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(2008) 337–346www.elsevier.com/locate/cellsig

Cellular Signalling 20

SERCA2b and 3 play a regulatory role in store-operated calcium entryin human platelets

Pedro C. Redondo b,⁎, Ginés M. Salido a, José A. Pariente a, Stewart O. Sage b, Juan A. Rosado a

a Department of Physiology of University of Extremadura, Cáceres, Spainb Department of Physiology, Development and Neuroscience of University of Cambridge, Cambridge, CB3 9ET, United Kingdom

Received 10 September 2007; accepted 18 October 2007Available online 26 October 2007

Abstract

Two agonist-releasable Ca2+stores have been identified in human platelets differentiated by the distinct sensitivity of their SERCA isoforms tothapsigargin (TG) and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Here we have examined whether the SERCA isotypes might be involved instore-operated Ca2+entry (SOCE) activated by the physiological agonist thrombin in human platelets. Ca2+-influx evoked by thrombin (0.01 U/mL)reached a maximum after 3 min, which was consistent with the decrease in the Ca2+content in the stores; afterwards, the extent of SOCE decreasedwith no correlation with the accumulation of Ca2+in the stores. Inhibition of SERCA2b, by 10 nM TG, and SERCA3, with 20 μM TBHQ,individually or simultaneously, accelerated Ca2+ store discharge and subsequently enhanced the extent of SOCE stimulated by thrombin. In addition,TG and TBHQ modified the time course of thrombin-evoked SOCE from a transient to a sustained increase in Ca2+ influx, which reveals a negativerole for SERCAs in the regulation of SOCE. This effect was consistent under conditions that inhibit Ca2+ extrusion by PMCA or the Na+/Ca2+

exchanger. Coimmunoprecipitation experiments revealed that thrombin stimulates direct interaction between SERCA2b and 3 with the hTRPC1channel, an effect that was found to be independent of SERCA activity. In summary, our results suggest that SERCA2b and 3 modulate thrombin-stimulated SOCE probably by direct interaction with the hTRPC1 channel in human platelets.© 2007 Elsevier Inc. All rights reserved.

Keywords: SERCAs; SOCE; Human platelet; 2,5-di-(tert-butyl)-1,4-hydroquinone; Thapsigargin

1. Introduction

Activation of store operated Ca2+ entry (SOCE) is a majormechanism for Ca2+ entry in non-excitable cells upon stimula-tion [1–11]. In human platelets, SOCE has been reported toconsist of two components: an early stage involving TRPC6channel gating [12] and the Na+/Ca2+ exchanger working inreverse mode [13], and a late stage involving a conformationalcoupling between the type II IP3 receptor (IP3RII), located in theintracellular stores, and the canonical transient receptor potentialchannel 1 (TRPC1) in the plasma membrane [4,5,12,14–19].Several mechanisms down-regulate SOCE in different cell

⁎ Corresponding author. Department of Physiology, Development andNeuroscience, University of Cambridge, Downing Site CB2 3EG, Cambridge,United Kingdom. Tel.: +44 1223 333870; fax: +44 1223 333840.

E-mail address: pcr@unex.es (P.C. Redondo).

0898-6568/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.cellsig.2007.10.019

types, including Ca2+ reuptake by the sarco-endoplasmic retic-ulum Ca2+-ATPases (SERCAs), which are responsible for storerefilling that, in turn, results in the termination of SOCE perhapsby a mechanism involving intraluminal calcium sensors, such asSTIM1 [20–24]. In addition, other mechanisms have beensuggested to be involved in the regulation of SOCE, includingcalmodulin, which may bind TRPC channels directly [25–29] oract via CaMKII to regulate TRPC channel gating [30–32]. OtherTRP channels, such as TRPV1, or the unrelated calcium chan-nels, KCNQ1 and Ca(v)1.2, have also been shown to be reg-ulated by calmodulin [32].

Inhibition of SERCAs by incubating cells with thapsigargin(TG) or 2,5 di-(tertbutyl)-1,4-hydroquinone (TBHQ) has beencommonly used as a strategy to activate SOCE in several celltypes. SERCAs continuously pump Ca2+ into the intracellularstores and their inhibition results in store depletion by passiveleakage, through themembrane, to the cytosol. In human platelets,two different SERCA isoforms, withmolecular masses of 100 and

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97 kDa, are expressed in two distinct Ca2+ stores [33–39]. The100 kDa isoform has been identified as SERCA 2b, and isinhibited by low concentrations of TG [36,37]. In contrast, the97 kDa isoform, identified as SERCA 3, is inhibited only byhigh concentrations of TG [34–37]. These SERCA isoformscan also be differentiated by the distinct sensitivity to TBHQ,so that SERCA 3, but not SERCA 2b, is sensitive to TBHQ[33,36,37].

Here we have investigated the time course of thrombin-induced Ca2+ entry in human platelets and the regulatory role oftwo different SERCA isoforms expressed in these cells. Ourresults indicate that, in addition to the inhibitory effect ofSERCA on SOCE by store refilling, both SERCA2b and 3negatively regulate thrombin-evoked Ca2+ entry by a mechan-ism that might involve interaction with hTRPC1 channels inthese cells.

2. Materials and methods

2.1. Materials

Fura-2 acetoxymethyl ester (fura-2/AM) from Texas Fluorescence (AustinTexas, U.S.A.). Apyrase (grade VII), aspirin, bovine serum albumin, ionomycin(Iono), dithiothreitol, thapsigargin (TG), thrombin (Thr), 2,5 di-(tertbutyl)-1,4-hydroquinone (TBHQ), sodium dodecyl sulphate (SDS), ionic detergent tween20, Na3VO4, N-methyl-D-glucamine hydrochloride (NMDG) and ECL reagentswere fromSigma (Poole, Dorset, UnitedKingdom). Anti-SERCA3 and SERCA2polyclonal antibodies were from Abcam (Cambridge, UK). Anti-IP3R type IIantibody was from Santa Cruz (Santa Cruz, CA, U.S.A.). Horseradish peroxide-conjugated donkey anti-rabbit IgG, horseradish peroxidase-conjugated sheepanti-mouse IgG and protein A-agarose were from Upstate Biotechnology (LakePlacid, NY, U.S.A.). Anti-hTRPC1 antibody was from Alomone Laboratories(Jerusalem, Israel). Wide range molecular weight markers were from AmershamPharmacia (Little Chalfont, Buckinghamshire, UK). All other reagents were ofanalytical grade.

2.2. Platelet preparation

Fura-2-loaded platelets were prepared as described previously [14] andapproved by Local Ethical Committees. Briefly, blood was obtained from drug-free healthy volunteers and mixed with one-sixth volume of acid/citrate dextroseanticoagulant containing (in mM): 85 sodium citrate, 78 citric acid and 111 D-glucose. Platelet-rich plasma was then prepared by centrifugation for 5 min at700 g and aspirin (100 μM) and apyrase (40 μg/ml) added. Platelet-rich plasmawas incubated at 37 °C with 2 μM fura-2/AM for 45 min. Cells were thencollected by centrifugation at 350 g for 20 min and resuspended in HEPES-buffered saline (HBS) containing (in mM): 145 NaCl, 10 HEPES, 10 D-glucose,5 KCl, 1 MgSO4, pH 7.45 and supplemented with 0.1% w/v bovine serumalbumin and 40 μg/ml apyrase. In some experiments 145 mM NMDG replacedNaCl to block the Na+/Ca2+exchanger working in forward mode [13].

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

Fluorescence was recorded from 2 mL aliquots of magnetically stirred cellsuspensions (108 cells/mL) at 37 °C using a fluorescence spectrophotometer withexcitation wavelengths of 340 and 380 nm and emission at 505 nm. Changes in [Ca2+]c were monitored using the fura-2340/380 fluorescence ratio and calibratedaccording to the method of Grynkiewicz and coworkers [40]. Ca2+release, Ca2+remaining in the stores (ascertained by adding TG+Iono to stimulated cells) andCa2+entry were estimated using the integral of the rise in [Ca2+]c for three min afterthe addition of the agonist or CaCl2, respectively, taking a sample every second[14,41]. In a number of experiments, Sr2+was used to monitor divalent cation entry.This was done to avoid complications arising from the stimulation of the plateletplasma membrane Ca2+-ATPase (PMCA) by the compounds used, since Sr2+is

transported with lower affinity than Ca2+by this Ca2+-ATPase [42]. Sr2+entry wasmeasured in a Ca2+-free HBS containing EGTA (100 μM) to minimize the effectsof contaminating Ca2+. Cytosolic Sr2+was monitored using the fura-2340/380 nmfluorescence ratio. Agonist-evoked Sr2+entry was calculated using the integral ofthe rise in the 340/380 nm fluorescence ratio for three min after addition of SrCl2.

2.4. Immunoprecipitation and Western blotting

Aliquots of platelet suspension (250 μL; 4×108 cell/mL) were lysed with anequal volume of lysis buffer, pH 7.2, containing 316 mMNaCl, 20 mM Tris, 2 mMEGTA, 0.2% SDS, 2% sodium deoxycholate, 2% Triton X-100, 2 mM Na3VO4,2mMphenylmethylsulfonyl fluoride, 100μg/ml leupeptin, and 10mMbenzamidine[43]. Aliquots (0.5 mL) were then immunoprecipitated by incubation 1.5 μg/mL ofwell anti-IP3R type II antibody or of anti-SERCA 3 antibody or anti-SERCA 2antibody andproteinA-agarose overnight at 4 °C. Immunoprecipitateswere resolvedby 8% SDS-PAGE, and separated proteins were transferred onto nitrocellulosemembranes. Immunodetection of hTRPC1 was achieved using the anti-hTRPC1antibody diluted 1:200 in TBST for 2 h. To detect the primary antibody, membraneswere incubated with horseradish peroxidase-conjugated donkey anti-goat IgGantibody diluted 1:5000 in TBSTsupplemented with 10% BSA for 1 h. Membraneswere then exposed to enhanced chemiluminescence (ECL) reagents for 5 min. Blotswere then exposed to a photographic film. Membrane reprobing for protein loadingcontrol was performed by removing bound antibodies by incubation for 30 min at50 °C with stripping buffer containing 100 mM 2-mercaptoethanol, 65.5 mM Tris,and 2% SDS, pH 6.7, followed byWestern blotting with the appropriate antibodies.Quantification of coupling between IP3RII or SERCA 2 or SERCA 3, with TRPC1was done by using scanning optical densitometry.

2.5. Statistical analysis

Data are expressed as mean±S.E.M. Analysis of statistical significance wasperformed using Student's unpaired t-test. For multiple comparisons, one-wayanalysis of variance combined with the Newman–Keuls tests was used. Pb0.05was considered significant.

3. Results

3.1. Time course of Ca2+release and entry induced by thrombinin human platelets

Fura 2-loaded platelets, suspended in a Ca2+-free medium(100 μM EGTA was added), were stimulated with a low con-centration of thrombin (0.01 U/mL), which does not activate non-capacitative Ca2+entry in platelets [44]. Thrombin transientlyincreased [Ca2+]c reaching an initial peak [Ca2+]c elevation of102.5±5.8 nM (Fig. 1, trace a). As shown in Fig. 1A–D: trace band in Fig. 1E: traces b, c and d, 300μMofCaCl2was added to theextracellular medium to evaluate thrombin-evoked SOCE at 1, 2,3, 5, 10, 15 and 18min after the addition of the agonist. Our resultsrevealed that the extent of SOCE reached a maximum 3 min afterstimulation with thrombin, afterwards SOCE decreased andreached a plateau 10 min after thrombin addition that wassustained for at least for a further 10 min (Fig. 1E traces b, c and dand Fig. 1F, black trace; ⁎Pb0.05 and ⁎⁎Pb0.01, n=6–8).

It is widely accepted that the Ca2+ content of the intracellularstores regulates the activation of SOCE [1–10]. Hence, weevaluated the amount of Ca2+ remaining in the stores at the sametimes that SOCEwas quantified above, by addition of TG (1 μM)plus Iono (50 nM), which induces extensive depletion of theintracellular Ca2+ pools in platelets [14], after stimulation withthrombin. As shown in Fig. 1F, grey trace, platelet stimulationwith thrombin induced an initial decrease in the amount of Ca2+

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stored in the intracellular compartments reaching aminimum after10 min, followed by a gradual increase. The stores were not fullyreplenished perhaps due to the presence of thrombin in themedium. Comparing the extent of SOCE and the amount of Ca2+

stored shows that although store depletion initiates SOCE, after3min there is not a direct relationship between the Ca2+ content inthe stores and the extent of SOCE, which must be regulated byother mechanisms.

Fig. 1. Time course of Ca2+ release and SOCE induced by thrombin. Fura-2 loaded hmL thrombin and CaCl2 (300 μM) was then added to the extracellular medium 1 min18 min (E, traces b, c and d) later to initiate Ca2+entry. In parallel experiments, a comthe Ca2+ content in the intracellular stores at the same times as above. Curves A-the Ca2+entry (black curve) or the Ca2+content of the stores (grey curve) estimate⁎Pb0.05 and ⁎⁎Pb0.01.

3.2. SERCA2b and 3 modulate SOCE induced by thrombin inhuman platelets

Platelets have at least two separate agonist-releasableCa2+storeswhich express different SERCA isoformswith distinct sensitivitiesto TG and TBHQ [37,38]. The major store, the dense tubularsystem (DTS) expresses SERCA2b, which is inhibited by lowconcentrations of TG and is insensitive to TBHQ, while the acidic

uman platelets suspended in a Ca2+-free medium were stimulated with 0.01 U/(A, trace b), 2 min (B, trace b), 3 min (C, trace b), 5 min (D, trace b), 10, 15 andbination of TG (1 μM)+Iono (50 nM) was added, instead of CaCl2, to evaluateE are representative of four to six independent experiments. F, data representd as described in the Materials and Methods and expressed as mean±S.E.M.

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stores expresses SERCA3,which shows low affinity for TG and issensitive to TBHQ [37,38]. Stimulation of human platelets withthrombin (0.01 U/mL) in combination with either a low con-centration of TG (10 nM), to inhibit SERCA2b, TBHQ (20 μM),to inhibit SERCA3, or both resulted in an initial peak [Ca2+]celevation of 132.2±25.4 nM, 135.3±10.1 nM and 157.4±15.2 nM, respectively (Figs. 2A, 3A and 4A, trace a). As ex-pected, [Ca2+]c returned to basal levels more slowly and remainedmore elevated than under control conditions (cells treated withthrombin alone; see Figs 2,3 and 4 vs Fig. 1).

Fig. 2. Effect of SERCA2b on thrombin-evoked SOCE. Fura-2 loaded human plateletcombination with 10 nM TG. CaCl2 (300 μM) was then added to the extracellular medb), 10, 15 and 18 min (E, traces b, c and d) to initiate Ca2+ entry. In parallel experimeevaluate the Ca2+ content in the intracellular stores at the same times as above. Curvesthe Ca2+ entry (black curve) or the Ca2+ content of the stores (grey curve) estimated a⁎Pb0.05 and ⁎⁎Pb0.01.

When the Ca2+ content in the intracellular pools was in-vestigated we found that inhibition of SERCA2b and 3 resultedin more rapid store depletion but after 5 min the amount of Ca2+

stored was comparable; however, no refilling was detected after15 min of stimulation in the presence of TG (Figs. 2 and 4F;n=4). In the presence of TBHQ a detectable Ca2+ store refillingwas detected after treatment with thrombin (Fig. 3F). Thereason of this discrepancy between when cells were stimulatedin the presence of TG or TBHQ might reside in the size of theCa2+ pools that express the different SERCA isoforms. We have

s suspended in a Ca2+-free medium were stimulated with 0.01 U/mL thrombin inium after 1 min (A, trace b), 2 min (B, trace b), 3 min (C, trace b), 5 min (D, tracents, a combination of TG (1 μM)+Iono (50 nM) was added, instead of CaCl2, toA–E are representative of four to six independent experiments. F, data represents described in the Materials and methods section and expressed as mean±S.E.M.

Fig. 3. Effect of SERCA3 on thrombin-evoked SOCE. Fura-2 loaded human platelets suspended in a Ca2+-free medium were stimulated with 0.01 U/mL thrombin incombinationwith 20 μMTBHQ, and CaCl2 (300 μM)was then added to the extracellular medium after 1 min (A, trace b), 2min (B, trace b), 3min (C, trace b), 5 min (D,trace b), 10, 15 and 18min (E, traces b, c and d) to initiate Ca2+ entry. In parallel experiments, a combination of TG (1 μM)+Iono (50 nM)was added, instead of CaCl2, toevaluate the Ca2+ content in the intracellular stores at the same times as above. Curves A–E are representative of four to six independent experiments. F, data representthe Ca2+ entry (black curve) or the Ca2+ content of the stores (grey curve) estimated as described in the Materials and methods section and expressed as mean±S.E.M.⁎Pb0.05 and ⁎⁎Pb0.01.

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previously reported that the DTS might contain approximately 5times more Ca2+ than the acidic stores [4], this might explainwhy we were unable to detect refilling in the acidic stores (whenTG abolished DTS replenishment) but detected DTS refilling(when TBHQ was in the medium).

As a result of the stimulationwith thrombin in combinationwithTG, TBHQor both, we found a greater Ca2+ release from the storesand SOCE was significantly increased at all times investigated(Figs. 2A–D, 3A–D and 4A–D, traces b and Figs. 2E, 3E and 4E,traces b, c and d; Pb0.05; n=4–6), compared to cells stimulatedwith thrombin alone. Interestingly, SOCE stimulated by thrombin

in the presence of TG or TBHQ reached a plateau that remainedfor at least 10min (Figs. 2F, 3F and 4F) in contrast to the transientactivation of SOCE by thrombin alone, thus suggesting a role forSERCA2b and 3 in the regulation of SOCE.

The Na+/Ca2+ exchanger and PMCA are the main mechanismsinvolved in Ca2+ extrusion in human platelets. In order to avoid anyinterference with Ca2+ extrusion we have performed a series ofexperiments where the extrusion mechanisms were blocked. Asshown previously, reducing Na+/Ca2+-exchanger activity bysuspending human platelets in a Na+-free medium resulted inreduced thrombin-evoked SOCE [13],whichwasmore sustained at

Fig. 4. Effect of simultaneous inhibition of SERCA2b and 3 on thrombin-evoked SOCE. Fura-2 loaded human platelets suspended in a Ca2+-free medium werestimulated with 0.01 U/mL thrombin in combination with 20 μM TBHQ and 10 nM TG, and CaCl2 (300 μM) was then added to the extracellular medium after 1 min(A, trace b), 2 min (B, trace b), 3 min (C, trace b), 5 min (D, trace b), 10, 15 and 18 min (E, traces b, c and d) to initiate Ca2+entry. In parallel experiments, a combinationof TG (1 μM)+Iono (50 nM) was added, instead of CaCl2, to evaluate the Ca2+ content in the intracellular stores at the same times as above. Curves A–E arerepresentative of four to six independent experiments. F, data represent the Ca2+ entry (black curve) or the Ca2+ content of the stores (grey curve) estimated as describedin the Materials and methods section and expressed as mean±S.E.M. ⁎Pb0.05 and ⁎⁎Pb0.01 and ⁎⁎⁎Pb0.001.

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late time points compared with that activated by thrombin alone(Fig. 5, trace b vs c). Monitoring the entry of Sr2+, which cannot betransported by the PMCA, in platelets where extrusion by the Na+/Ca2+-exchangerwas impaired using aNa+-freemedium, resulted intransient thrombin-induced cation entry that was paralleled by thetime course of SOCE (Fig. 5, trace a vs b). Unfortunately, thebiophysics of fura-2 fluorescence after binding with Ca2+ or Sr2+

are not identical so that only the time courses are comparable.As shown above, in the presence of the SERCA inhibitors TG

and TBHQ the extent of cation entry induced by thrombin was

significantly greater (Fig. 5, trace b vs e; Pb0.05). In cells wherethe Na+/Ca2+-exchanger was inhibited (Fig. 5, trace f) the timecourse of thrombin-induced SOCE reached a plateau after 3 minthat was maintained for at least 15 min. In addition, the extent ofSOCE induced by thrombin in combination with TG+TBHQwassignificantly greaterwhen theNa+/Ca2+-exchangerwas inactivated(Fig. 5, trace e vs f; Pb0.05). However, the pattern of Sr2+entry,when extrusion through the Na+/Ca2+-exchanger and PMCAwasimpaired (Fig. 5, trace d), was similar to that observed when theextrusion mechanisms were functional (Fig. 5, trace d vs e).

Fig. 5. Role of calcium extrusion in SOCE in human platelet. Human plateletswere suspended in Ca2+-free HBS (traces a, b, d and e) or in Ca2+and Na+-freeHBS (traces a, c, d and f), and then stimulated with 0.01 U/mL of thrombin(traces a, b and c) alone or in presence of the SERCAs inhibitors, TBHQ(20 μM) and TG (10 nM) (traces d, e and f). 300 μM CaCl2 (traces b, c, e and f)or SrCl2 (traces a and d) was added to the extracellular medium 1, 2, 3, 5, 10, 15and 18 min after thrombin (alone or in combination with TG+TBHQ) to initiatecation entry. Data represent the mean±S.E.M. of six to eight independentexperiment performed in different donors.

Fig. 6. SERCAs modulate the coupling between IP3RII and hTRPC1 in plate-lets. Human platelets suspended in a free-Ca2+-HBS (100 μMEGTAwas added)were stimulated for 3 min with thrombin (0.01 U/mL) in the absence or presenceof 10 nM TG plus 20 μMTBHQ and then lysed. Aliquots (500 μL) of whole celllysates were immunoprecipitated with 1.5 μg/ mL of anti-IP3RII antibody andanalysed by Western blotting using anti-hTRPC1 antibody. Reprobing of themembranes was done using an anti-IP3RII antibody to assess that the similaramount of protein has been loaded in all the lanes. Positions of molecular massmarkers are shown on the right. These results are representative of six to eightindependent experiments performed using different donors.

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3.3. SERCA isoforms modulate thrombin-evoked couplingbetween type II IP3R and hTRPC1

Coupling between IP3RII and hTRPC1 has been described tobe a key step during the activation of SOCE in human platelets[4,14,16,17]. In order to evaluate whether the effect of SERCAsactivities on the time-course of SOCE is due to modulation ofthe coupling between IP3RII and hTRPC1 we explored theeffect of the SERCA inhibitors on the coupling tested bycoimmunoprecipitation of hTRPC1 with IP3RII. Cells werestimulated with thrombin alone or in the presence of TG(10 nM) in combination with TBHQ (20 μM). As shown inFig. 6, platelet stimulation with thrombin (0.01 U/mL) inducedcoupling between IP3RII and hTRPC1. In the presence of TGand TBHQ, to prevent SERCA2b and 3 function, respectively,thrombin-evoked coupling was enhanced (Fig. 6), suggesting arole for SERCAs in the modulation of the coupling betweenhTRPC1 and the type II IP3R.

3.4. SERCA2b and 3 bind to hTRPC1 after stimulation withthrombin

It has been reported that hTRPC1 in resting human plateletsis part of a heteromultimeric complex with TRPC4 and TRPC5[45]. However, the identity of other proteins that may belong tothe complex in stimulated platelets remains unknown. Wetherefore tested for coupling between the SERCA isoforms 2b

and 3 and hTRPC1 by looking for co-immunoprecipitation fromplatelet lysates. Immunoprecipitation and subsequent SDS-PAGE and Western blotting were conducted using platelets atrest or stimulated with 0.01 U/mL thrombin in the absence ofextracellular Ca2+ (100 μM EGTA was added). As shown inFig. 7, treatment of platelets with thrombin (0.01 U/mL) for3 min increased the interaction between SERCA2b or 3 and

Fig. 7. Coimmunoprecipitation of SERCA2b and SERCA 3 with TRPC1 in thrombin stimulated platelets. Human platelets suspended in a Ca2+-free HBS (100 μMEGTAwas added) were stimulated for 3 min with thrombin (0.01 U/mL) in absence or presence of TG plus TBHQ and then lysed. 500 μL of whole cell lysates wereimmunoprecipitated with 1.5 μg/mL of anti-SERCA3 (A; n=4–6) or anti-SERCA2b antibody (B; n=4) and analyzed by Western blotting using anti-hTRPC1antibody. Reprobing of the membranes was done using anti-SERCA 2 or 3 antibody to assess that the similar amount of protein has been loaded in all the lanes.Positions of molecular mass markers are shown on the right. These results are representative of four to six independent experiments performed using different donors.

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hTRPC1 by 123.3±79.4% or 161.7±53.9%, as detected bycoimmunoprecipitation (Fig. 7B and A, respectively; n=4–6).Inhibition of SERCA2b and 3 activity by treatment with TG(10 nM) in combination with TBHQ (20 μM) did not signif-icantly modified the coupling between SERCA3 or SERCA 2band hTRPC1 (Fig. 7; PN0.05; n=4–6).

4. Discussion

Ca2+ entry through store-operated channels is regulated byseveral mechanisms. These include biochemical processes, suchas the Ca2+ content of the intracellular Ca2+ stores, which istransmitted to the Ca2+ channels through different intracellularpathways involving the protein STIM1[20–24], and mechan-isms regulating channel gating, such as phosphatidylinositol 4,5-bisphosphate [12] or calmodulin [32]. Biophysical factors,including the electrochemical gradient across the plasma mem-

brane, which provides the driving force for Ca2+ entry, alsoinfluence Ca2+ entry as does the effect of Ca2+ removal from thecytosol, either by Ca2+ reuptake by SERCA, Ca2+ extrusionthrough the Na+/Ca2+ exchanger and PMCA or Ca2+ bufferingby mitochondria [47–49].

In the present study we have investigated the role of theSERCAs, the main mechanism for Ca2+ uptake in human platelets,in the regulation of SOCE in these cells. Our results indicate thatthrombin, at concentrations that do not show detectable non-capacitative Ca2+ entry [44], transiently stimulate SOCE, reachinga maximum within 3 min of stimulation. At later times SOCEactivation decreased, reaching a plateau 10 min after thrombinaddition. Specific inhibition of SERCA2b and 3 was achieved by alow concentration of TG orwith TBHQ, respectively, as previouslydescribed [4,33,34,39,50]. Inactivation of SERCA2b and 3 eitherseparately or simultaneously, was found to accelerate Ca2+ storedischarge, as expected due to the impairment of Ca2+ store refilling

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that opposes thrombin-evoked Ca2+ release. In addition, SERCAinactivation results in two separate modifications in thrombin-induced SOCE: an enhancement of the extent of SOCE stimulatedby thrombin and a modification in the time course of thrombin-evoked SOCE from a transient to a sustained increase in Ca2+

influx. The enhanced SOCE observed in the presence of SERCAinhibitors could be attributed to the effect of these inhibitors preventstore refilling and then stimulating SOCE. However, this can onlypartially explain our findings. The Ca2+ content in the stores 10minafter thrombin addition was similar in the absence or presence ofTG and/or TBHQ (the integral of the rise in [Ca2+]c, taking a datapoint every second, induced by extensive depletion of theintracellular stores using TG (1 μM) plus Iono (50 nM) wasabout 6.0 μM s in all cases) and the extent of SOCE wassignificantly different in the absence or presence of the SERCAinhibitors (the integral of the rise in [Ca2+]c, taking a data pointevery second, observed after the addition of 300 μM CaCl2 was9.0 μM s for thrombin alone and 15.0, 17.0 and 23.0 μM s afterstimulation with thrombin in the presence of TG, TBHQ or both,respectively). These findings, together with the change in thepattern of thrombin-evoked SOCE, reveal a negative role forSERCAs in the regulation of SOCE in human platelets. Thiseffect was consistent under conditions that inhibit Ca2+ extrusionby PMCA or the Na+/Ca2+ exchanger, which suggest that thisobservation was not due to changes in Ca2+ extrusion during theinhibition of SERCAs. The regulatory effect of SERCAs onSOCE have been recently described in HEK-293 cells, whereTRPC7 and TRPC3 have been shown to be regulated bySERCA due to its ability to reduce the high Ca2+ microdomainsgenerated after channel opening, which exert a negative feedbackover the permeability of the channel [51].

We have reported that a conformational coupling between theIP3RII and hTRPC1 is involved in the activation of SOCE inhuman platelets [4,5,12,52]. Here we show that inhibition ofSERCAs enhanced the interaction between IP3RII to hTRPC1evoked by thrombin, which may indicate that SERCAs nega-tively regulate the coupling and thus SOCE in these cells. Inaddition, our results show physical interaction of SERCA2b and3 with hTRPC1, which can only be mediated by the closeapposition of the intracellular Ca2+ stores and the plasmamembrane in platelets. The interaction between SERCAsand hTRPC1 does not require SERCA activation since it wasnot altered by treatment with the SERCA inhibitors TG andTBHQ. SERCA2b has been reported to co-localize withhTRPC1 channels in mouse cortical astrocytes [46], where as-sociation was detected in the restricted space between the mem-branes of the stores and the plasma membrane after thestimulation of SOCE. However, we show for the first timeinteraction between SERCA3 and hTRPC1. With the coimmu-noprecipitation experiments used we cannot unequivocallyconfirm whether there is a direct interaction between theSERCA isoforms and hTRPC1 or whether this association occursthrough other structural elements belonging to the hTRPC1protein complex.

In conclusion, we provide evidence for the involvement ofboth SERCA2b and 3 in the regulation of SOCE stimulatedby the physiological agonist thrombin in human platelets. The

negative role of SERCA2b and 3 in SOCE might be mediatedby interaction with the hTRPC1 channel subunit in these cells.

Acknowledgements

Supported by Ministerio de Educación y Ciencia-DirecciónGeneral de Investigatión (Grant BFU2004-00165). PCR is sup-ported by a postdoctoral fellowship from the Consejería deInfraestructura y Desarrollo Tecnológico-Junta de Extremadura(POS05A003).

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