC6abolished the enhancing effect of PIP2 1,2-dioctanoyl (3 μM) onthrombin-evoked Ca2+ entry ( pb0.01; n=5). Thus, thrombin-induced Ca2+ entry in cells electrotransjected with the anti-hTRPC6 antibody was similar in the absence and presence of thePIP2 analogue (Fig. 8D and E). This effect was prevented whenthe antibody was incubated previously for 1 h in the presence of

ion entry. (A–D)Human platelets (109 cells/mL)were electropermeabilized in aGeneated in the absence or presence of 1 μg/mL anti-hTRPC6 antibody for an additionalspended in HBS containing 200 μMCaCl2. At the time of experiment 250 μMEGTAdD) followed by addition ofCaCl2 (A andB) orMnCl2 (C andD) (final concentrationodifications in [Ca2+]c were monitored using the 340/380 nm ratio and traces wereelectrotransjected or not with anti-hTRPC6 antibody) treated with TG or TBHQ. Fornm, the isoemissive wavelength. (E–G) Human platelets (109 cells/mL) were

ctroporation, cellswere incubated in the absence or presence of 1μg/mLanti-hTRPC650×g for 20min and resuspended in HBS containing 200 μMCaCl2. Cells were thens indicated. At the time of experiment 250 μMEGTAwas added. Platelets were thenwed by addition of CaCl2 (final concentration 300μM) to initiateCa2+ entry. Changesxperiments.

Fig. 10. Electrotransjection of anti-hTRPC6 antibody attenuates OAG-evokednon-capacitative entry. (A) Human platelets (109 cells/mL) were electropermea-bilized in a Gene Pulser as described in Materials and methods. Followingelectroporation, cells were incubated in the absence or presence of 1 μg/mL anti-hTRPC6 antibody for an additional 60 min at 37 °C, as indicated, loaded withfura-2, centrifuged at 350×g for 20 min and resuspended in HBS containing200 μM CaCl2. At the time of experiment 1 mM CaCl2 was added. Plateletswere treated with 100 μM OAG. (B) Fura-2-loaded human platelets wereincubated for 30 min with 15 μg/mL of anti-hTRPC1 antibody (anti-hTRPC1ab: OAG) or the vehicle (OAG and PIP2: OAG). Cells were treated in a mediumcontaining 1 mM Ca2+ with PIP2 1,2-dioctanoyl (3 μM; PIP2: OAG) or thevehicle (HBS; OAG and anti-hTRPC1 ab: OAG) and then stimulated with100 μM OAG. Changes in [Ca2+]c were monitored as described above. Tracesare representative of four separate experiments.


the control antigen peptide (1 μg/mL; data not shown). Thesefindings strongly suggest that the enhancing effect of PIP2 onCa2+ entry involves a positive regulation of hTRPC6 in humanplatelets. In support of this hypothesis we have also found thattreatment of platelets with N-(p-amylcinnamoyl)anthranilicacid, which has been shown to directly block hTRPC6 channels[44], significantly reduced thrombin-evoked Ca2+-entry by 30%(Fig. 8F; pb0.01; n=5) and abolished the enhanced effectinduced by PIP2 (Fig. 8G vs. Fig. 8C).

3.7. Electrotransjection of anti-hTRPC6 antibody reducesTG- and TBHQ-induced Ca2+ entry

We have further investigated the role of hTRPC6 in TG- andTBHQ-evoked Ca2+ entry in human platelets. As shown inFig. 9A and C, electrotransjection of cells with anti-hTRPC6antibody significantly reduced Ca2+ and Mn2+ entry induced bytreatment with 10 nM TG by 30±5% and 50±4% ( pb0.01;n=5). Similar results were observed when platelets werestimulated with 20 μM TBHQ. Electrotransjection of the anti-hTRPC6 antibody significantly attenuated TBHQ-evoked Ca2+

andMn2+ entry by 33±5% and 45±6% (Fig. 9B and D; pb0.01;n=5).

3.8. Relative roles of hTRPC1 and hTRPC6 in SOCE inducedby depletion of DTS or the acidic stores

We have investigated the relative roles of hTRPC6 andhTRPC1 in SOCE induced by 10 nM TG (to deplete the DTS),20 μM TBHQ (to discharge the acidic stores) or 1 μM TG incombination with 50 nM ionomycin (to induce extensivedepletion of both stores). As shown in Fig. 9E–G, electro-transjection of cells with anti-hTRPC6 antibody significantlyreduced Ca2+ entry induced by TG, TBHQ or TG+ionomycin by35±1%, 24±4% and 28±5%, respectively (pb0.01; n=5).Incubation with the anti-hTRPC1 antibody significantly attenu-ated Ca2+ entry induced by TG, TBHQ or TG+ ionomycin by42±3%, 25±4% and 38±4%, respectively ( pb0.01; n=5),while impairment of hTRPC1 and hTRPC6 channel functionsignificantly inhibited Ca2+ entry induced by TG, TBHQ or TG+ionomycin by 65±2%, 48 ±4% and 63±2%, respectively(pb0.01; n=5).

3.9. Electrotransjection of anti-hTRPC6 antibody attenuatesOAG-evoked non-capacitative Ca2+ entry

We have further investigated the role of hTRPC6 in non-capacitative Ca2+ entry induced by OAG, a DAG analogue.Electrotransjection of cells with anti-hTRPC6 antibody signif-icantly reduced Ca2+ entry induced by OAG by 70% (Fig. 10;pb0.05; n=4). In contrast, neither incubation with anti-TRPC1antibody nor treatment with PIP2 1,2-dioctanoyl altered OAG-evoked Ca2+ entry in human platelets (Fig. 10; n=4). Thesefindings suggest that hTRPC6, but not hTRPC1, is involved innon-capacitative Ca2+ entry induced by OAG. In addition, ourresults indicate that PIP2 does not modify non-capacitative Ca2+

entry supporting that the stimulation of Ca2+ influx by thrombin

or store depletion by TG or TBHQ induced by PIP2 was specificfor SOCE.

4. Discussion

The nature of the mechanisms underlying store-operated Ca2+

entry represents one of the fundamental and still unansweredquestions in cellular Ca2+ homeostasis. Among other messengersinvolved in the regulation of Ca2+ entry, PIP2 has been presentedas a candidate to positively or negatively modulate the function ofion channels, which require binding of PIP2 for activation. Apartfrom the Drosophila TRP and TRPL channels, recent evidencehas suggested that several members of the mammalian TRPchannels are regulated by PIP2. Among these channels TRPV1has been shown to be activated by PIP2 depletion [45]. Inaddition, TRPM family members are inactivated by PIP2hydrolysis or incubation with PIP2 antibodies, suggesting thatPIP2 depletion might be a signal for channel inactivation [13,46].The regulation of TRPC channels by PIP2 has also beensuggested. TRPC3 is retained in the plasma membrane through


a multimolecular complex involving PLC and PIP2 [9], whichsuggest that PIP2 and TRPC3 channels are involved in signalmicrodomains.

Using a water-soluble PIP2 analogue, PIP2 1,2-dioctanoyl, wehave found that PIP2 does not induce Ca

2+ mobilization in non-stimulated platelets nor increases OAG-induced non-capacitativeCa2+ entry. However, PIP2 enhances Ca

2+ entry stimulated by thephysiological agonist thrombin, which has been shown to induceCa2+ influx into human platelets secondarily to the emptying ofintracellular Ca2+ stores [47], as well as divalent cation entryinduced by depletion of the DTS or the acidic organelles, the twoCa2+ stores described in human platelets [38]. The effect inducedby PIP2 on Ca2+ entry is not due to inhibition of the ability ofplatelets to remove Ca2+ from the cytosol since it has a negligibleeffect, if any, in the restoration of [Ca2+]c to low resting levelswhen a rise in [Ca2+]c was induced by depletion of theintracellular Ca2+ stores using the SERCA inhibitor, TG, and alow concentration of ionomycin. Our results indicate that PIP2does not alter Ca2+ accumulation into the stores but reduces theability of thrombin to mobilise Ca2+ from the intracellular Ca2+

pools. This phenomenon might be attributed to the constitutiveinhibition of the IP3 receptors by direct interaction with PIP2.Studies in rat cerebellum have reported that IP3 receptors forms astable inhibitory complex with endogenous PIP2 so thatdisruption of IP3 receptor (IP3R)-PIP2 interaction by specificanti-PIP2 antibodies resulted in 3–4-fold increase in IP3R activityand apparent affinity for IP3. In support of this, exogenouslyadded PIP2 or the water-soluble analogue PIP2 1,2-dioctanoyl,block IP3 binding to IP3R and inhibits IP3R activity [48]. Thisinteraction explains the inhibitory effect of PIP2 1,2-dioctanoyl onthrombin-induced release of Ca2+ from the intracellular stores.Addition of exogenous PIP2 under our conditions might attenuatePIP2 depletion once PLC has been activated by thrombin, and anumber of IP3 receptors might still be blocked by PIP2.

To further support the involvement of PIP2 in Ca2+ entry inplatelets we investigated the effect decreasing the levels of PIP2either by PIP2 resynthesis blockade, using Li+, a potent inhibitorof the inositol monophosphatase that hydrolyses IP to inositol[49], by introducing a monoclonal anti-PIP2 antibody, a procedurethat has been widely used to analyse the functional significance ofPIP2 [50–53] or by screening the PIP2 negatively charged headgroups using poly-lysine. As expected, these experimental pro-cedures reduce the ability of thrombin to mobilise Ca2+ from theintracellular stores, although they had no effect on Ca2+ storage bythe cells. In addition, these assays reduced Ca2+ entry induced bythrombin or selective depletion of the DTS or the acidic stores.Previous studies by us showing the inhibitory effect of Li+ onSOCE were interpreted as a requirement of SOCE for basal levelsof IP3 to bind the IP3 receptors [16]. Now these results might beinterpreted as evidence for the requirement of PIP2 for the re-gulation of cation channel function. In fact, xestospongin C, an IP3receptor antagonist that blocks IP3-induced Ca2+ release withoutinteracting with the IP3-binding site [54], was able to inhibit TG-induced SOCE in platelets [16], which suggest a role for the IP3receptor but not for IP3 binding in the activation of SOCE.

We have previously shown that hTRPC1 is involved in theconduction of cation entry during the activation of SOCE in

human platelets [5,55]. In addition, here we provide evidencefor the functional relevance of hTRPC6 in Ca2+ and Mn2+ entryinduced by thrombin or depletion of both Ca2+ stores, the DTSand the acidic stores. Using specific antibodies for hTRPC1 and6 proteins we have found that both proteins might be involvedin the formation of independent cation channels, since treatmentwith these antibodies showed additive inhibitory effects on Ca2+

entry. Impairment of hTRPC6 but not of hTRPC1 functionprevented PIP2-induced increase in Ca2+ entry, thereforesuggesting that PIP2 positively regulates hTRPC6.

PIP2 has also been shown to regulate TRPC6 in neurones,although, in this case, the channel is activated by hydrolysis ofPIP2 [15]. The regulation of TRP channels by PIP2 might have aremarkable effect in cellular Ca2+ homeostasis through the for-mation ofmolecular complexes in signalmicrodomains involvingdifferent proteins, such as PLC,which upon activation by receptoroccupation would decrease the level of PIP2, therefore activatingchannels that are negatively regulated and inactivating channelsthat are positively regulated by PIP2.

Taken together, our data and those of others suggest thathTRPC6 might play a dual role in human platelets, as a non-capacitative Ca2+ entry channel, regulated by DAG (or theanalogue OAG) as previously reported in platelets [43] and othercells [56] and also as a component of the capacitative or store-operated Ca2+ channels, which is likely regulated by cellular PIP2levels. The latter is supported by several findings: (1) The lack ofeffect of PIP2 per se on Ca

2+ mobilization both in the absence andpresence of extracellular Ca2+, which suggests that PIP2 does notinduce non-capacitative Ca2+ entry. (2) PIP2 induces stimulationof Ca2+ entry mediated by the physiological agonist thrombin orby selective depletion of the dense tubular system by 10 nM TGand the acidic stores, using 20μMTBHQ.Considering the lack ofnon-capacitative Ca2+ entry induced by PIP2 this effect isexpected to be mediated through the regulation of SOCE. Wecannot rule out the possibility that the increase in [Ca2+]c inducedby these agonists, specially thrombin, leads to the secretion ofplatelet agonists that, in turn, might activate non-capacitativeCa2+

entry, although we have recently reported that the rise in [Ca2+]cinduced by 10 nMTGor 20μMTBHQ is unable per se to activateplatelet functions, such as aggregation, which would be expectedto be activated upon platelet agonist secretion [57]. In fact, rises in[Ca2+]c induced by TG reaching over 400 μM, higher than thoseinduced by 10 nMTGeven in the presence of extracellularCa2+ inour experiments, have been shown to be unable to activate plateletATP secretion [58]. (3) Stimulation of thrombin-, TG-, andTBHQ-evoked divalent cation entry by PIP2 is impaired byelectrotransjection of anti-hTRPC6 antibody, which suggests arole for hTRPC6 in PIP2-mediated effect, and (4) electrotransjec-tion of anti-hTRPC6 antibody impaired divalent cation entryinduced by TG and TBHQ, which suggest a role for hTRPC6 inSOCE.

As reported for other TRP proteins [12,14], PIP2 mightregulate hTRPC6 channel function by interaction with the C-terminal domain, which is the region where the epitope recog-nised by the anti-hTRPC6 antibody used is located. Differentmechanisms have been proposed to account for the potentiationof TRP channel activity by PIP2, including that PIP2 interaction


with TRP channels might lead to stabilisation of the latter inan open channel configuration. In addition, PIP2 might disruptthe interaction between the channel and an inhibitorymolecule [59].

We hypothesize that activation of PLC-coupled receptorsplays a dual effect on hTRPC6 channel activity. First of all, PLCactivation leads to IP3 production, which releases Ca2+ fromintracellular stores, leading to the activation of SOCE throughhTRPC6 and other channels, a process that is positively reg-ulated by PIP2. On the other hand, PLC-dependent hydrolysis ofPIP2 evokes desensitization of hTRPC6, thereby limiting itsactivity as described for other PIP2-regulated TRP channels[14]. PIP2 might therefore be considered a useful tool to ex-amine the role of TRPC6 in these cells. Although speculative,the transient activation of hTRPC6 might be involved in theinitial steps of activation of SOCE in platelets, where twofunctionally early and late phases have been reported [60].

Acknowledgments

This work was supported by Junta de Extremadura-Consejeríade Sanidad y Consumo (SCSS0619) and MECGrant (BFU2007-60104). I.J. was supported by a Valhondo Calaf Foundation. P.C.R. was supported by Junta de Extremadura-Consejería deInfraestructura y Desarrollo Tecnológico (POS05A003). Wethank Mercedes Gómez Blázquez for her technical assistance.

References

[1] R.C. Hardie, TRP channels and lipids: from Drosophila to mammalianphysiology, J. Physiol. 578 (2007) 9–24.

[2] N. Hellwig, N. Albrecht, C. Harteneck, G. Schultz, M. Schaefer, Homo-and heteromeric assembly of TRPV channel subunits, J. Cell Sci. 118(2005) 917–928.

[3] P.D. Wes, J. Chevesich, A. Jeromin, C. Rosenberg, G. Stetten, C. Montell,TRPC1, a human homolog of a Drosophila store-operated channel, Proc.Natl. Acad. Sci. U. S. A. 92 (1995) 9652–9656.

[4] L. Birnbaumer, TRPC4 knockout mice: the coming of age of TRP channelsas gates of calcium entry responsible for cellular responses, Circ. Res. 91(2002) 1–3.

[5] J.A. Rosado, S.L. Brownlow, S.O. Sage, Endogenously expressed Trp1 isinvolved in store-mediated Ca2+ entry by conformational coupling inhuman platelets, J. Biol. Chem. 277 (2002) 42157–42163.

[6] B.B. Singh, X. Liu, J. Tang, M.X. Zhu, I.S. Ambudkar, Calmodulinregulates Ca2+-dependent feedback inhibition of store-operated Ca2+ influxby interaction with a site in the C terminus of TrpC1, Mol. Cell 9 (2002)739–750.

[7] F. Zeng, S.Z. Xu, P.K. Jackson, D. McHugh, B. Kumar, S.J. Fountain, D.J.Beech, Human TRPC5 channel activated by a multiplicity of signals in asingle cell, J. Physiol. 559 (2004) 739–750.

[8] G. Vazquez, G.S. Bird, Y. Mori, J.W. Putney Jr., Native TRPC7 channelactivation by an inositol trisphosphate receptor-dependent mechanism,J. Biol. Chem. 281 (2006) 25250–25258.

[9] J. Soboloff,M. Spassova, T. Hewavitharana, L.P. He, P. Luncsford,W.Xu, K.Venkatachalam, D. van Rossum, R.L. Patterson, D.L. Gill, TRPC channels:integrators of multiple cellular signals, Handb. Exp. Pharmacol. 179 (2007)575–591.

[10] T. Gudermann, T. Hofmann, M. Mederos y Schnitzler, A. Dietrich,Activation, subunit composition and physiological relevance of DAG-sensitive TRPC proteins, Novartis Found. Symp. 258 (2004) 103–118.

[11] F. Qin, Regulation of TRP ion channels by phosphatidylinositol-4,5-bisphosphate, Handb. Exp. Pharmacol. 179 (2007) 509–525.

[12] E.D. Prescott, D. Julius, A modular PIP2 binding site as a determinant ofcapsaicin receptor sensitivity, Science 300 (2003) 1284–1288.

[13] L.W. Runnels, L. Yue, D.E. Clapham, The TRPM7 channel is inactivatedby PIP2 hydrolysis, Nat. Cell Biol. 4 (2002) 329–336.

[14] B. Nilius, F. Mahieu, J. Prenen, A. Janssens, G. Owsianik, R. Vennekens,T. Voets, The Ca2+-activated cation channel TRPM4 is regulated byphosphatidylinositol 4,5-biphosphate, EMBO J. 25 (2006) 467–478.

[15] P. Delmas, Assembly and gating of TRPC channels in signallingmicrodomains, Novartis Found. Symp. 258 (2004) 75–89.

[16] J.A. Rosado, S.O. Sage, Coupling between inositol 1,4,5-trisphosphatereceptors and human transient receptor potential channel 1 whenintracellular Ca2+ stores are depleted, Biochem. J. 350 (2000) 631–635.

[17] E. den Dekker, D.G.Molin, G. Breikers, R. van Oerle, J.W. Akkerman, G.J.van Eys, J.W. Heemskerk, Expression of transient receptor potential mRNAisoforms and Ca2+ influx in differentiating human stem cells and platelets,Biochim. Biophys. Acta 1539 (2001) 243–255.

[18] S.L. Brownlow, S.O. Sage, Transient receptor potential protein subunitassembly and membrane distribution in human platelets, Thromb.Haemost. 94 (2005) 839–845.

[19] S.Z. Xu, D. Beech, TrpC1 is a membrane-spanning subunit of store-operated Ca2+ channels in native vascular smooth muscle cells, Circ. Res.88 (2001) 84–87.

[20] D. Krizaj, Compartmentalization of calcium entry pathways in mouse rods,Eur. J. Neurosci. 22 (2005) 3292–3296.

[21] S. Thebault, A. Zholos, A. Enfissi, C. Slomianny, E. Dewailly, M.Roudbaraki, J. Parys, N. Prevarskaya, Receptor-operated Ca2+ entrymediated by TRPC3/TRPC6 proteins in rat prostate smooth muscle (PS1)cell line, J. Cell. Physiol. 204 (2005) 320–328.

[22] J.A. Rosado, S.O. Sage, Phosphoinositides are required for store-mediatedcalcium entry in human platelets, J. Biol. Chem. 275 (2000) 9110–9113.

[23] J.A. Rosado, T. Porras, M. Conde, S.O. Sage, Cyclic nucleotides modulatestore-mediated calcium entry through the activation of protein-tyrosinephosphatases and altered actin polymerization in human platelets, J. Biol.Chem. 276 (2001) 15666–15675.

[24] G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2+ indicatorswith greatly improved fluorescence properties, J. Biol. Chem. 260 (1985)3440–3450.

[25] S.O. Sage, J.E. Merritt, T.J. Hallam, T.J. Rink, Receptor-mediated calciumentry in fura-2-loaded human platelets stimulated with ADP and thrombin.Dual-wavelengths studies with Mn2+, Biochem. J. 258 (1989) 923–926.

[26] J.A. Rosado, S. Jenner, S.O. Sage, A role for the actin cytoskeleton in theinitiation and maintenance of store-mediated calcium entry in humanplatelets. Evidence for conformational coupling, J. Biol. Chem. 275 (2000)7527–7533.

[27] J.W. Heemskerk, M.A. Feijge, L. Henneman, J. Rosing, H.C. Hemker, TheCa2+-mobilizing potency of alpha-thrombin and thrombin-receptor-activating peptide on human platelets—concentration and time effects ofthrombin-induced Ca2+ signaling, Eur. J. Biochem. 249 (1997) 547–555.

[28] J.A. Rosado, S.O. Sage, Farnesylcysteine analogues inhibit store-regulatedCa2+ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton, Biochem. J. 347 (2000) 183–192.

[29] N. Ben-Amor, P.C. Redondo, A. Bartegi, J.A. Pariente, G.M. Salido, J.A.Rosado, A role for 5,6-epoxyeicosatrienoic acid in calcium entry by denovo conformational coupling in human platelets, J. Physiol. 570 (2006)309–323.

[30] J.A. Rosado, S.O. Sage, Regulation of plasma membrane Ca2+-ATPase bysmall GTPases and phosphoinositides in human platelets, J. Biol. Chem.275 (2000) 19529–19535.

[31] A. Dhar, S.D. Shukla, Electrotransjection of pp60v-src monoclonalantibody inhibits activation of phospholipase C in platelets. A newmechanism for platelet-activating factor responses, J. Biol. Chem. 269(1994) 9123–9127.

[32] M.C. Scrutton, D.E. Knight, K.S. Authi, Preparation and uses ofsemipermeabilized platelets, in: S.P. Watson, K.S. Authi (Eds.), Platelets,The Practical Approach Series, IRL Press, Oxford, 1996, pp. 47–66.

[33] J.J. Lopez, G.M. Salido, J.A. Pariente, J.A. Rosado, Interaction of STIM1with endogenously expressed human canonical TRP1 upon depletion ofintracellular Ca2+ stores, J. Biol. Chem. 281 (2006) 28254–28264.


[34] J.A. Rosado, J.J. Lopez, A.G. Harper, M.T. Harper, P.C. Redondo, J.A.Pariente, S.O. Sage, G.M. Salido, Two pathways for store-mediatedcalcium entry differentially dependent on the actin cytoskeleton in humanplatelets, J. Biol. Chem. 279 (2004) 29231–29235.

[35] B. Papp, A. Enyedi, K. Paszty, T. Kovacs, B. Sarkadi, G. Gardos, C.Magnier, F. Wuytack, J. Enouf, Simultaneous presence of two distinctendoplasmic-reticulum-type calcium-pump isoforms in human cells.Characterization by radio-immunoblotting and inhibition by 2,5-di-(t-butyl)-1,4-benzohydroquinone, Biochem. J. 288 (1992) 297–302.

[36] R. Bobe, R. Bredoux, F. Wuytack, R. Quarck, T. Kovacs, B. Papp, E.Corvazier, C. Magnier, J. Enouf, The rat platelet 97-kDa Ca2+ ATPaseisoform is the sarcoendoplasmic reticulum Ca2+ ATPase 3 protein, J. Biol.Chem. 269 (1994) 1417–1424.

[37] L. Cavallini, M. Coassin, A. Alexandre, Two classes of agonist-sensitiveCa2+ stores in platelets, as identified by their differential sensitivity to 2,5-di-(tert-butyl)-1,4-benzohydroquinone and thapsigargin, Biochem. J. 310(1995) 449–452.

[38] J.J. Lopez, C. Camello-Almaraz, J.A. Pariente, G.M. Salido, J.A. Rosado,Ca2+ accumulation into acidic organelles mediated by Ca2+- and vacuolarH+-ATPases in human platelets, Biochem. J. 390 (2005) 243–252.

[39] J.J. Lopez, P.C. Redondo, J.A. Pariente, G.M. Salido, J.A. Rosado, Twodistinct Ca2+ compartments show differential sensitivity to thrombin, ADPand vasopressin in human platelets, Cell. Signal. 18 (2005) 373–381.

[40] T.J. Rink, S.O. Sage, Stimulated calcium efflux from fura-2-loaded humanplatelets, J. Physiol. 393 (1987) 513–524.

[41] S.L. Shyng, C.G. Nichols, Membrane phospholipid control of nucleotidesensitivity of KATP channels, Science 282 (1998) 1138–1141.

[42] D. Oliver, C.C. Lien, M. Soom, T. Baukrowitz, P. Jonas, B. Fakler,Functional conversion between A-type and delayed rectifier K+ channelsby membrane lipids, Science 304 (2004) 265–270.

[43] S.R. Hassock,M.X. Zhu, C. Trost, V. Flockerzi, K.S. Authi, Expression androle of TRPC proteins in human platelets: evidence that TRPC6 forms thestore-independent calcium entry channel, Blood 100 (2002) 2801–2811.

[44] C. Harteneck, H. Frenzel, R. Kraft, N-(p-amylcinnamoyl)anthranilic acid(ACA): a phospholipase A(2) inhibitor and TRP channel blocker, Cardiovasc.Drug Rev. 25 (2007) 61–75.

[45] H.H. Chuang, E.D. Prescott, H. Kong, S. Shields, S.E. Jordt, A.I. Basbaum,M.V. Chao, D. Julius, Bradykinin and nerve growth factor release thecapsaicin receptor from PtdIns(4,5)P2-mediated inhibition, Nature 411(2001) 957–962.

[46] R.C. Hardie, Regulation of TRP channels via lipid second messengers,Annu. Rev. Physiol. 65 (2003) 735–759.

[47] M.T. Alonso, J. Alvarez, M. Montero, A. Sanchez, J. Garcia-Sancho,Agonist-induced Ca2+ influx into human platelets is secondary to theemptying of intracellular Ca2+ stores, Biochem. J. 280 (1991) 783–789.

[48] V.D. Lupu, E. Kaznacheyeva, U.M. Krishna, J.R. Falck, I. Bezprozvanny,Functional coupling of phosphatidylinositol 4,5-bisphosphate to inositol1,4,5-trisphosphate receptor, J. Biol. Chem. 273 (1998) 14067–14070.

[49] M.J. Berridge, Unlocking the secrets of cell signaling, Annu. Rev. Physiol.67 (2005) 1–21.

[50] M. Takahashi, S. Toyoshima, A. Miyazawa, T. Horikoshi, T. Yoshioka,Regulation of c-MYC protein expression in the developing rat cerebellum byphosphoinositide turnover, Biochem. Biophys. Res. Commun. 197 (1993)278–286.

[51] D. Tran, P. Gascard, B. Berthon, K. Fukami, T. Takenawa, F. Giraud, M.Claret, Cellular distribution of polyphosphoinositides in rat hepatocytes,Cell. Signal. 5 (1993) 565–581.

[52] I.H. Ho, R.D.Murrell-Lagnado,Molecular mechanism for sodium-dependentactivation of G protein-gated K+ channels, J. Physiol. 520 (1999) 645–651.

[53] E. Kaznacheyeva, A. Zubov, K. Gusev, I. Bezprozvanny, G.N. Mozhayeva,Activation of calcium entry in human carcinoma A431 cells by storedepletion and phospholipase C-dependent mechanisms converge on ICRAC-like calcium channels, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 148–153.

[54] J. Gafni, J.A. Munsch, T.H. Lam, M.C. Catlin, L.G. Costa, T.F. Molinski, I.N.Pessah, Xestospongins: potent membrane permeable blockers of the inositol1,4,5-trisphosphate receptor, Neuron 19 (1997) 723–733.

[55] J.A. Rosado, P.C. Redondo, S.O. Sage, J.A. Pariente, G.M. Salido, Store-operated Ca2+ entry: Vesicle fusion or reversible trafficking and de novoconformational coupling? J. Cell. Physiol. 205 (2005) 262–269.

[56] P.C. Leung, K.T. Cheng, C. Liu, W.T. Cheung, H.Y. Kwan, K.L. Lau, Y.Huang, X. Yao, Mechanism of non-capacitative Ca2+ influx in responseto bradykinin in vascular endothelial cells, J. Vasc. Res. 43 (2006)367–376.

[57] I. Jardin, N. Ben Amor, J.M. Hernandez-Cruz, G.M. Salido, J.A. Rosado,Involvement of SNARE proteins in thrombin-induced platelet aggregation:evidence for the relevance of Ca2+ entry, Arch. Biochem. Biophys. (2007),doi:10.1016/j.abb.2007.04.038.

[58] L. Turetta, E. Bazzan, K. Bertagno, E. Musacchio, R. Deana, Role of Ca2+

and protein kinase C in the serotonin (5-HT) transport in human platelets,Cell Calcium 31 (2002) 235–244.

[59] T. Voets, B. Nilius, Modulation of TRPs by PIPs, J. Physiol. 582 (2007)939–944.

[60] S. Jenner, S.O. Sage, Two pathways for store-mediated calcium entry inhuman platelets, Platelets 11 (2000) 215–221.

Documents

Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets