Inositol 1,4,5-trisphosphate receptor subtypes are differentially distributed between smooth muscle and endothelial layers of rat arteries

In blood vessels, the ability to control vascular tone depends on extracellular calcium entry and the release of calcium from inositol 1,4,5-trisphosphate receptor (IP3R)-gated stores located in both the endothelial and smooth muscle cells of the vascular wall. Therefore, we examined mRNA expression and protein distribution of IP3R subtypes in intact aorta, basilar and mesenteric arteries of the rat. IP3R1 mRNA was predominantly expressed in all three arteries. Immunohistochemistry showed that IP3R1 was present in both the muscle and endothelial cell layers, while IP3R2 and IP3R3 were largely restricted to the endothelium. Weak expression of IP3R2 was observed in the smooth muscle of the basilar artery. Co-localisation studies of IP3R subtypes with known cellular elements showed no association of any of the three subtypes with the endothelial cell plasma membrane, but a close association between the subtypes and actin filaments was observed in all cell layers. IP3R2 was found to be present near the endothelial cell nucleus. We are the first to demonstrate differential IP3R subtype distribution between the cell layers of the intact vascular wall and hypothesise that this may underlie the diversity of IP3R-dependent responses, such as vasoconstriction, vasodilation and vasomotion, displayed by arteries.     

Calcium regulation of inositol 1,4,5-trisphosphate receptors

Ca2+ exerts both a stimulatory and inhibitory effect on type-I IP3R channel activity. However, the structural determinants of Ca2+ sensing in IP3Rs are not fully understood. Previous studies by others have identified eight domains of the type-I IP3R that bind 45Ca2+ when expressed as GST-fusion proteins. We have mutated six highly conserved acidic residues within the second of these domains (aa378-450) in the full-length IP3R and measured the Ca2+ regulation of IP3-mediated Ca2+ release in COS-7 cells. 45Ca2+ flux assays measured with a maximal [IP3] (1 microM) indicate that one of the mutants retained a Ca2+ sensitivity that was not significantly different from control (E411Q), three of the mutants show an enhanced Ca2+ inhibition (D426N, E428Q and E439Q) and two of the mutants were relatively insensitive to Ca2+ inhibition (D442N and D444N). IP3 dose-response relationships indicated that the sensitivity to Ca2+ inhibition and affinity for IP3 were correlated for three of the constructs. Other mutants with enhanced IP3 sensitivity (e.g. R441Q and a type-II/I IP3R chimera) were also less sensitive to Ca2+ inhibition. We conclude that the acidic residues within the aa378-450 segment are unlikely to represent a single functional Ca2+ binding domain and do not contribute to Ca2+ activation of the receptor. The different effects of the mutations may be related to their location within two clusters of acidic residues identified in the crystal structure of the ligand-binding domain [I. Bosanac, J.R. Alattia, T.K. Mal, et al., Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand, Nature 420 (2002) 696-700]. The data support the view that all IP3R isoforms may display a range of Ca2+ sensitivities that are determined by multiple sites within the protein and markedly influenced by the affinity of the receptor for IP3.     

Inositol 1,4,5-trisphosphate receptor type 1 phosphorylation and regulation by extracellular signal-regulated kinase

Type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is a widely expressed intracellular calcium-release channel found in many cell types. The operation of IP(3)R1 is regulated through phosphorylation by multiple protein kinases. Extracellular signal-regulated kinase (ERK) has been found involved in calcium signaling in distinct cell types, but the underlying mechanisms remain unclear. Here, we present evidence that ERK1/2 and IP(3)R1 bind together through an ERK binding motif in mouse cerebellum in vivo as well as in vitro. ERK-phosphorylating serines (Ser 436) was identified in mouse IP(3)R1 and Ser 436 phosphorylation had a suppressive effect on IP(3) binding to the recombinant N-terminal 604-amino acid residues (N604). Moreover, phosphorylation of Ser 436 in R(224-604) evidently enhance its interaction with the N-terminal "suppressor" region (N223). At last, our data showed that Ser 436 phosphorylation in IP(3)R1 decreased Ca(2+) releasing through IP(3)R1 channels.     

Inositol 1,4,5-trisphosphate 3-kinase A is a novel microtubule-associated protein: PKA-dependent phosphoregulation of microtubule binding affinity

Inositol 1,4,5-trisphosphate 3-kinase A (IP(3)K-A) is a brain specific and F-actin-binding protein. We recently demonstrated that IP(3)K-A modulates a structural reorganization of dendritic spines through F-actin remodeling, which is required for synaptic plasticity and memory formation in brain. However, detailed functions of IP(3)K-A and its regulatory mechanisms involved in the neuronal cytoskeletal dynamics still remain unknown. In the present study, we identified tubulin as a candidate of IP(3)K-A-binding protein through proteomic screening. By various in vitro and in vivo approaches, we demonstrated that IP(3)K-A was a novel microtubule-associated protein (MAP), and the N terminus of IP(3)K-A was a critical region for direct binding to tubulin in dendritic shaft of hippocampal neurons. Moreover, PKA phosphorylated Ser-119 within IP(3)K-A, leading to a significant reduction of microtubule binding affinity. These results suggest that PKA-dependent phosphorylation and microtubule binding of IP(3)K-A are involved in its regulatory mechanism for activity-dependent neuronal events such as local calcium signaling and its synaptic targeting.     

Calcium release of heat-shocked porcine oocytes induced by thimerosal or inositol 1,4,5-trisphosphate (IP3)

The objective of this study was to determine the effect of heat shock (HS) on the Ca(2+) release and the subsequent development in matured porcine oocytes. Oocytes were matured in vitro and randomly allocated to different heat treatments at 41.5 degrees C for 1 (HS1h), 2 (HS2h) or 4h (HS4h). Control groups of oocytes were cultured for 0 or 4h without HS (39 degrees C, C0h, C4h). In Experiment 1 (eight replicates), matured oocytes were activated by thimerosal (200 microM, 10 min) following HS. Among all heated groups, maximal intracellular calcium concentration ([Ca(2+)](i)) was the highest in the HS2h. The lowest [Ca(2+)](i) peak among HS groups was observed in the HS4h, but it was higher than that in the non-heated C4h group (P<0.05). In Experiment 2 (12 replicates), each matured oocyte was injected with IP(3) (0.5mM) and the Ca(2+) transient was recorded. The peak [Ca(2+)](i) in the C4h group was still the lowest among all groups (P<0.05). Total Ca(2+) release in HS2h appeared the highest among all treatments, and it was significantly higher than that in HS1h and C4h groups (P<0.05). In order to clarify the effect of incubation time in vitro (Experiment 3), matured oocytes were cultured at 39 degrees C for 0, 2 and 4h prior to treatment with thimerosal or injected with IP(3) (three replicates). The Ca(2+) release of matured oocytes declined with the prolonged culture (P<0.05). Finally, the development of HS-oocytes was evaluated after parthenogenetic activation (Experiment 4, three replicates), and the proportion of embryos developing to the blastocysts were lower (P<0.05) in the HS groups (31+/-7% to 33+/-1%) than in the control groups (52+/-11% to 56+/-9%). We conclude that HS alters the Ca(2+)-releasing ability of matured pig oocytes, and that heat-shocked oocytes with greater Ca(2+) release incur a low developmental competence after parthenogenetic activation.     

Inositol 1,4,5-trisphosphate receptor contains multiple cavities and L-shaped ligand-binding domains

Calcium concentrations are strictly regulated in all biological cells, and one of the key molecules responsible for this regulation is the inositol 1,4,5-trisphosphate receptor, which was known to form a homotetrameric Ca(2+) channel in the endoplasmic reticulum. The receptor is involved in neuronal transmission via Ca(2+) signaling and for many other functions that relate to morphological and physiological processes in living organisms. We analysed the three-dimensional structure of the ligand-free form of the receptor based on a single-particle technique using an originally developed electron microscope equipped with a helium-cooled specimen stage and an automatic particle picking system. We propose a model that explains the complex mechanism for the regulation of Ca(2+) release by co-agonists, Ca(2+), inositol 1,4,5-trisphosphate based on the structure of multiple internal cavities and a porous balloon-shaped cytoplasmic domain containing a prominent L-shaped density which was assigned by the X-ray structure of the inositol 1,4,5-trisphosphate binding domain.     

Na+-sensitive component of 3-O-methylglucose uptake in frog skeletal muscle

Summary A Na⁺-sensitive uptake of 3-O-methylglucose (3-O-MG), a nonmetabolized sugar, was characterized in frog skeletal muscle. A removal of Na⁺ from the bathing solution reduced 3-O-MG uptake, depending on the amount of Na⁺ removed. At a 3-O-MG concentration of 2mm, the Na⁺-sensitive component of uptake in Ringer's solution was estimated to be about 26% of the total uptake. The magnitude of Na⁺-sensitive component sigmoidally increased with an increase of 3-O-MG in bathing solution, whereas in Na⁺-free Ringer's solution the uptake was proportional to the concentration. The half saturation of the Na⁺-sensitive component was at a 3-O-MG concentration of about 13mm, and the Hill coefficient was 1.4 to 1.6. Phlorizin (5mm), a potent inhibitor specific for Na⁺-coupled glucose transport, reduced the uptake in a solution containing Na⁺ to the level in Na⁺-free Ringer's solution. Glucose of concentrations higher than 20mm suppressed 3-O-MG uptake to a level slightly lower than that in Na⁺-free Ringer's solution. These observations indicate that there are Na⁺-coupled sugar transport systems in frog skeletal muscle which are shared by both glucose and 3-O-MG.     

Post-translational Regulation of the Type III Inositol 1,4,5-Trisphosphate Receptor by miRNA-506

The type III isoform of the inositol 1,4,5-trisphosphate receptor (InsP₃R3) is apically localized and triggers Ca²⁺ waves and secretion in a number of polarized epithelia. However, nothing is known about epigenetic regulation of this InsP3R isoform. We investigated miRNA regulation of InsP₃R3 in primary bile duct epithelia (cholangiocytes) and in the H69 cholangiocyte cell line, because the role of InsP₃R3 in cholangiocyte Ca²⁺ signaling and secretion is well established and because loss of InsP₃R3 from cholangiocytes is responsible for the impairment in bile secretion that occurs in a number of liver diseases. Analysis of the 3′-UTR of human InsP₃R3 mRNA revealed two highly conserved binding sites for miR-506. Transfection of miR-506 mimics into cell lines expressing InsP₃R3–3′UTR-luciferase led to decreased reporter activity, whereas co-transfection with miR-506 inhibitors led to enhanced activity. Reporter activity was abrogated in isolated mutant proximal or distal miR-506 constructs in miR-506-transfected HEK293 cells. InsP₃R3 protein levels were decreased by miR-506 mimics and increased by inhibitors, and InsP₃R3 expression was markedly decreased in H69 cells stably transfected with miR-506 relative to control cells. miR-506-H69 cells exhibited a fibrotic signature. In situ hybridization revealed elevated miR-506 expression in vivo in human-diseased cholangiocytes. Histamine-induced, InsP₃-mediated Ca²⁺ signals were decreased by 50% in stable miR-506 cells compared with controls. Finally, InsP₃R3-mediated fluid secretion was significantly decreased in isolated bile duct units transfected with miR-506, relative to control IBDU. Together, these data identify miR-506 as a regulator of InsP₃R3 expression and InsP₃R3-mediated Ca²⁺ signaling and secretion.     

Metabolism of inositol 1,4,5-trisphosphate in squid photoreceptors

Summary Inositol 1,4,5-trisphosphate (InsP₃) is rapidly formed in squid photoreceptors in response to light, where it is converted sequenctially into inositol bisphosphate (InsP₂) and inositol monophosphate (InsP₁). All of the InsP₃ appears to be degraded to inositol 1,4-bisphosphate via an InsP₃-phosphatase, which is characterized in this study. The enzyme is water-soluble and present in the light-transducing distal segments of squid photoreceptors. It has a K_m of 50 M for InsP₃, requires Mg⁺⁺ for its activity, is maximally active at neutral pH, specifically hydrolyses the 5-phosphate and is inhibited by 2,3-diphosphoglycerate. In these respects, InsP₃-phosphatase of squid is very similar to the enzymes of other cells. Since no InsP₄ or more highly phosphorylated inositols are found in squid photoreceptors, the InsP₃-phosphatase may be important in the regulation of InsP₃ concentration within these cells.Abbreviations InsP ₁ , InsP ₂ , InsP ₃ , InsP ₄ , InsP ₆ inositol monobis-, tris-, tetrakis-, hexakisphosphate, respectively - 2,3-DPG 2,3-diphosphoglycerate - EDTA ethylene diamine tetraacetic acid - DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMSF phenylmethylsulfonyl fluoride     

Ligand-induced conformational changes via flexible linkers in the amino-terminal region of the inositol 1,4,5-trisphosphate receptor

Cytoplasmic Ca2+ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), which functions as a Ca2+ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP(3)R have been reported; those of the IP(3)-binding core (IP(3)R(CORE)) with bound IP(3), and the suppressor domain. This study examines the structural effects of ligand binding on an IP(3)R construct, designated IP(3)R(N), that contains both the IP(3)-binding core and the suppressor domain. Our circular dichroism results reveal that the IP(3)-bound and IP(3)-free states have similar secondary structure content, consistent with preservation of the overall fold within the individual domains. Thermal denaturation data show that, while IP(3) has a large effect on the stability of IP(3)R(CORE), it has little effect on IP(3)R(N), indicating that the suppressor domain is critical to the stability of IP(3)R(N). The NMR data for IP(3)R(N) provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP(3)-bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP(3)R(N) undergoes a change in average conformation in response to IP(3) binding and the presence of Ca2+ in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of IP(3)R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP(3) binding drives the conformational equilibrium toward more compact structures, while the presence of Ca2+ drives it to a more extended set.     

Metabotropic receptor activation,desensitization and sequestration-I: modelling calcium and inositol 1,4,5-trisphosphate dynamics following receptor activation

A mathematical account is given of the processes governing the time courses of calcium ions (Ca2+), inositol 1,4,5-trisphosphate (IP(3)) and phosphatidylinositol 4,5-bisphosphate (PIP(2)) in single cells following the application of external agonist to metabotropic receptors. A model is constructed that incorporates the regulation of metabotropic receptor activity, the G-protein cascade and the Ca2+ dynamics in the cytosol. It is subsequently used to reproduce observations on the extent of desensitization and sequestration of the P(2)Y(2) receptor following its activation by uridine triphosphate (UTP). The theory predicts the dependence on agonist concentration of the change in the number of receptors in the membrane as well as the time course of disappearance of receptors from the plasmalemma, upon exposure to agonist. In addition, the extent of activation and desensitization of the receptor, using the calcium transients in cells initiated by exposure to agonist, is also predicted. Model predictions show the significance of membrane PIP(2) depletion and resupply on the time course of IP(3) and Ca2+ levels. Results of the modelling also reveal the importance of receptor recycling and PIP(2) resupply for maintaining Ca2+ and IP(3) levels during sustained application of agonist.     

An inositol 1,4,5-trisphosphate-6-kinase activity in pea roots

A soluble extract from pea (Pisum sativum L.) roots, when incubated with ATP and inositol 1,4,5-trisphosphate, produced an inositol tetrakisphosphate. The chromatographic properties of this inositol tetrakisphosphate, and of the products formed by its chemical degradation, identify it as inositol 1,4,5,6-tetrakisphosphate. No evidence was obtained for a 3-phosphorylation of inositol 1,4,5-trisphosphate. The importance of these observations with respect to inositol phosphates and calcium signalling in higher plants, is discussed.Abbreviations HPLC high-performance liquid chromatography - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - InsP₄ inositol tetrakisphosphate J.A.C. gratefully acknowledges support from the Agricultural and Food Research Council, U.K., Plant Molecular Biology Initiative.     

Inhibition of mitochondrial calcium uptake rather than efflux impedes calcium release by inositol-1,4,5-trisphosphate-sensitive receptors

Mitochondria modulate cellular Ca²⁺ signals by accumulating the ion via a uniporter and releasing it via Na⁺- or H⁺-exchange. In smooth muscle, inhibition of mitochondrial Ca²⁺ uptake inhibits Ca²⁺ release from the sarcoplasmic reticulum (SR) via inositol-1,4,5-trisphosphate-sensitive receptors (IP₃R). At least two mechanisms may explain this effect. First, localised uptake of Ca²⁺ by mitochondria may prevent negative feedback by cytosolic Ca²⁺ on IP₃R activity, or secondly localised provision of Ca²⁺ by mitochondrial efflux may maintain IP₃R function or SR Ca²⁺ content. To distinguish between these possibilities the role of mitochondrial Ca²⁺ efflux on IP₃R function was examined. IP₃ was liberated in freshly isolated single colonic smooth muscle cells and mitochondrial Na⁺–Ca²⁺ exchanger inhibited with CGP-37157 (10 μM). Mitochondria accumulated Ca²⁺ during IP₃-evoked [Ca²⁺]_c rises and released the ion back to the cytosol (within 15 s) when mitochondrial Ca²⁺ efflux was active. When mitochondrial Ca²⁺ efflux was inhibited by CGP-37157, an extensive and sustained loading of mitochondria with Ca²⁺ occurred after IP₃-evoked Ca²⁺ release. IP₃-evoked [Ca²⁺]_c rises were initially unaffected, then only slowly inhibited by CGP-37157. IP₃R activity was required for inhibition to occur; incubation with CGP-37157 for the same duration without IP₃ release did not inhibit IP₃R. CGP-37157 directly inhibited voltage-gated Ca²⁺ channel activity, however SR Ca²⁺ content was unaltered by the drug. Thus, the gradual decline of IP₃R function that followed mitochondrial Na⁺–Ca²⁺ exchanger inhibition resulted from a gradual overload of mitochondria with Ca²⁺, leading to a reduced capacity for Ca²⁺ uptake. Localised uptake of Ca²⁺ by mitochondria, rather than mitochondrial Ca²⁺ efflux, appears critical for maintaining IP₃R activity.     

Dual regulation of calcium mobilization by inositol 1,4, 5-trisphosphate in a living cell

Changes in cytosolic free calcium ([Ca(2+)](i)) often take the form of a sustained response or repetitive oscillations. The frequency and amplitude of [Ca(2+)](i) oscillations are essential for the selective stimulation of gene expression and for enzyme activation. However, the mechanism that determines whether [Ca(2+)](i) oscillates at a particular frequency or becomes a sustained response is poorly understood. We find that [Ca(2+)](i) oscillations in rat megakaryocytes, as in other cells, results from a Ca(2+)-dependent inhibition of inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release. Moreover, we find that this inhibition becomes progressively less effective with higher IP(3) concentrations. We suggest that disinhibition, by increasing IP(3) concentration, of Ca(2+)-dependent inhibition is a common mechanism for the regulation of [Ca(2+)](i) oscillations in cells containing IP(3)-sensitive Ca(2+) stores.     

Differential expression of type 2 and type 3 inositol 1,4,5-trisphosphate receptor mRNAs in various mouse tissues: in situ hybridization study

The inositol 1,4,5-trisphosphate receptor (IP₃R) is an intracellular Ca²⁺ release channel responsible for mobilizing stored Ca²⁺. Three different receptor types have been molecularly cloned, and their genes have been classified into a family. The gene for the type 1 receptor (IP₃R1) is predominantly expressed in cerebellar Purkinje neurons, but its gene product is localized widely in a variety of tissues; however, there is little information on what types of cells express the other two receptor types, type 2 and type 3 (IP₃R2 and IP₃R3, respectively). We studied the expression of the IP₃R gene family in various mouse tissues by in situ hybridization histochemistry. Compared with IP₃R1, the levels of expression of IP₃R2 and IP₃R3 mRNAs were low in all of the tissues tested. IP₃R2 mRNA was localized in the intralobular duct cells of the submandibular gland, the urinary tubule cells of the kidney, the epithelial cells of epididymal ducts and the follicular granulosa cells of the ovary, while the IP₃R3 mRNA was distributed in gastric cells, salivary and pancreatic acinar cells and the epithelium of the small intestine. All of these cells which express either IP₃R2 or IP₃R3 mRNA are known to have a secretory function in which IP₃/Ca²⁺ signalling has been shown to be involved, and thus either IP₃R2 or IP₃R3 may be a prerequisite to secretion in these cells.     

Inositol 1,4,5-trisphosphate receptor-isoform diversity in cell death and survival

Cell-death and -survival decisions are critically controlled by intracellular Ca^2 + homeostasis and dynamics at the level of the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) play a pivotal role in these processes by mediating Ca^2 + flux from the ER into the cytosol and mitochondria. Hence, it is clear that many pro-survival and pro-death signaling pathways and proteins affect Ca^2 + signaling by directly targeting IP₃R channels, which can happen in an IP₃R-isoform-dependent manner. In this review, we will focus on how the different IP₃R isoforms (IP₃R1, IP₃R2 and IP₃R3) control cell death and survival. First, we will present an overview of the isoform-specific regulation of IP₃Rs by cellular factors like IP₃, Ca^2 +, Ca^2 +-binding proteins, adenosine triphosphate (ATP), thiol modification, phosphorylation and interacting proteins, and of IP₃R-isoform specific expression patterns. Second, we will discuss the role of the ER as a Ca^2 + store in cell death and survival and how IP₃Rs and pro-survival/pro-death proteins can modulate the basal ER Ca^2 + leak. Third, we will review the regulation of the Ca^2 +-flux properties of the IP₃R isoforms by the ER-resident and by the cytoplasmic proteins involved in cell death and survival as well as by redox regulation. Hence, we aim to highlight the specific roles of the various IP₃R isoforms in cell-death and -survival signaling. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Modulation of the activity of the transverse tubule Mg2+ATPase from frog skeletal muscle by a monoclonal antibody in vitro

We have established several hybridoma lines that produce monoclonal antibodies against transverse tubule (t-tubule) proteins from frog skeletal muscle. The specificity of these antibodies was characterized by ELISA and Western immunoblotting with purified t-tubule, sarcoplasmic reticulum and partially purified sarcolemmal membranes. One of the monoclonal antibodies (2/34.4) recognizes a band of 109 000 Da in immunoblots. When purified frog t-tubule vesicles were preincubated with this antibody we observed an increase in the rate of the Mg²⁺-ATPase enzyme (up to six fold) which was dependent on antibody concentration. Immunocytological experiments done on cryostat sections of frog muscle indicate that the antigen recognized by this antibody is localized mainly at the level of the t-tubules (I band) and to a lesser extent at the sarcolemma. These results indicate that monoclonal antibody 2/34.4 recognizes the t-tubule Mg²⁺-ATPase and modulates its activity. This antibody should be useful as a probe on studies designed to study the physiological function of the enzyme.Abbreviations t-tubules transverse-tubules - mAb monoclonal antibody - SR sarcoplasmic reticulum - SL sarcolemma     

Functional role of the calmodulin- and inositol 1,4,5-trisphosphate receptor-binding (CIRB) site of TRPC6 in human platelet activation

Background

All identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP₃Rs)-binding (CIRB) site involved in the regulation of TRPC channel function.

Objectives

To assess the basis of CaM/IP₃Rs binding to the CIRB site of TRPC6 and its role in platelet physiology.

Methods

Protein association was detected by co-immunoprecipitation and Western blotting, Ca²⁺ mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry.

Results

Co-immunoprecipitation of TRPC6 with CaM or the IP₃Rs at different cytosolic free Ca²⁺ concentrations ([Ca²⁺]_c) indicates that the association between these proteins is finely regulated by cytosolic Ca²⁺ via association of CaM and displacement of the IP₃Rs at high [Ca²⁺]_c. Thrombin-stimulated association of TRPC6 with CaM or the IP₃Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca²⁺-dependent and -independent mechanisms. Incorporation of an anti-TRPC6 C-terminal antibody, whose epitope overlaps the CIRB region, impaired the dynamics of the association of TRPC6 with CaM and the IP₃Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca²⁺ entry in the presence of low or high, respectively, extracellular Ca²⁺ concentrations, as well as altered thrombin-evoked platelet aggregation.

Conclusions

Our results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca²⁺ entry and aggregation through its interaction with CaM and IP₃Rs.