Endogenously Expressed Trp1 Is Involved in Store-mediated Ca2+ Entry by Conformational Coupling in Human Platelets*

Physical interaction between transient receptor potential (Trp) channels and inositol 1,4,5-trisphosphate receptors (IP3Rs) has been presented as a candidate mechanism for the activation of store-mediated Ca2+ entry. The role of a human homologue of Drosophila transient receptor potential channel, hTrp1, in the conduction of store-mediated Ca2+ entry was examined in human platelets. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 557–571 of hTrp1, inhibited both store depletion-induced Ca2+ and Mn2+ entry in a concentration-dependent manner. Stimulation of platelets with the physiological agonist thrombin activated coupling between the IP3 receptor type II and endogenously expressed hTrp1. This event was reversed by refilling of the internal Ca2+ stores but maintained after removal of the agonist if the stores were not allowed to refill. Inhibition of IP3 recycling using Li+ or inhibition of IP3Rs with xestospongin C or treatment with jasplakinolide, to stabilize the cortical actin filament network, abolished thrombin-induced coupling between hTrp1 and IP3R type II. Incubation with the anti-hTrp1 antibody inhibited thrombin-evoked Ca2+ entry without affecting Ca2+ release from intracellular stores. These results provide evidence for the involvement of hTrp1 in the activation of store-mediated Ca2+ entry by coupling to IP3R type II in normal human cells.

The molecular basis of the activation and maintenance of SMCE is not fully understood, and two main questions still remain: elucidation of the mechanism of activation of SMCE after depletion of the internal Ca 2ϩ stores and identification of the channel that mediates extracellular Ca 2ϩ entry. Several hypotheses have been proposed to account for the activation of SMCE, which fall into two main categories: those suggesting a diffusible messenger that gates plasma membrane channels and those suggesting a conformational coupling between elements in the endoplasmic reticulum (possibly the IP 3 receptors) and the plasma membrane (3,6). Recently, a modification of the conformational coupling model has been proposed to operate in several cell types. This "secretion-like coupling model" is based on the trafficking and coupling of portions of the endoplasmic reticulum with the plasma membrane, where the actin cytoskeleton plays an important role (7)(8)(9).
Studies aiming to identify the channels involved in the conduction of SMCE have focused attention on human homologues of Drosophila transient receptor potential (Trp) channels (see Ref. 6). Recent studies have provided evidence that relates Trp proteins to SMCE channels. Functional expression of Trp proteins enhances SMCE in several mammalian cells, including COS cells (10) and human salivary gland cells (11). A second argument for Trp channels mediating SMCE is provided by antisense studies showing interference with the expression of trp sequences affects the activation of SMCE (10).
Human platelets have been shown to express mRNA for Trp1 and its splice variant Trp1A (12). In addition, Trp1 proteins have been detected in platelets using an anti-hTrp1 antibody specific for the amino acid residues 557-571 (13). Both Trp1 and Trp1A have been shown to form non-selective cation channels regulated by store depletion in a number of expression systems (14,15). In agreement with the conformational coupling hypothesis, functional IP 3 receptors (IP 3 Rs) have been shown to be required for the activation of SMCE (13,16), and physical interaction between IP 3 receptors and Trp channels has been reported in several transfected cell lines (17)(18)(19)(20) and in platelets endogenously expressing Trp1 (13). In human platelets, this coupling is activated by depletion of the intracellular Ca 2ϩ stores (13). We report here that physiological agonists also stimulate coupling of IP 3 receptors to hTrp1 in human platelets and that incubation with anti-Trp1 antibody resulted in a dramatic decrease in agonist-or store depletionevoked Ca 2ϩ entry, providing strong evidence for the involvement of Trp1 in the SMCE in normal human cells.
Platelet Preparation and Leukocyte Separation-Experiments were carried out on human blood platelets or peripheral blood leukocytes obtained from healthy drug-free volunteers with local ethical committee approval. Platelet-rich plasma was prepared by centrifugation for 5 min at 700 ϫ g and aspirin (100 M) and apyrase (40 g/ml) added as described previously (21). Peripheral blood leukocytes were obtained by gradient density centrifugation using HISTOPAQUE-1119 and 1077 Medium (22). Fura-2-loaded platelets, co-loaded with dimethyl BAPTA when required, were prepared as described previously (13) and resuspended in HEPES-buffered saline (HBS) containing 145 mM NaCl, 10 mM HEPES, 10 mM D-glucose, 5 (23). Mn 2ϩ influx was monitored as a quenching of fura-2 fluorescence at the isoemissive wavelength, 360 nm, presented on an arbitrary linear scale (24).
Thrombin-evoked Ca 2ϩ influx was estimated as the integral of the rise in [Ca 2ϩ ] i above basal for 150 s after addition of thrombin in the presence of external Ca 2ϩ , corrected by subtraction of the integral over the same period of stimulation in the absence of external Ca 2ϩ (with 100 M EGTA). When SMCE was stimulated after store depletion using TG, Ca 2ϩ influx was estimated using the integral of the rise in [Ca 2ϩ ] i for 150 s after addition of CaCl 2 .
Measurement of [Sr 2ϩ ] i -Sr 2ϩ was used to monitor non-capacitative cation entry to avoid complications arising from stimulation of the platelet plasma membrane Ca 2ϩ ATPase by thrombin (25) since Sr 2ϩ is carried with lower affinity than Ca 2ϩ by the platelet plasma membrane Ca 2ϩ ATPase (26). Sr 2ϩ entry was determined in Ca 2ϩ -free HBS containing EGTA (100 M) to minimize the effects of contaminating Ca 2ϩ . Cytosolic Sr 2ϩ was monitored using the fura-2 340-nm/380-nm fluorescence ratio.
Immunofluorescence-Samples of platelet suspension (200 l; 2 ϫ 10 8 cells/ml) were transferred to 200 l of ice-cold 3% (w/v) formaldehyde in phosphate-buffered saline for 10 min and then incubated for 1 h with 1 g/ml anti-hTrp1 antibody. The platelets were then collected by centrifugation and washed twice in phosphate-buffered saline containing 137 mM NaCl, 2.7 mM KCl, 5.62 mM Na 2 HPO 4 , 1.09 mM NaH 2 PO 4 , 1.47 mM KH 2 PO 4 , pH 7.2, and supplemented with 0.5% (w/v) bovine serum albumin. To detect the primary antibody, samples were incubated with 0.02 g/ml FITC-conjugated donkey anti-rabbit IgG antibody for 1 h and washed twice in phosphate-buffered saline. Fluorescence was measured using a fluorescence spectrophotometer (PerkinElmer Life Sciences). Samples were excited at 496 nm, and emission was at 516 nm. When permeabilized platelets were required, the cells were incubated for 10 min with 0.025% (v/v) Nonidet P-40 detergent.
Immunoprecipitation and Western Blotting-500-l aliquots of platelet suspension (2 ϫ 10 9 cell/ml) were lysed with an equal volume of lysis buffer, pH 7.2, containing 316 mM NaCl, 20 mM Tris, 2 mM EGTA, 0.2% SDS, 2% sodium deoxycholate, 2% Triton X-100, 2 mM Na 3 VO 4 , 2 mM phenylmethylsulfonyl fluoride, 100 g/ml leupeptin, and 10 mM benzamidine. Aliquots (1 ml) were then immunoprecipitated by incubation with 2 g of anti-hTrp1 polyclonal antibody (Alomone), 2 g of T1E3 polyclonal antibody, or 3 g of anti-IP 3 R type II polyclonal antibody and protein A-agarose overnight at 4°C. Immunoprecipitates were resolved by 8% SDS-PAGE, and separated proteins were transferred onto nitrocellulose membranes. Immunodetection of hTrp1 or IP 3 R type II was achieved using the anti-hTrp1 or T1E3 antibodies diluted 1:200 or 1:600 in TBST, respectively, or with anti-IP 3 RII diluted 1:500 in TBST for 3 h. To detect the primary antibody, blots were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody or horseradish peroxidase-conjugated donkey anti-goat IgG antibody diluted 1:10,000 in TBST, washed six times in TBST, and exposed to enhanced chemiluminescence reagents for 1 min. Blots were then exposed to preflashed photographic film. For reprobing, membranes were incubated for 30 min at 50°C in stripping buffer containing 100 mM 2-mercaptoethanol, 65.5 mM Tris, and 2% SDS, pH 6.7. Membranes were then washed and Western blotting was performed as described previously.
Statistical Analysis-Analysis of statistical significance was performed using Student's t test. For multiple comparisons, one-way analysis of variance combined with the Dunnett test was used.

RESULTS
Thrombin Stimulates Coupling between hTrp1 and IP 3 R Type II-Recent studies have reported expression of hTrp1 mRNA in human platelets and megakaryocytes (12). Using a commercial antibody that specifically recognizes the sequence hTrp1 557-571 , which is present only in hTrp1 and not in other reported hTrp proteins (27), a single protein band of ϳ100 kDa displayed reactivity toward the antibody in human platelets (Fig. 1A, lanes 1 and 2) (13). The specificity of the commercial antibody was tested with the anti-Trp1 antibody T1E3, which has been shown to be a specific and powerful tool in the investigation of mammalian Trp1 proteins (28). As shown in Fig. 1A, after immunoprecipitation with the commercial antibody, T1E3 displayed reactivity toward exactly the same protein band, suggesting that both antibodies specifically recognize Trp1 in human platelets. Both antibodies recognized comparable amounts of Trp1 in either resting or store-depleted platelets ( Fig. 1A; n ϭ 4). To further investigate whether these antibodies specifically recognized the same protein, both antibodies were used on the same samples. As shown in Fig. 1A, after immunoprecipitation with the commercial antibody, probing with the same antibody revealed reactivity against a protein band of ϳ100 kDa (Fig. 1B). Reprobing with the T1E3 antibody revealed reactivity toward the same protein (Fig. 1C). Proteins in this or other molecular mass ranges were not detected in human peripheral blood leukocytes (Fig. 1D, lanes 2 and 3), where hTrp1 mRNA expression has not been detected (29).
We have recently reported that depletion of the intracellular Ca 2ϩ stores using thapsigargin stimulates coupling between endogenously expressed hTrp1 and the IP 3 R type II in human platelets independently of rises in [Ca 2ϩ ] i (13). As shown in Fig.  1A (lanes 5 and 6), immunoprecipitation with anti-IP 3 R type II followed by SDS-PAGE and Western blotting with T1E3 confirmed de novo coupling between hTrp1 and IP 3 R type II when the intracellular Ca 2ϩ stores were depleted using TG. To investigate the physiological significance of this coupling, the effect of thrombin was investigated. Normal platelets or platelets loaded with the intracellular Ca 2ϩ chelator dimethyl BAPTA were stimulated with 0.1 units/ml thrombin in the absence of extracellular Ca 2ϩ (100 M EGTA was added) and lysed 3 min later. Control platelets were compared with cells heavily loaded with the Ca 2ϩ chelator dimethyl BAPTA so as to differentiate between Ca 2ϩ -and store depletion-dependent responses (30). We have recently demonstrated that dimethyl BAPTA loading prevents thrombin-evoked [Ca 2ϩ ] i elevations (9). After immunoprecipitation with anti-hTrp1 antibody, Western blotting revealed the presence of IP 3 R type II in samples from thrombin-treated but not resting platelets ( Fig. 2A, upper panel; n ϭ 4). Thrombin-induced coupling between hTrp1 and IP 3 R type II was independent of elevations in [Ca 2ϩ ] i since it was also observed in dimethyl BAPTA-loaded platelets. Western blotting with anti-hTrp1 confirmed a similar content of this protein in all lanes ( Fig. 2A, lower panel).
We also conducted converse experiments, immunoprecipitating platelet lysates with the anti-IP 3 R type II antibody and detecting for the presence of hTrp1. Consistent with the above results, after immunoprecipitation with anti-IP 3 R type II, hTrp1 was detected in samples from thrombin-treated but not resting cells (Fig. 2B, upper panel; n ϭ 4). Again, thrombin evoked coupling in both control and dimethyl BAPTA-loaded platelets.
To investigate whether the coupling between IP 3 RII and hTrp1 is disassembled by removal of thrombin, we have performed a series of experiments in which, after platelet stimulation with thrombin, the agonist was washed away. In HBS containing 1 mM Ca 2ϩ , thrombin (0.1 units/ml) evoked a rise in [Ca 2ϩ ] i in platelets (Fig. 3A, part i) due to release of Ca 2ϩ from the stores and Ca 2ϩ entry. However, after removal of thrombin and incubation of the cells in HBS (1 mM Ca 2ϩ added to allow refilling of the stores), Ca 2ϩ entry was inactivated (Fig. 3A, part ii). Consistent with this, we found that IP 3 RII and hTrp1 couple in response to thrombin stimulation, whereas removal of the agonist under conditions that allow refilling of the Ca 2ϩ stores clearly reversed the extent of coupling (Fig. 3A, part iii). If, after stimulation with thrombin, the agonist was washed away and, in the absence of added Ca 2ϩ , the thrombin antagonist PPACK (1 M, sufficient to completely block any rise in [Ca 2ϩ ] i evoked by 0.1 units/ml thrombin) was added to inactivate residual traces of the agonist, the coupling was maintained (Fig. 3A, part iii).
We have shown previously that store depletion-induced Ca 2ϩ entry and coupling requires both IP 3 and activation of IP 3 Rs and is blocked by stabilization of the cortical actin filaments (9,13,31). To examine whether the mechanism of activation of the coupling between IP 3 RII and hTrp1 by thrombin has the same requirements, we investigated the role of IP 3 and IP 3 R activation by using Li ϩ or the inhibitor of IP 3 R function Xest C. As reported previously (13), treatment of human platelets for 30 min with 20 M Xest C or for 2 h with 10 mM Li ϩ abolished thrombin-evoked release of Ca 2ϩ from the intracellular stores. As shown in Fig. 3B, incubation of human platelets with Xest C  3 and 4), or anti-IP3R type II (lanes 5 and 6) and analyzed by Western blotting (WB) using T1E3 antibody. As shown in B and C, human platelets were incubated in the absence or presence of 1 M TG plus 50 nM Iono, as indicated, for 3 min and then lysed. Whole cell lysates were immunoprecipitated with anti-hTrp1 antibody from Alomone and analyzed by Western blotting using the same antibody (A). After removing the antibodies, the membranes were reprobed using the T1E3 antibody (C). As shown in D, human platelet lysates (500 g/ml) or peripheral blood leukocyte (p.b.l.) lysates (500 and 750 g/ml) were analyzed by SDS-PAGE and Western blotting using the Alomone anti-hTrp1 antibody. The position of hTrp1 is indicated on the left. Positions of molecular mass markers are shown on the right. These results are representative of at least three other experiments. or Li ϩ completely blocked the coupling between IP 3 RII and hTrp1. These findings confirm that IP 3 and functional IP 3 RII are required for the coupling with the hTrp1 protein.
To assess the role of the cortical actin filaments in thrombininduced coupling between IP 3 RII and hTrp1, we investigated the effect of JP. We have reported previously that JP reduced thrombin-induced Ca 2ϩ entry without affecting Ca 2ϩ release from the stores (32). Consistent with this, treatment of human platelets with 10 M JP for 30 min abolished the coupling between IP 3 RII and hTrp1 induced by thrombin (Fig. 3C).
Location of the Epitope Recognized by the Anti-hTrp1 Antibody-The commercial antibody used to perform the majority of our studies specifically recognizes the peptide sequence QLYDKGYTSKEQKDC, which corresponds to the amino acid residues 557-571 of the human hTrp1 protein. This antibody has been shown to be effective in Western blotting, immunoprecipitation, and immunolocalization studies (13,27,33,34). The sequence 557-571 has been shown to be located between the transmembrane domains 5 and 6; thus, it would be expected to be located at the external face of the PM (29,35). To further investigate the location of the epitope recognized by the anti-hTrp1 antibody, we performed a series of immunofluorescence experiments. As shown in Fig. 4, incubation of fixed, non-permeabilized platelets in suspension with 1 g/ml anti-hTrp1 antibody followed by detection using a FITC-conjugated secondary antibody revealed the presence of hTrp1 proteins in the platelet membranes (Fig. 4, column b; n ϭ 4), indicating that the epitope is located extracellularly. The fluorescence observed was not due to nonspecific binding of the secondary antibody as demonstrated by the lower fluorescence detected in samples incubated with this antibody alone (Fig. 4, column a;  n ϭ 4). The increase in fluorescence detected after incubation with the anti-hTrp1 antibody was almost completely prevented when the antibody was incubated previously for 1 h in the presence of the control antigen peptide (CAP) (1 g/ml; Fig. 4, column e; n ϭ 4), confirming the antibody specificity. To investigate whether store depletion induced increased expression of the hTrp1 protein in the PM, we repeated the experimental protocol using TG-treated cells. Treatment of human platelets with 200 nM TG for 3 min in a Ca 2ϩ -free medium slightly increased the detection of hTrp1 in the membrane; however, this rise was not significant (Fig. 4, column c; p ϭ 0.39; n ϭ 4). Similar results were obtained when the studies were performed in cells permeabilized with the detergent Nonidet P-40, con-

FIG. 3. Characterization of thrombin-induced coupling between hTrp1 and IP 3 RII in human platelets.
As shown in A, fura-2-loaded human platelets were resuspended in a Ca 2ϩ -free medium. Elevations in [Ca 2ϩ ] i were monitored using the 340-nm/380-nm ratio and calibrated in terms of [Ca 2ϩ ] i . At the time of the experiment, 1 mM CaCl 2 was added. Cells were then stimulated in the absence (part iii, C) or presence of 0.1 units/ml thrombin (part i; part iii, T; part iii, R; and part iii, A). After removal of thrombin, cells were incubated with HBS, and either 1 mM CaCl 2 was added (to allow refilling of the intracellular Ca 2ϩ stores; part ii and part iii, R) or 1 M PPACK was added without CaCl 2 to inactivate residual traces of thrombin and maintain Ca 2ϩ store depletion (part iii, A). As shown in B, platelets were incubated either in the absence (lanes 1 and 2) or the presence of 10 mM Li ϩ for 2 h (lanes 3 and 4) or 20 M Xest C for 30 min (lanes 5 and 6). Cells were then stimulated with thrombin (0.1 units/ml). As shown in C, platelets were incubated either in the absence (lanes 1 and 2) or the presence of 10 M JP for 30 min (lanes 3 and 4). Cells were then stimulated with thrombin (0.1 units/ml). Samples were taken 5 s before and 3 min after the addition of thrombin and lysed. Whole cell lysates were immunoprecipitated with anti-IP 3 R type II polyclonal antibody and analyzed by Western blotting using anti-hTrp1 antibody (Alomone). These results are representative of three independent experiments. firming that the anti-hTrp1 antibody recognizes an epitope located on the external face of the PM (Fig. 4, column d; p ϭ 0.49; n ϭ 4).
Incubation with Anti-hTrp1 Antibody Inhibits TG-induced Store-mediated Ca 2ϩ and Mn 2ϩ Entry-Since the sequence 557-571 is located in the pore-forming region between the fifth transmembrane domain and region VII of hTrp1 (29,30), we investigated whether the antibody to this sequence could block channel function. To assess this, human platelet suspensions were incubated for 10 min with increasing concentrations of the anti-hTrp1 antibody (1.5-15 g/ml) followed by depletion of the intracellular Ca 2ϩ stores using TG to activate SMCE.
In a Ca 2ϩ -free medium, TG evoked a prolonged elevation of [Ca 2ϩ ] i in platelets due to leakage of Ca 2ϩ from intracellular stores. Subsequent addition of Ca 2ϩ (300 M) to the external medium induced a sustained increase in [Ca 2ϩ ] i , indicative of SMCE (Fig. 5A). Incubation with the anti-hTrp1 antibody for 10 min significantly reduced SMCE by 34 Ϯ 3, 46 Ϯ 3, 60 Ϯ 2, and 76 Ϯ 2% (n ϭ 4) at concentrations of 1.5, 5, 10, and 15 g/ml, respectively ( Fig. 5B; p Ͻ 0.01). The effect of the highest concentration used in this study (15 g/ml) was prevented when the antibody was incubated previously for 1 h in the presence of the CAP (15 g/ml). To investigate the specificity of this assay, the effect of incubation with an antibody directed to a protein not related to hTrp proteins or any other platelet protein was tested. Incubation of platelets for 10 min in the presence of 15 g/ml anti-mouse IgG antibody following the protocol used for the anti-hTrp1 antibody was unable to block TG-induced Ca 2ϩ entry either in the absence or presence of the CAP (15 g/ml; Fig. 5C; n ϭ 3). SMCE has been widely demonstrated to occur through nonselective cation channels (see Ref. 24). Thus, we have used Mn 2ϩ to evaluate the effect of incubation with anti-hTrp1 antibody on TG-evoked divalent cation entry. This cation can be used as a surrogate for Ca 2ϩ entry given its quenching effect on fura-2 fluorescence at the isoemissive wavelength, 360 nm (24). Addition of Mn 2ϩ (300 M) to platelets with TG-depleted intracellular Ca 2ϩ stores resulted in a sustained quenching of fluorescence (Fig. 5D, trace a) as compared with undepleted cells (Fig. 5D, trace f). When platelets were incubated for 10 min with anti-hTrp1 antibody, Mn 2ϩ entry was attenuated. The initial rate of Mn 2ϩ -evoked fluorescence quenching in platelets incubated with the antibody and then treated with TG was significantly decreased to 85 Ϯ 4, 67 Ϯ 4, 54 Ϯ 4, and 33 Ϯ 7% (n ϭ 4) of the initial rate observed in cells treated with TG alone, at concentrations of 1.5, 5, 10, and 15 g/ml, respectively ( Fig. 5D; p Ͻ 0.01) These results indicate that the anti-hTrp1 antibody blocked divalent cation entry in human platelets in a concentration-dependent manner.
Anti-hTrp1 Antibody Inhibits Thrombin-evoked Ca 2ϩ Entry-As shown in Fig. 6, A and B, incubation of fura-2-loaded human platelets for 10 min with 15 g/ml anti-hTrp1 antibody resulted in a substantial inhibition of the elevation in [Ca 2ϩ ] i evoked by the physiological agonist thrombin (0.05 units/ml) in medium containing 1 mM Ca 2ϩ (n ϭ 4; p Ͻ 0.001). In contrast, incubation with 15 g/ml anti-mouse IgG antibody did not interfere with thrombin-induced Ca 2ϩ entry ( Fig. 6C; n ϭ 3). Since we have recently demonstrated that thrombin is able to activate non-capacitative cation entry in platelets (32), we used a low concentration of the agonist (0.05 units/ml) to reduce this effect and avoid interference with thrombin-evoked store depletion-mediated Ca 2ϩ entry. As shown in Fig. 6G, trace b, addition of thrombin (0.05 units/ml) to platelets, in which Sr 2ϩ entry had already been stimulated by complete depletion of the intracellular Ca 2ϩ stores by treatment with TG (1 M) and Iono (50 nM), was unable to caused a further Sr 2ϩ entry. In contrast, a higher concentration of thrombin (10 units/ml) induced noncapacitative Sr 2ϩ entry (Fig. 6G, trace a). Preincubation of platelets with 15 g/ml anti-hTrp1 antibody clearly reduced store depletion-induced Sr 2ϩ entry without affecting non-capacitative Sr 2ϩ entry stimulated by thrombin (10 units/ml; Fig.  6G, trace c). The nature of the channels involved in the noncapacitative cation entry by thrombin needs further attention.
In the absence of external Ca 2ϩ (100 M EGTA added), incubation with anti-hTrp1 or anti-mouse IgG antibodies did not alter the thrombin-evoked rise in [Ca 2ϩ ] i , indicating no effect on the release of Ca 2ϩ from intracellular stores (Fig. 6, D-F). These findings suggest that this treatment was selectively blocking Ca 2ϩ entry over internal release. If we consider the entry of Ca 2ϩ stimulated by thrombin (correcting the signal for the release of Ca 2ϩ from intracellular stores (see "Experimental Procedures")), incubation with anti-hTrp1 antibody significantly inhibited thrombin-induced Ca 2ϩ influx by 77 Ϯ 9% (n ϭ 4; p Ͻ 0.001).

DISCUSSION
The mechanism that activates SMCE after depletion of the intracellular Ca 2ϩ stores is not fully understood, and identification of the channels involved in this process would assist in the advancement of knowledge of this signaling mechanism. Recent studies have presented compelling evidence for the involvement of mammalian homologues of the Drosophila Trp channels in SMCE in certain cells (9,15,36). We have reported previously that hTrp1 couples with the IP 3 R type II in platelets after depletion of the internal Ca 2ϩ stores using TG (13). The anti-hTrp1 antibody used in these studies specifically recognizes the sequence hTrp1 557-571 , which is present in hTrp1 and no other proteins (27). Using this antibody, hTrp1 proteins were detected in human platelets, where hTrp1 mRNA expression has been demonstrated previously (12), but not in human leukocytes lacking hTrp1 mRNA expression (29). In the present study, we report that thrombin was able to induce coupling between the IP 3 R type II and naturally expressed hTrp1 proteins in human platelets. This coupling was reversed by removal of the agonist and, as for store depletion-induced coupling between IP 3 R type II and hTrp1 proteins, requires IP 3 and functional IP 3 R and some degree of reorganization of the cortical actin barrier. To our knowledge, this is the first time that agonist-stimulated coupling between hTrp proteins and IP 3 Rs has been reported, suggesting that direct coupling between hTrp1 and IP 3 R type II is a physiological mechanism by which SMCE could be activated in platelets.
Reports that store depletion results in coupling between hTrp1 and IP 3 R type II in platelets provide only circumstantial evidence for the involvement of hTrp1 in SMCE. Thus, using an antibody that specifically recognizes the amino acid sequence 557-571 of hTrp1, we have further investigated the involvement of hTrp1 in the mediation of SMCE in platelets. Since the sequence 557-571 is located in the pore region of the hTrp1 channel (29), one might expect that binding of this antibody to the hTrp1 channel could interfere with its channel function. Our results showing that incubation of platelets with the anti-hTrp1 antibody inhibits Ca 2ϩ entry provide strong evidence for the involvement of hTrp1 proteins in the normal response of platelets to stimuli that increase IP 3 levels and activate storemediated Ca 2ϩ entry. Human platelets express mRNA for hTrp1 and its splice variant hTrp1A (12). Both isoforms contain the sequence hTrp1 557-571 (12) and therefore are recognized by the antibody used. hTrp1 and hTrp1A have been shown to form non-selective cation channels regulated by store depletion in different expression systems (14,15). Whether hTrp1 or hTrp1A forms the channel alone, or whether the Trp cation channel in platelets might be composed of a multisubunit structure due to the formation of homomultimeric or heteromultimeric Trp1-based channels, remains to be resolved. Since the antibody interferes with the entry of Ca 2ϩ , Sr 2ϩ , and Mn 2ϩ , the channel involving hTrp1 in platelets appears to be a nonselective cation channel. Such a non-selective channel may explain earlier reports that TG evokes a rise in cytosolic [Na ϩ ] in human platelets (37).
The inhibition of Ca 2ϩ entry by the anti-hTrp1 antibody occurs as a result of specific binding to the sequence 557-571 since previous incubation of the antibody with the control antigen peptide prevented both detection of the hTrp1 protein in the PM and the interference with Ca 2ϩ entry. The effect of incubation with anti-hTrp1 antibody is not mediated by nonspecific binding of the antibody to different proteins in the PM since the same treatment performed with an antibody not related to Trp or platelet proteins had no effect. These observations demonstrate that the antibody and the vehicle itself are not acting as Ca 2ϩ chelators or affecting any other component of the Ca 2ϩ entry pathway. In addition, incubation of platelets with the anti-hTrp1 antibody did not alter the ability of thrombin to activate its receptor in the PM, as indicated by the lack of effect of this treatment on thrombin-evoked release of Ca 2ϩ from the intracellular stores. Moreover, anti-hTrp1 antibodytreated cells retained their ability to respond to Ca 2ϩ -mobilizing agents such as TG or IP 3 generated after stimulation with thrombin, which indicates that this treatment did not affect the ability of platelets to store Ca 2ϩ in intracellular stores or release Ca 2ϩ from intracellular stores.
In conclusion, the results presented here indicate that naturally expressed hTrp1 channels are components of agonistand store depletion-activated cation entry channels, which par-ticipate in the normal response of platelets to physiological stimuli. In agreement with previous studies in several cell types (19), including platelets (13), activation of this channel might require coupling with the IP 3 receptor located in the membrane of the endoplasmic reticulum, which might be the sensor and transducer of the Ca 2ϩ content in the intracellular stores (38).