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Intact store-operated calcium entry (SOCE) mechanisms ensure the maintenance of Ca 2+ homeostasis in cardiomyocytes while their dysregulation promotes the development of cardiomyopathies. To better understand this calcium handling process in cardiomyocytes, we sought to identify unknown protein partners of stromal interaction molecule 1 (STIM1), a main regulatory protein of SOCE. We identified the muscle-related coiled-coil protein (MURC), also known as Cavin-4, as a candidate and showed that MURC interacts with STIM1 in cardiomyocytes. This interaction occurs via the HR1 and ERM domains of MURC and STIM1, respectively. Our results also demonstrated that the overexpression of MURC in neonatal rat ventricular myocytes (NRVM) is sufficient to potentiate SOCE and that its HR1 domain is required to mediate this effect. Interestingly, the R140W-MURC mutant, a missense variant of the HR1 domain associated with human dilated cardiomyopathy, exacerbates the SOCE increase in NRVM. Although the endogenous expression of STIM1 and Ca 2+ channel Orai1 is not modulated under these conditions, we showed that MURC increases the interaction between these proteins under resting conditions. Our study provides novel evidence that MURC regulates SOCE by interacting with STIM1 in cardiomyocytes. In addition, we identified a first potential mechanism by which the R140W mutation of MURC may contribute to calcium mishandling and the development of cardiomyopathies., (Copyright © 2019. Published by Elsevier B.V.)

STIM1 plays a crucial role in Ca(2+) homeostasis, particularly in replenishing the intracellular Ca(2+) store following its depletion. In cardiomyocytes, the Ca(2+) content of the sarcoplasmic reticulum must be tightly controlled to sustain contractile activity. The presence of STIM1 in cardiomyocytes suggests that it may play a role in regulating the contraction of cardiomyocytes. The aim of the present study was to determine how STIM1 participates in the regulation of cardiac contractility. Atomic force microscopy revealed that knocking down STIM1 disrupts the contractility of cardiomyocyte-derived HL-1 cells. Ca(2+) imaging also revealed that knocking down STIM1 causes irregular spontaneous Ca(2+) oscillations in HL-1 cells. Action potential recordings further showed that knocking down STIM1 induces early and delayed afterdepolarizations. Knocking down STIM1 increased the peak amplitude and current density of T-type voltage-dependent Ca(2+) channels (T-VDCC) and shifted the activation curve toward more negative membrane potentials in HL-1 cells. Biotinylation assays revealed that knocking down STIM1 increased T-VDCC surface expression and co-immunoprecipitation assays suggested that STIM1 directly regulates T-VDCC activity. Thus, STIM1 is a negative regulator of T-VDCC activity and maintains a constant cardiac rhythm by preventing a Ca(2+) overload that elicits arrhythmogenic events., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Mature T-lymphocytes undergo spontaneous apoptosis in the biobreeding diabetes-prone strain of rats due to the loss of the functional GIMAP5 (GTPase of the immune-associated nucleotide-binding protein 5) protein. The mechanisms underlying the pro-survival function of GIMAP5 in T-cells have not yet been elucidated. We have previously shown that GIMAP5 deficiency in T-cells impairs Ca2+ entry via plasma membrane channels following exposure to thapsigargin or stimulation of the T-cell antigen receptor. In the present study we report that this reduced Ca2+ influx in GIMAP5-deficient T-cells is associated with the inability of their mitochondria to sequester Ca2+ following capacitative entry, which is required for sustained Ca2+ influx via the plasma membrane channels. Consistent with a role for GIMAP5 in regulating mitochondrial Ca2+, overexpression of GIMAP5 in HEK (human embryonic kidney)-293 cells resulted in increased Ca2+ accumulation within the mitochondria. Disruption of microtubules, but not the actin cytoskeleton, abrogated mitochondrial Ca2+ sequestration in primary rat T-cells, whereas both microtubules and actin cytoskeleton were needed for the GIMAP5-mediated increase in mitochondrial Ca2+ in HEK-293 cells. Moreover, GIMAP5 showed partial colocalization with tubulin in HEK-293 cells. On the basis of these findings, we propose that the pro-survival function of GIMAP5 in T-lymphocytes may be linked to its requirement to facilitate microtubule-dependent mitochondrial buffering of Ca2+ following capacitative entry.

Although most signaling responses initiated by tumor necrosis factor-alpha (TNF-alpha) occur in a Ca(2+)-independent fashion, TNF-alpha receptor signaling augments Ca(2+) entry induced by Galpha(q/11) G-protein coupled receptors (GPCRs) in endothelial cells and increases trans-endothelial permeability. The signaling events involved in GPCR-induced Ca(2+) influx have been characterized and involve store-operated Ca(2+) entry facilitated by the Ca(2+) permeable ion channel, transient receptor potential canonical 4 (TRPC4). Little is known about the mechanisms by which TNF-alpha receptor signaling augments GPCR-induced Ca(2+) entry. TNF-alpha Receptor Ubiquitous Signaling and Scaffolding protein (TRUSS) is a tumor necrosis factor receptor-1 (TNF-R1)-associated protein whose gene name is TRPC4-associated protein (TRPC4AP). The goal of our study was to test the hypothesis that TRUSS serves to link TNF-R1 and GPCR-signaling pathways at the level of TRPC4 by: (i) determining if TRUSS and TNF-R1 interact with TRPC4, and (ii) investigating the role of TRUSS, TNF-R1, and TRPC4 in GPCR-induced Ca(2+) signaling. Here, we show that TRUSS and TNF-R1 interact with a sub-family of TRPC channels (TRPC1, 4, and 5). In addition, we show that TRUSS and TNF-R1 function together with TRPC4 to elevate endoplasmic reticulum Ca(2+) filling in the context of reduced endoplasmic reticulum Ca(2+) storage initiated by G-protein coupled m1 muscarinic acetylcholine receptor (m1AchR) signaling. Together, these findings suggest that TNF-R1, TRUSS, and TRPC4 augment Ca(2+) loading of endoplasmic reticulum Ca(2+) stores in the context of m1AchR stimulation and provide new insights into the mechanisms that connect TNF-R1 to GPCR-induced Ca(2+) signaling., ((c) 2010 Wiley-Liss, Inc.)

The inositol 1,4,5-trisphosphate receptor (IP(3)R) is a Ca(2+) release channel that plays a pivotal role in regulating intracellular Ca(2+) levels in resting cells. Three isoforms of IP(3)Rs have been identified, and they all possess a large regulatory domain that covers about 60% of the protein. This regulation is accomplished by interaction with small molecules, posttranslational modifications, and mostly protein-protein interactions. In our search for new binding partners of the IP(3)R, we found that 90-kDa heat-shock protein (Hsp90) binds to the IP(3)R. This interaction increased on stimulation of HEK293T6.11 cells with insulin but not with G(q) protein-coupled receptor (G(q)PCR) agonists. Moreover, the Hsp90 inhibitor geldanamycin (GA) disrupted the interaction between Hsp90 and the IP(3)R. Pretreatment of HEK293T6.11 cells with GA greatly increased the intracellular Ca(2+) release induced by a G(q)PCR agonist. Insulin alone did not induce any intracellular Ca(2+) release. However, insulin diminished the intracellular Ca(2+) release induced by a G(q)PCR agonist. Interestingly, GA abolished the inhibitory effect of insulin on G(q)PCR-induced intracellular Ca(2+) release. Furthermore, in our search for a mechanistic explanation to this phenomenon, we found that inhibition of kinases activated downstream of the insulin receptor greatly increased the interaction between Hsp90 and the IP(3)R. Of greater interest, we found that the simultaneous inhibition of mammalian target of rapamycin and the Src kinase almost completely disrupted the interaction between Hsp90 and the IP(3)R. These results demonstrate that insulin promotes the interaction of Hsp90 with the IP(3)R to dampen its Ca(2+) release activity by a complex mechanism involving mammalian target of rapamycin and the Src kinase.

Calcium channels play important roles in cellular signalling. TRP (transient receptor potential) channels form a superfamily of calcium channels through which Ca(2+) enters the cell. TRPs have six transmembrane segments with a putative pore between the fifth and the sixth segments, and assemble in tetrameric complexes to form functional Ca(2+) channels. They are thus similar to K(V) (voltage-gated potassium channel) channels in terms of structure and molecular determinants that promote subunit assembly. In this review, the molecular determinants mediating the assembly of Drosophila TRP, TRPC (TRP canonical), TRPV (TRP vanilloid) and K(V) channels are described.

Smooth muscle cells in arterioles have pivotal roles in the determination of blood pressure and distribution of local blood flow. The cells exhibit calcium entry in response to passive store depletion, but the mechanisms and relevance of this phenomenon are poorly understood. Previously, a role for canonical transient receptor potential 1 (TRPC1) was indicated, but heterologous expression studies showed TRPC1 to have poor function in isolation, suggesting a requirement for additional proteins. Here we test the hypothesis that TRPC5 is such an additional protein, because TRPC5 forms heteromultimeric channels with TRPC1, and RNA encoding TRPC5 is present in arterioles. Recordings were from arteriolar fragments freshly isolated from rabbit pial membrane. Ionic current in response to store depletion has properties like that of the TRPC1/TRPC5 heteromultimer, and so the effect of the E3-targeted, externally acting, anti-TRPC5 blocking antibody (T5E3) was explored. T5E3 suppressed calcium entry in store-depleted arterioles but had no effect in the absence of store depletion. T5E3 preadsorbed to its antigenic peptide did not inhibit calcium entry. TRPC6 is commonly detected in smooth muscle and is present in the arterioles, but T5E3 had no effect on TRPC6. The data suggest that calcium entry occurring in response to passive store depletion in smooth muscle cells of arterioles involves TRPC5 as well as TRPC1.

Transient receptor potential canonical (TRPC) channels are associated with calcium entry activity in nonexcitable cells. TRPCs can form homo- or heterotetrameric channels, in which case they can assemble together within a subfamily groups. TRPC1, 4, and 5 represent one group, and TRPC3, 6, and 7 represent the other. The molecular determinants involved in promoting subunit tetramerization are not known. To identify them, we generated chimeras by swapping the different domains of TRPC4 with the same regions in TRPC6. We showed that TRPC4 coimmunoprecipitated with the chimeras containing the ankyrin repeats and coiled-coil domains of TRPC4 into TRPC6. However, chimeras containing only the ankyrin repeats or only the coiled-coil domain of TRPC4 did not coimmunoprecipitate with TRPC4. We also showed that a second domain of interaction composed of the pore region and the C-terminal tail is involved in the oligomerization of TRPC4. However, chimeras containing only the pore region or only the C-terminal tail of TRPC4 did not coimmunoprecipitate with TRPC4. Furthermore, we showed that the N terminus of TRPC6 coimmunoprecipitated with the C terminus of TRPC6. Overexpression in HEK293T cells of chimeras that contained an N terminus and a C terminus from different subfamily groups increased intracellular calcium entry subsequent to stimulation of G(q) protein-coupled receptors. These results suggest that two types of interactions are involved in the assembly of the four subunits of the TRPC channel. The first interaction occurs between the N termini and involves two regions. The second interaction occurs between the N terminus and the C terminus and does not appear to be necessary for the activity of TRPCs.

Mutations in the presenilin (PS) genes are linked to the development of early-onset Alzheimer's disease by a gain-of-function mechanism that alters proteolytic processing of the amyloid precursor protein (APP). Recent work indicates that Alzheimer's-disease-linked mutations in presenilin1 and presenilin2 attenuate calcium entry and augment calcium release from the endoplasmic reticulum (ER) in different cell types. However, the regulatory mechanisms underlying the altered profile of Ca(2+) signaling are unknown. The present study investigated the influence of two familial Alzheimer's-disease-linked presenilin2 variants (N141I and M239V) and a loss-of-function presenilin2 mutant (D263A) on the activity of the transient receptor potential canonical (TRPC)6 Ca(2+) entry channel. We show that transient coexpression of Alzheimer's-disease-linked presenilin2 mutants and TRPC6 in human embryonic kidney (HEK) 293T cells abolished agonist-induced TRPC6 activation without affecting agonist-induced endogenous Ca(2+) entry. The inhibitory effect of presenilin2 and the Alzheimer's-disease-linked presenilin2 variants was not due to an increase in amyloid beta-peptides in the medium. Despite the strong negative effect of the presenilin2 and Alzheimer's-disease-linked presenilin2 variants on agonist-induced TRPC6 activation, conformational coupling between inositol 1,4,5-trisphosphate receptor type 3 (IP(3)R3) and TRPC6 was unaffected. In cells coexpressing presenilin2 or the FAD-linked presenilin2 variants, Ca(2+) entry through TRPC6 could still be induced by direct activation of TRPC6 with 1-oleoyl-2-acetyl-sn-glycerol (OAG). Furthermore, transient coexpression of a loss-of-function PS2 mutant and TRPC6 in HEK293T cells enhanced angiotensin II (AngII)- and OAG-induced Ca(2+) entry. These results clearly indicate that presenilin2 influences TRPC6-mediated Ca(2+) entry into HEK293 cells.

Mammalian homologues of the Drosophila transient receptor potential channel (TRPC) are involved in Ca(2+) entry following agonist stimulation of nonexcitable cells. Seven mammalian TRPCs have been cloned but their mechanisms of activation and/or regulation are still the subject of intense research efforts. It has already been shown that calmodulin (CaM) can regulate the activity of Drosophila TRP and TRPL and, more recently, CaM has been shown to interact with mammalian TRPCs. In this study, TRPC6 stably transfected into HEK-293 cells was used to investigate the possible influence of CaM on TRPC6-dependent Ca(2+) entry. Overexpression of TRPC6 in mammalian cells is known to enhance agonist-induced Ca(2+) entry, but not thapsigargin-induced Ca(2+) entry. Here, we show that CaM inhibitors (calmidazolium and trifluoperazine) abolish receptor-operated Ca(2+) entry (ROCE) without affecting thapsigargin-operated Ca(2+) entry and that the activity of CaM is dependent on complexation with Ca(2+). We also show that Ca(2+)-CaM binds to TRPC6 and that the binding can be abolished by CaM inhibitors. These results indicate that CaM is involved in the modulation of ROCE.

Homologues of Drosophilia transient receptor potential (TRP) have been proposed to be unitary subunits of plasma membrane ion channels that are activated as a consequence of active or passive depletion of Ca(2+) stores. In agreement with this hypothesis, cells expressing TRPs display novel Ca(2+)-permeable cation channels that can be activated by the inositol 1,4,5-trisphosphate receptor (IP3R) protein. Expression of TRPs alters cells in many ways, including up-regulation of IP3Rs not coded for by TRP genes, and proof that TRP forms channels of these and other cells is still missing. Here, we document physical interaction of TRP and IP3R by coimmunoprecipitation and glutathione S-transferase-pulldown experiments and identify two regions of IP3R, F2q and F2g, that interact with one region of TRP, C7. These interacting regions were expressed in cells with an unmodified complement of TRPs and IP3Rs to study their effect on agonist- as well as store depletion-induced Ca(2+) entry and to test for a role of their respective binding partners in Ca(2+) entry. C7 and an F2q-containing fragment of IP3R decreased both forms of Ca(2+) entry. In contrast, F2g enhanced the two forms of Ca(2+) entry. We conclude that store depletion-activated Ca(2+) entry occurs through channels that have TRPs as one of their normal structural components, and that these channels are directly activated by IP3Rs. IP3Rs, therefore, have the dual role of releasing Ca(2+) from stores and activating Ca(2+) influx in response to either increasing IP3 or decreasing luminal Ca(2+).

hTrp3 is a human homologue of the Drosophila gene responsible for a transient receptor potential (trp) mutation. When stably expressed in HEK293 cells, hTrp3 formed ion channels that were active under resting conditions but could be further stimulated by carbachol or ATP via endogenous muscarinic or purinergic receptors, respectively. Agonist evoked currents reversed polarity near 0 mV in physiological ionic conditions and were associated with a significant increase in the current variance. These results suggest the involvement of a non-selective cation channel with relatively large unitary amplitude. Consistent with this, resolved unitary events had a conductance of approximately 60 pS in the negative voltage range and an extrapolated reversal potential near 0 mV.

Hormonal stimulation of Gq-protein coupled receptors triggers Ca2+ mobilization from internal stores. This is followed by a Ca2+ entry through the plasma membrane. Drosophila Trp and Trpl proteins have been implicated in Ca2+ entry and three mammalian homologues of Drosophila Trp/Trpl, hTrp1, hTrp3 and bTrp4 (also bCCE) have been cloned and expressed. Using mouse brain RNA as template, we report here the polymerase chain reaction-based cloning and functional expression of a novel Trp, mTrp6. The cDNA encodes a protein of 930 amino acids, the sequence of which is 36.8, 36.3, 43.1, 38.6, and 74. 1% identical to Drosophila Trp and Trpl, bovine Trp4, and human Trp1 and Trp3, respectively. Transient expression of mTrp6 in COS.M6 cells by transfection of the full-length mTrp6 cDNA increases Ca2+ entry induced by stimulation of co-transfected M5 muscarinic acetylcholine receptor with carbachol (CCh), as seen by dual wavelength fura 2 fluorescence ratio measurements. The mTrp6-mediated increase in Ca2+ entry activity was blocked by SKF-96365 and La3+. Ca2+ entry activity induced by thapsigargin was similar in COS cells transfected with or without the mTrp6 cDNA. The thapsigargin-stimulated Ca2+ entry could not be further stimulated by CCh in control cells but was markedly increased in mTrp6-transfected cells. Records of whole cell transmembrane currents developed in response to voltage ramps from -80 to +40 mV in control HEK cells and HEK cells stably expressing mTrp6 revealed the presence of a muscarinic receptor responsive non-selective cation conductance in Trp6 cells that was absent in control cells. Our data support the hypothesis that mTrp6 encodes an ion channel subunit that mediates Ca2+ entry stimulated by a G-protein coupled receptor, but not Ca2+ entry stimulated by intracellular Ca2+ store depletion.

During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca2+ homeostasis and the physiological roles of CCE.

Summary: Capacitative calcium entry (CCE) describes CA2+ influx into cells that replenishes CA2+ stores emptied through the action of IP3 and other agents. It is an essential component of cellular responses to many hormones and growth factors. The molecular basis of this form of Ca2+ entry is complex and may involve more than one type of channel. Studies on visual signal transduction in Drosophila led to the hypothesis that a protein encoded in trp may be a component of CCE channels. We reported the existence of six trp-related genes in the mouse genome. Expression in L cells of small portions of these genes in antisense orientation suppressed CCE. Expression in COS cells of two full-length cDNAs encoding human trp homologs, Htrp1 and Htrp3, increased CCE. This identifies mammalian gene products that participate in CCE. We propose that trp homologs are subunits of CCE channels, not unlike those of classical voltage-gated ion channels.

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for Ca2+ release from a non-mitochondrial intracellular store. An important discrepancy has been observed between the affinity measured in binding studies (Kd) and the apparent affinity obtained in Ca2+ mobilization studies (EC50). It has been proposed that this discrepancy could be due to different experimental conditions used for Ca2+ mobilization studies and for InsP3 binding studies. With the fluorescent indicator Fura-2, we studied InsP3-induced Ca2+ release activity at 7 degrees C and at 37 degrees C, in bovine adrenal cortex microsomes. Under both conditions, the Ca2+ releasing effect of InsP3 (1 microM) was completed within about 2 s, as a result of the quantal process of InsP3 receptor action. The apparent affinity (EC50) observed for InsP3-induced Ca2+ release at 7 degrees C and at 37 degrees C were 0.64 +/- 0.2 microM and 0.9 +/- 0.2 microM respectively. InsP3 degradation studies, at 37 degrees C, indicated that less than 10% of [3H]-InsP3 was degraded within the first 10 s of incubation. InsP3 association rates were evaluated, at low temperature, with increasing concentrations of [3H]-InsP3. These kinetic studies revealed a direct relationship between the initial rate of association (Vi) and InsP3 concentration. From this relationship, we evaluated that the concentration of InsP3 needed to occupy half of the binding sites within the first second of incubation was 271 nM. We conclude that the discrepancy between Kd and EC50 is related to a kinetic constraint dictated by the quantal process by which InsP3 releases Ca2+.

In a wide variety of cells, inositol 1,4,5-trisphosphate (InsP3) is an important second messenger involved in the regulation of intracellular Ca2+ concentration. InsP3 interacts with specific receptors and triggers the release of sequestered Ca2+ from an internal store. We have synthesized a structural analogue of InsP3 by phosphorylation of the free hydroxyl groups of 1,2,4-benzenetriol with dibenzylphosphorochloridate. The product benzene 1,2,4-trisphosphate (BzP3) was shown to interact with InsP3 receptor and InsP3 metabolizing enzymes of bovine adrenal cortex. BzP3 competitively blocked InsP3 binding to adrenal cortex microsomes with a half-maximal efficiency at 34 microM. This affinity was about 10,000 times lower than that of InsP3 for its receptor. The Ca2+ releasing activity of BzP3 on the same microsomal preparation was monitored with the fluorescent indicator fura-2. BzP3 had no agonistic effect on this activity but it was able to inhibit InsP3-induced Ca2+ release in a dose-dependent manner. The activity of InsP3 phosphatase was also studied. BzP3 inhibited the activity of the phosphatase with a half-maximal efficiency of 32 microM. BzP3 was also able to inhibit the activity of the cytosolic InsP3 kinase with a half-maximal efficiency of 6.1 microM. These results show that BzP3 is interacting with the three specific recognition sites for InsP3 in the bovine adrenal cortex. The inhibitory effect of this compound is relatively more potent on the metabolizing enzymes than on the Ca(2+)-mobilizing receptor.

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for Ca2+ release from an internal store whose nature and location remains undefined. To get more information on this intracellular Ca2+ store, a post-nuclear particulate fraction was prepared from bovine adrenal cortex and its Ca2+ uptake and release activities were monitored with the fluorescent indicator Fura-2. In the presence of Mg2+ (2 mM), the particulate preparation showed high ATP-dependent Ca2+ sequestering activity and decreased the ambient Ca2+ concentration to about 150 nM. In the absence of Mg2+, Ca2+ was still sequestered but less efficiently, reaching a level around 170 nM. In the presence of Mg2+, the Ca2+ released by a maximal dose of InsP3 (2 microM) was completely resequestered whereas in the absence of Mg2+, no resequestration occurred even after complete degradation of InsP3. The use of selective agents such as oligomycin, saponin, ionomycin and biliary salts indicated that Ca2+ was stored in three different pools which are distinct from the mitochondria and from inside-out membrane vesicles. Our data also indicate that InsP3 releases Ca2+ from a pool which is filled up by a Mg2(+) -dependent Ca2+ ATPase.

In a wide variety of cells, inositol-1,4,5-triphosphate is a second messenger that interacts with specific intracellular receptors and triggers the release of sequestered Ca2+ from an intracellular store. We have looked at the influence of heparin on the action and metabolism of inositol-1,4,5-triphosphate in the bovine adrenal cortex. Heparin blocked inositol-1,4,5-trisphosphate binding with half-maximal efficiency around 10 micrograms/ml. Scatchard analyses revealed that heparin did not change the affinity but decreased the number of available binding sites. The Ca2+-releasing activity of inositol-1,4,5-trisphosphate was monitored with the fluorescent indicator, fura-2. Heparin blocked this activity with half-maximal effeciency around 10 micrograms/ml. The effect of heparin could be overcome by a supramaximal dose of inositol-1,4,5-trisphosphate (25 microM). The activity of inositol-1,4,5-trisphosphate-3-kinase from bovine adrenal cortex cytosol was also studied. Heparin inhibited the activity of the kinase with a half-maximal effeciency around 0.4 microgram/ml. Lineweaver-Burk plots revealed that this potent effect was noncompetitive. Finally, we observed that heparin is without effect on inositol-1,4,5-trisphosphate-5-phosphatase (at concentrations as high as 2 mg/ml). These results are consistent with the suggestion that the binding sites for inositol-1,4,5-trisphosphate are the intracellular receptors responsible for the Ca2+-mobilizing effects of inositol-1,4,5-trisphosphate. These results also show that the kinase, the phosphatase, and the receptor are three different molecular entities, which are affected in a different manner by heparin.