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

Abstract

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. 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. store-mediated calcium entry transient receptor potential human Trp inositol 1,4,5-trisphosphate inositol 1,4,5-trisphosphate receptor d-Phe-Pro-Arg-choromethylketone dihydrochloride thapsigargin ionomycin xestospongin C jasplakinolide HEPES-buffered saline plasma membrane Tris-buffered saline/0.1% Tween 20 control antigen peptide bis(o-aminophenoxy)-ethane-N,N,N′,N′-tetra-acetic acid fluorescein isothiocyanate Cytosolic Ca2+ is a ubiquitous modulator of a large number of cellular processes such as contraction, secretion, cell growth, and cell death (for example, Ref. 1Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14669-14671Crossref PubMed Scopus (139) Google Scholar). Elevations in cytosolic free Ca2+ concentration ([Ca2+]i) can be mediated by release of Ca2+ from the intracellular Ca2+ stores and entry of Ca2+ across the plasma membrane (2Rink T.J. Sage S.O. Annu. Rev. Physiol. 1990; 52: 431-439Crossref PubMed Scopus (319) Google Scholar, 3Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1294) Google Scholar). In nonexcitable cells, store-mediated Ca2+ entry (SMCE),1 a mechanism regulated by the filling state of the intracellular Ca2+stores, is a major pathway for Ca2+ influx (4Putney Jr., J.W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1264) Google Scholar, 5Sage S.O. Exp. Physiol. 1997; 82: 807-823Crossref PubMed Scopus (62) Google Scholar). 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 Ca2+ stores and identification of the channel that mediates extracellular Ca2+ 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 IP3 receptors) and the plasma membrane (3Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1294) Google Scholar, 6Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1050) Google Scholar). 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 (7Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 8Rosado J.A. Sage S.O. J. Physiol. (Lond.). 2000; 526: 221-229Crossref Scopus (136) Google Scholar, 9Rosado J.A. Jenner S. Sage S.O. J. Biol. Chem. 2000; 275: 7527-7533Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Studies aiming to identify the channels involved in the conduction of SMCE have focused attention on human homologues ofDrosophila transient receptor potential (Trp) channels (see Ref. 6Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1050) Google Scholar). 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 (10Zhu X. Jiang M. Peyton M.J. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar) and human salivary gland cells (11Liu X.B. Wang W.C. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). 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 (10Zhu X. Jiang M. Peyton M.J. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar). Human platelets have been shown to express mRNA for Trp1 and its splice variant Trp1A (12den Dekker E. Molin D.G. Breikers G. van Oerle R. Akkerman J.W. van Eys G.J. Heemskerk J.W. Biochim. Biophys. Acta. 2001; 1539: 243-255Crossref PubMed Scopus (69) Google Scholar). In addition, Trp1 proteins have been detected in platelets using an anti-hTrp1 antibody specific for the amino acid residues 557–571 (13Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar). Both Trp1 and Trp1A have been shown to form non-selective cation channels regulated by store depletion in a number of expression systems (14Zitt C. Zobel A. Obukhov A.G. Hartneck C. Kalkbrenner F. Lückhoff A. Schiltz G. Neuron. 1996; 16: 1189-1196Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 15Sinkins W.G. Estacion M. Schilling W.P. Biochem. J. 1998; 331: 331-339Crossref PubMed Scopus (93) Google Scholar). In agreement with the conformational coupling hypothesis, functional IP3receptors (IP3Rs) have been shown to be required for the activation of SMCE (13Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar, 16Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (534) Google Scholar), and physical interaction between IP3 receptors and Trp channels has been reported in several transfected cell lines (17Kiselyov K., Xu, X. Mozhayeva G.N. Kuo T. Pessah I. Mignery G.A. Zhu X. Birnbaumer L. Muallem S. Nature. 1998; 396: 478-482Crossref PubMed Scopus (563) Google Scholar, 18Kiselyov K. Mignery G.A. Zhu M.X. Muallem S. Mol. Cell. 1999; 4: 423-429Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 19Birnbaumer L. Boulay G. Brown D. Jiang M. Dietrich A. Mikoshiba K. Zhu X. Qin N. Recent Prog. Horm. Res. 2000; 55: 127-161PubMed Google Scholar, 20Lockwich T.P. Liu X. Singh B.B. Jadlowiec J. Weiland S. Ambudkar I.S. J. Biol. Chem. 2000; 275: 11934-11942Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar) and in platelets endogenously expressing Trp1 (13Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar). In human platelets, this coupling is activated by depletion of the intracellular Ca2+ stores (13Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar). We report here that physiological agonists also stimulate coupling of IP3 receptors to hTrp1 in human platelets and that incubation with anti-Trp1 antibody resulted in a dramatic decrease in agonist- or store depletion-evoked Ca2+ entry, providing strong evidence for the involvement of Trp1 in the SMCE in normal human cells. Fura-2 acetoxymethyl ester (fura-2/AM) was from Texas Fluorescence (Austin, TX). Apyrase (grade VII), aspirin, bovine serum albumin, paraformaldehyde, Nonidet P-40, thrombin, HISTOPAQUE-1119, HISTOPAQUE-1077, and thapsigargin (TG) were from Sigma. Xestospongin C (Xest C), ionomycin (Iono), andd-Phe-Pro-Arg-choromethylketone dihydrochloride (PPACK) were from Calbiochem. Dimethyl bis(o-aminophenoxy)-ethane-N,N,N′,N′-tetra-acetic acid (BAPTA), acetoxymethyl ester, and jasplakinolide (JP) were from Molecular Probes (Leiden, The Netherlands). Anti-hTrp1 polyclonal antibody and control antigen peptide were obtained from Alomone Laboratories (Jerusalem, Israel). T1E3 antibody was kindly provided by Dr. David J. Beech (School of Biomedical Sciences, University of Leeds, UK). FITC-conjugated donkey anti-rabbit IgG antibody, anti-IP3R type II polyclonal antibody (C-20), and horseradish peroxidase-conjugated donkey anti-goat IgG antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody and anti-mouse IgG antibody were from Amersham Biosciences. All other reagents were of analytical grade. 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 × g and aspirin (100 μm) and apyrase as J.A. D. Sage S.O. Biochem. J. 2000; PubMed Scopus Google Scholar). blood leukocytes were obtained by centrifugation using and A. J. Google Scholar). platelets, with were prepared as (13Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar) and in HEPES-buffered saline 5 and with bovine serum and Fluorescence was from of at using a with of and and at in were using the and in of G. M. J. Biol. Chem. Full Text PDF PubMed Scopus Google Scholar). Mn2+ influx was as a of at the presented on an S.O. T.J. T.J. Biochem. J. PubMed Scopus Google Scholar). Ca2+ influx was as the of the in for after of thrombin in the of by of the the of in the of SMCE was after store depletion using Ca2+ influx was using the of the in for after of was to cation entry to from of the plasma membrane by thrombin T.J. Sage S.O. J. Physiol. (Lond.). Scopus Google Scholar) is carried with Ca2+ by the plasma membrane Ca2+ E. G. M. E. PubMed Scopus (93) Google Scholar). entry was in (100 μm) to the of Cytosolic was using the of × were to of in saline for min and for with anti-hTrp1 The platelets were by centrifugation and in saline and with bovine serum the antibody, were with FITC-conjugated donkey anti-rabbit IgG antibody for and in Fluorescence was using a were at and was at platelets were the cells were for min with Nonidet of × were with an of 20 and were by incubation with of anti-hTrp1 polyclonal antibody of T1E3 polyclonal antibody, or of anti-IP3R type II polyclonal antibody and at were by and proteins were of hTrp1 or IP3R type II was using the anti-hTrp1 or T1E3 or in or with in for the antibody, were with horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody or horseradish peroxidase-conjugated donkey anti-goat IgG antibody in in and to reagents for were to were for min at in and were and was as of was using of with the was Recent studies have reported expression of hTrp1 mRNA in human platelets and (12den Dekker E. Molin D.G. Breikers G. van Oerle R. Akkerman J.W. van Eys G.J. Heemskerk J.W. Biochim. Biophys. Acta. 2001; 1539: 243-255Crossref PubMed Scopus (69) Google Scholar). a antibody that recognizes the sequence which is in hTrp1 and not in other reported proteins O'Connell B. R. T. C. Zhu X. Birnbaumer L. Ambudkar I.S. J. Physiol. 1999; PubMed Google Scholar), a of the antibody in human platelets (13Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar). The of the antibody was with the anti-Trp1 antibody which has been shown to be a specific and in the of mammalian Trp1 proteins Beech Res. 2001; PubMed Scopus Google Scholar). shown in after with the antibody, T1E3 the suggesting that both Trp1 in human platelets. Both of Trp1 in or platelets the both were on the shown in after with the antibody, with the antibody a of with the T1E3 antibody the in this or other molecular were not detected in human peripheral blood leukocytes and where hTrp1 mRNA expression has not been detected J. A. C. G. C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). We have reported that depletion of the intracellular Ca2+ stores using thapsigargin coupling between endogenously expressed hTrp1 and the IP3R type II in human platelets of in (13Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar). shown in A 5 and with anti-IP3R type II by and with T1E3 between hTrp1 and IP3R type II the intracellular Ca2+ stores were using the physiological of this the of thrombin was platelets or platelets with the intracellular Ca2+ were with thrombin in the of extracellular Ca2+ (100 was and min platelets were with cells with the Ca2+ as to between and store Sage S.O. Exp. Physiol. PubMed Scopus Google Scholar). We have that thrombin-evoked J.A. Jenner S. Sage S.O. J. Biol. Chem. 2000; 275: 7527-7533Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). with anti-hTrp1 antibody, the of IP3R type II in from but not platelets coupling between hTrp1 and IP3R type II was of in was also in platelets. with anti-hTrp1 a of this in We also with the anti-IP3R type II antibody and for the of hTrp1. with the after with anti-IP3R type hTrp1 was detected in from but not cells thrombin coupling in both control and platelets. the coupling between and hTrp1 is by removal of thrombin, have a of in after with thrombin, the agonist was In thrombin a in in platelets to release of Ca2+ from the stores and Ca2+ entry. after removal of thrombin and incubation of the cells in Ca2+ to refilling of the Ca2+ entry was with that and hTrp1 in to thrombin removal of the agonist that refilling of the Ca2+ stores reversed the of coupling after with thrombin, the agonist was in the of the thrombin to in by was to of the the coupling was maintained We have shown that store depletion-induced and coupling both IP3 and activation of IP3Rs and is by of the cortical actin J.A. Jenner S. Sage S.O. J. Biol. Chem. 2000; 275: 7527-7533Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar, J.A. Sage S.O. Biochem. J. 2001; PubMed Scopus Google Scholar). the mechanism of activation of the coupling between and hTrp1 by thrombin has the the role of IP3 and IP3R activation by using Li+ or the of IP3R C. reported (13Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar), treatment of human platelets for min with 20 C or for with Li+ abolished thrombin-evoked release of Ca2+ from the intracellular stores. shown in incubation of human platelets with C or Li+ the coupling between and hTrp1. These that IP3 and functional required for the coupling with the hTrp1 the role of the cortical actin in thrombin-induced coupling between and hTrp1, the of We have reported that thrombin-induced Ca2+ entry without affecting Ca2+ release from the stores J.A. Sage S.O. J. Physiol. (Lond.). 2000; Scopus Google Scholar). with treatment of human platelets with for min abolished the coupling between and hTrp1 by thrombin The antibody to the of studies recognizes the peptide sequence which to the amino acid residues 557–571 of the human hTrp1 This antibody has been shown to be in and studies (13Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar, O'Connell B. R. T. C. Zhu X. Birnbaumer L. Ambudkar I.S. J. Physiol. 1999; PubMed Google Scholar, M. T. M. M. K. A. M. E. K. T. A. Penner R. M. T. J. Exp. PubMed Scopus (177) Google Scholar, S. and J. Physiol. Scholar). The sequence 557–571 has been shown to be between the 5 and be to be at the of the J. A. C. G. C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, X. Peyton M. Birnbaumer L. 1995; PubMed Scopus Google Scholar). the of the by the anti-hTrp1 antibody, a of shown in incubation of platelets in with anti-hTrp1 antibody by using a FITC-conjugated antibody the of hTrp1 proteins in the that the is The was not to of the antibody as by the detected in with this antibody The in detected after incubation with the anti-hTrp1 antibody was the antibody was for in the of the control antigen peptide the antibody store depletion expression of the hTrp1 in the the using cells. of human platelets with for min in a the of hTrp1 in the this was not results were obtained the studies were in cells with the Nonidet P-40, that the anti-hTrp1 antibody recognizes an on the of the the sequence 557–571 is in the between the and of hTrp1 J. A. C. G. C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, Sage S.O. Exp. Physiol. PubMed Scopus Google Scholar), the antibody to this sequence channel human were for min with of the anti-hTrp1 antibody by depletion of the intracellular Ca2+ stores using to In a a of in platelets to of Ca2+ from intracellular stores. of Ca2+ μm) to the a in of SMCE 5 Incubation with the anti-hTrp1 antibody for min SMCE by and at of and 5 The of the concentration in this was the antibody was for in the of the the of this the of incubation with an antibody to a not to proteins or other was Incubation of platelets for min in the of anti-mouse IgG antibody the for the anti-hTrp1 antibody was to in the or of the SMCE has been to non-selective cation channels (see Ref. S.O. T.J. T.J. Biochem. J. PubMed Scopus Google Scholar). have Mn2+ to the of incubation with anti-hTrp1 antibody on cation entry. This cation can be as a for Ca2+ entry its on at the S.O. T.J. T.J. Biochem. J. PubMed Scopus Google Scholar). of Mn2+ μm) to platelets with intracellular Ca2+ stores resulted in a of 5 as with cells 5 platelets were for min with anti-hTrp1 antibody, Mn2+ entry was The of in platelets with the antibody and with was to and of the in cells with at of and 5 These results that the anti-hTrp1 antibody cation entry in human platelets in a concentration-dependent manner. shown in and incubation of human platelets for min with anti-hTrp1 antibody resulted in a inhibition of the in by the physiological agonist thrombin in In incubation with anti-mouse IgG antibody not with thrombin-induced Ca2+ entry have that thrombin is to cation entry in platelets J.A. Sage S.O. J. Physiol. (Lond.). 2000; Scopus Google Scholar), a concentration of the agonist to this and interference with thrombin-evoked store Ca2+ entry. shown in of thrombin to platelets, in which entry been by depletion of the intracellular Ca2+ stores by treatment with μm) and was to a entry. In a concentration of thrombin entry of platelets with anti-hTrp1 antibody store depletion-induced entry without affecting entry by thrombin The of the channels involved in the cation entry by thrombin In the of Ca2+ (100 incubation with anti-hTrp1 or anti-mouse IgG not the thrombin-evoked in on the release of Ca2+ from intracellular stores These that this treatment was Ca2+ entry internal the entry of by thrombin the for the release of Ca2+ from intracellular stores (see incubation with anti-hTrp1 antibody inhibited thrombin-induced Ca2+ influx by The mechanism that SMCE after depletion of the intracellular Ca2+ stores is not fully understood, and identification of the channels involved in this in the of of this Recent studies have presented evidence for the involvement of mammalian homologues of the Drosophila Trp channels in SMCE in cells J.A. Jenner S. Sage S.O. J. Biol. Chem. 2000; 275: 7527-7533Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 15Sinkins W.G. Estacion M. Schilling W.P. Biochem. J. 1998; 331: 331-339Crossref PubMed Scopus (93) Google Scholar, G. Brown Qin N. Jiang M. Dietrich A. Zhu M.X. Birnbaumer M. Mikoshiba K. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). We have reported that hTrp1 with the IP3R type II in platelets after depletion of the internal Ca2+ stores using (13Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar). The anti-hTrp1 antibody in studies recognizes the sequence which is in hTrp1 and other proteins O'Connell B. R. T. C. Zhu X. Birnbaumer L. Ambudkar I.S. J. Physiol. 1999; PubMed Google Scholar). this antibody, hTrp1 proteins were detected in human platelets, where hTrp1 mRNA expression has been (12den Dekker E. Molin D.G. Breikers G. van Oerle R. Akkerman J.W. van Eys G.J. Heemskerk J.W. Biochim. Biophys. Acta. 2001; 1539: 243-255Crossref PubMed Scopus (69) Google Scholar), but not in human leukocytes hTrp1 mRNA expression J. A. C. G. C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). In the report that thrombin was to coupling between the IP3R type II and expressed hTrp1 proteins in human platelets. This coupling was reversed by removal of the agonist as for store depletion-induced coupling between IP3R type II and hTrp1 IP3 and functional IP3R and of of the cortical actin this is the that coupling between proteins and IP3Rs has been suggesting that coupling between hTrp1 and IP3R type II is a physiological mechanism by which SMCE be activated in platelets. that store depletion results in coupling between hTrp1 and IP3R type II in platelets provide evidence for the involvement of hTrp1 in using an antibody that recognizes the amino acid sequence 557–571 of hTrp1, have the involvement of hTrp1 in the of SMCE in platelets. the sequence 557–571 is in the of the hTrp1 channel J. A. C. G. C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), that of this antibody to the hTrp1 channel with its channel results showing that incubation of platelets with the anti-hTrp1 antibody Ca2+ entry provide strong evidence for the involvement of hTrp1 proteins in the normal of platelets to that IP3 and store-mediated Ca2+ entry. Human platelets express mRNA for hTrp1 and its splice variant (12den Dekker E. Molin D.G. Breikers G. van Oerle R. Akkerman J.W. van Eys G.J. Heemskerk J.W. Biochim. Biophys. Acta. 2001; 1539: 243-255Crossref PubMed Scopus (69) Google Scholar). Both the sequence (12den Dekker E. Molin D.G. Breikers G. van Oerle R. Akkerman J.W. van Eys G.J. Heemskerk J.W. Biochim. Biophys. Acta. 2001; 1539: 243-255Crossref PubMed Scopus (69) Google Scholar) and by the antibody hTrp1 and have been shown to form non-selective cation channels regulated by store depletion in expression systems (14Zitt C. Zobel A. Obukhov A.G. Hartneck C. Kalkbrenner F. Lückhoff A. Schiltz G. Neuron. 1996; 16: 1189-1196Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 15Sinkins W.G. Estacion M. Schilling W.P. Biochem. J. 1998; 331: 331-339Crossref PubMed Scopus (93) Google Scholar). hTrp1 or the channel or the Trp cation channel in platelets be of a to the of or to be the antibody with the entry of and the channel hTrp1 in platelets to be a non-selective cation a non-selective channel that a in cytosolic in human platelets M. N. A. J. Physiol. (Lond.). Scopus Google Scholar). The inhibition of Ca2+ entry by the anti-hTrp1 antibody as a of specific to the sequence 557–571 incubation of the antibody with the control antigen peptide both of the hTrp1 in the and the interference with Ca2+ entry. The of incubation with anti-hTrp1 antibody is not mediated by of the antibody to proteins in the the treatment with an antibody not to Trp or proteins These that the antibody and the not as Ca2+ or affecting other of the Ca2+ entry In addition, incubation of platelets with the anti-hTrp1 antibody not the of thrombin to its receptor in the as by the of of this treatment on thrombin-evoked release of Ca2+ from the intracellular stores. anti-hTrp1 cells to to such as or after with thrombin, which that this treatment not the of platelets to store Ca2+ in intracellular stores or release intracellular stores. In the results presented here that expressed hTrp1 channels of agonist- and store cation entry which in the normal of platelets to physiological In agreement with studies in several cell L. Boulay G. Brown D. Jiang M. Dietrich A. Mikoshiba K. Zhu X. Qin N. Recent Prog. Horm. Res. 2000; 55: 127-161PubMed Google Scholar), including platelets (13Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar), activation of this channel coupling with the IP3 receptor in the membrane of the endoplasmic which be the and of the Ca2+ in the intracellular stores 1990; PubMed Scopus Google Scholar). We Dr. David J. Beech for the of the T1E3