Analysis of TRPC3-interacting proteins by tandem mass spectrometry

Mammalian transient receptor potential canonical (TRPC) channels are a family of nonspecific cation channels that are activated in response to stimulation of phospholipase C (PLC)-dependent hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate. Despite extensive studies, the mechanism(s) involved in regulation of mammalian TRPC channels remains unknown. Presence of various protein-interacting domains in TRPC channels have led to the suggestion that they associate with proteins that are involved in their function and regulation. This study was directed toward identifying the proteins associated with native TRPC3 using a shotgun proteomic approach. Anti-TRPC3 antibody was used to immunoprecipitate TRPC3 from solubilized rat brain crude membranes under conditions that allow retention of TRPC3 function. Proteins in the TRPC3 (using anti-TRPC3 antibody) and control (using rabbit IgG) immunoprecipitates were separated by SDS-PAGE, the gel was sectioned, and the resolved proteins were digested by trypsin in situ. After extraction of the peptides, the peptides were separated by HPLC and sequences derived by MS/MS. Analysis of the data revealed 64 specific TRPC3-associated proteins which can be grouped in terms of their cellular location and involvement in specific cellular function. Many of the proteins identified have been previously reported as TRPC3-regulatory proteins, such as IP3Rs and vesicle trafficking proteins. In addition, we report novel putative TRPC3-interacting proteins, including those involved in protein endocytosis and neuronal growth. To our knowledge, this is the first comprehensive proteomic analysis of a native TRPC channel. These data reveal potential TRPC3 regulatory proteins and provide novel insights of the mechanism(s) regulating TRPC3 channels as well as the possible cellular functions where the channel might be involved.     

Regulation of TRPC6 channel activity by tyrosine phosphorylation

Various hormonal stimuli and growth factors activate the mammalian canonical transient receptor potential (TRPC) channel through phospholipase C (PLC) activation. However, the precise mechanism of the regulation of TRPC channel activity remains unknown. Here, we provide the first evidence that direct tyrosine phosphorylation by Src family protein-tyrosine kinases (PTKs) is a novel mechanism for modulating TRPC6 channel activity. We found that TRPC6 is tyrosine-phosphorylated in COS-7 cells when coexpressed with Fyn, a member of the Src family PTKs. We also found that Fyn interacts with TRPC6 and that the interaction is mediated by the SH2 domain of Fyn and the N-terminal region of TRPC6 in a phosphorylation-independent manner. In addition, we demonstrated the physical association of TRPC6 with Fyn in the mammalian brain. Moreover, we showed that stimulation of the epidermal growth factor receptor induced rapid tyrosine phosphorylation of TRPC6 in COS-7 cells. This epidermal growth factor-induced tyrosine phosphorylation of TRPC6 was significantly blocked by PP2, a specific inhibitor of Src family PTKs, and by a dominant negative form of Fyn, suggesting that the direct phosphorylation of TRPC6 by Src family PTKs could be caused by physiological stimulation. Furthermore, using single channel recording, we showed that Fyn modulates TRPC6 channel activity via tyrosine phosphorylation. Thus, our findings demonstrated that tyrosine phosphorylation by Src family PTKs is a novel regulatory mechanism of TRPC6 channel activity.     

Desensitization of canonical transient receptor potential channel 5 by protein kinase C

The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channel in mammalian cells. TRPC5 is desensitized rapidly after activation by G protein-coupled receptor. Herein we report our investigation into the desensitization of mTRPC5 and localization of the molecular determinants of this desensitization using mutagenesis. TRPC5 was initially activated by muscarinic stimulation using 100 microM carbachol (CCh) and then decayed rapidly even in the presence of CCh (desensitization). Increased EGTA or omission of MgATP in the pipette solution slowed the rate of this desensitization. The protein kinase C (PKC) inhibitors, 1 microM chelerythrine, 100 nM GF109203X, or PKC peptide inhibitor (19-36), inhibited this desensitization of TRPC5 activated by 100 microM CCh. When TRPC5 current was activated by intracellular GTPgammaS, PKC inhibitors prevented TRPC5 desensitization and the mutation of TRPC5 T972 to alanine slowed the desensitization process dramatically. We conclude that the desensitization of TRPC5 occurs via PKC phosphorylation and suggest that threonine at residue 972 of mouse TRPC5 might be required for its phosphorylation by PKC.     

Design,synthesis and characterization of novel N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors leading to the identification of the selective compound,AC1903

The transient receptor potential cation channel 5 (TRPC5) has been previously shown to affect podocyte survival in the kidney. As such, inhibitors of TRPC5 are interesting candidates for the treatment of chronic kidney disease (CKD). Herein, we report the synthesis and biological characterization of a series of N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors. Work reported here evaluates the benzimidazole scaffold and substituents resulting in the discovery of AC1903, a TRPC5 inhibitor that is active in multiple animal models of CKD.     

The role of transient receptor potential channel blockers in human gastric cancer cell viability

Transient receptor potential cation channel, subfamily M, receptor 7 (TRPM7) is a ubiquitous divalent-selective ion channel with its own kinase domain. Human gastric cancer cells express the TRPM7 channel, and the presence of this channel is essential for cell survival. Recent studies have suggested that 5-lipoxygenase (5-LOX) inhibitors are potent blockers of the TRPM7 channels. The aim of this study was to show the effects of 5-LOX inhibitors on the growth and survival of gastric cancer cells. Among 5-LOX inhibitors, nordihydroguaiaretic acid (NDGA), 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), and 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2,2-dimethylpropanoic acid (MK886) were potent blockers of TRPM7-like currents in gastric cancer cells and also induced cell death. However, zileuton was ineffective in suppressing TRPM7-like current activity and inducing cell death. Moreover, a specific transient receptor potential cation channel, subfamily C, member 3 (TRPC3) inhibitor, a pyrazole compound (Pyr3), and a specific melastatin TRP (TRPM4) inhibitor, 9-phenanthrol, did not affect TRPM7-like currents or induce cell death. We conclude that TRPM7 has an important role in the growth and survival of gastric cancer cells and a likely potential target for the pharmacological treatment of gastric cancer.     

Canonical Transient Receptor Potential Channel 3 Contributes to Febrile Seizure Inducing Neuronal Cell Death and Neuroinflammation

Febrile seizure (FS) counts as the most common seizures symptom in children undergoing recurrent seizures, posing a high risk to developing subsequent temporal lobe epilepsy. Canonical transient receptor potential channel (TRPC) members are identified as the FS-related genes in hyperthermia prone rats. However, the role of TRPC3 in hyperthermia-induced FS rats remains unclear. In the present study, we investigated whether TRPC3 functionally contributes to the development of FSs. Elevated TRPC3 mRNA and protein levels was detected in hyperthermia-induced FS rats and rat hippocampal neuron cells. The specific inhibitor of TRPC3, Pyr3, remarkably attenuated the susceptibility and severity of seizures, neuronal cell death, and neuroinflammation in FS rats. Conversely, NCX3 activation was apparently suppressed in rats subjected to recurrent FS and rat hippocampal neuron cells. The expression of NCX3 was up-regulated after TRPC3 inhibition in vivo and in vitro. Furthermore, an interaction between TRPC3 and NCX3 was detected by co-immunoprecipitation. Inhibition of TRPC3 suppressed intracellular Ca²⁺ levels in hyperthermia-treated hippocampal neuronal cells. In conclusion, our findings supported that TRPC3 functions as a critical regulator of seizure susceptibility and targeting TRPC3 may be a new therapeutic strategy for FS.     

Potentiation of TRPC5 by protons

Mammalian members of the classical transient receptor potential channel subfamily (TRPC) are Ca(2+)-permeable cation channels involved in receptor-mediated increases in intracellular Ca(2+). TRPC4 and TRPC5 form a group within the TRPC subfamily and are activated in a phospholipase C-dependent manner by an unidentified messenger. Unlike most other Ca(2+)-permeable channels, TRPC4 and -5 are potentiated by micromolar concentrations of La(3+) and Gd(3+). This effect results from an action of the cations at two glutamate residues accessible from the extracellular solution. Here, we show that TRPC4 and -5 respond to changes in extracellular pH. Lowering the pH increased both G protein-activated and spontaneous TRPC5 currents. Both effects were already observed with small reductions in pH (from 7.4 to 7.0) and increased up to pH 6.5. TRPC4 was also potentiated by decreases in pH, whereas TRPC6 was only inhibited, with a pIC(50) of 5.7. Mutation of the glutamate residues responsible for lanthanoid sensitivity of TRPC5 (E543Q and E595Q) modified the potentiation of TRPC5 by acid. Further evidence for a similarity in the actions of lanthanoids and H(+) on TRPC5 is the reduction in single channel conductance and dramatic increase in channel open probability in the presence of either H(+) or Gd(3+) that leads to larger integral currents. In conclusion, the high sensitivity of TRPC5 to H(+) indicates that, in addition to regulation by phospholipase C and other factors, the channel may act as a sensor of pH that links decreases in extracellular pH to Ca(2+) entry and depolarization.     

Expression of functional receptor-coupled TRPC3 channels in DT40 triple receptor InsP3 knockout cells

The TRPC3 channel, an intensively studied member of the widely expressed transient receptor potential (TRP) family, is a Ca(2+)-conducting channel activated in response to phospholipase C-coupled receptors. Despite scrutiny, the receptor-induced mechanism to activate TRPC3 channels remains unclear. Evidence indicates TRPC3 channels interact directly with intracellular inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) and that channel activation is mediated through coupling to InsP(3)Rs. TRPC3 channels were expressed in DT40 chicken B lymphocytes in which all three InsP(3)R genes were deleted (DT40InsP(3)R-k/o). Endogenous B-cell receptors (BCR) coupled through Syk kinase to phospholipase C-gamma (PLC-gamma) activated the expressed TRPC3 channels in both DT40w/t and DT40InsP(3)R-k/o cells. The diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) also activated TRPC3 channels independently of InsP(3)Rs. BCR-induced TRPC3 activation was blocked by the PLC enzymic inhibitor, U-73122, and also blocked by wortmannin-induced PLC substrate depletion. Neither U-73122 nor wortmannin modified either OAG-induced TRPC3 activation or store-operated channel activation in DT40 cells. Cotransfection of cells with both G protein-coupled M5 muscarinic receptors and TRPC3 channels resulted in successful M5 coupling to open TRPC3 channels mediated by PLC-beta. We conclude that TRPC3 channels are activated independently of InsP(3)Rs through DAG production resulting from receptor-mediated activation of either PLC-gamma or PLC-beta.     

Selective association of TRPC channel subunits in rat brain synaptosomes

TRPC genes encode a ubiquitous family of ion channel proteins responsible for Ca(2+) influx following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. These channels may be localized to large multimeric signaling complexes via association with PDZ-containing scaffolding proteins. Based on sequence homology, the TRPC channel family can be divided into two major subgroups: TRPC1, -C4, and -C5 and TRPC3, -C6, and -C7. Although TRPC channels are thought to be tetramers, the actual subunit composition remains unknown. To determine subunit arrangement, individual TRPC channel pairs were heterologously expressed in Sf9 insect cells and immunoprecipitated using affinity-purified rabbit polyclonal antibodies specific for each channel subtype. Reciprocal co-immunoprecipitations showed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate but that cross-association between the two major subgroups does not occur. Additionally, the interaction between each TRPC channel and the PDZ-containing protein, INAD (protein responsible for the inactivation-no-after-potential Drosophila mutant), was examined. TRPC1, -C4, and -C5 co-immunoprecipitated with INAD, whereas TRPC3, -C6, and -C7 did not. To define channel subunit interactions in vivo, immunoprecipitations were performed from isolated rat brain synaptosomal preparations. The results revealed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate in both cortex and cerebellum but that cross-association between the two major subgroups does not occur. These results demonstrate that TRPC channels are present in nerve terminals and provide the first direct evidence for selective assembly of channel subunits in vivo.     

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.     

E3-targeted anti-TRPC5 antibody inhibits store-operated calcium entry in freshly isolated pial arterioles

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.     

Inhibition of transient receptor potential canonical channels impairs cytokinesis in human malignant gliomas

Abstract.  Objectives : Glial-derived primary brain tumours, gliomas, are among the fastest growing malignancies and present a huge clinical challenge. Research suggests an important, yet poorly understood, role of ion channels in growth control of normal and malignant cells. In this study, we sought to functionally characterize Transient Receptor Potential Canoncial (TRPC) channels in glioma cell proliferation. TRPC channels form non-selective cation channels that have been suggested to represent a Ca²⁺ influx pathway impacting cellular growth. Materials and Methods : Employing a combination of molecular, biochemical and biophysical techniques, we characterized TRPC channels in glioma cells. Results : We showed consistent expression of four channel family members (TRPC-1, -3, -5, -6) in glioma cell lines and acute patient-derived tissues. These channels gave rise to small, non-voltage-dependent cation currents that were blocked by the TRPC inhibitors GdCl₃, 2-APB, or SKF96365. Importantly, TRPC channels contributed to the resting conductance of glioma cells and their acute pharmacological inhibition caused an ~10 mV hyperpolarization of the cells' resting potential. Additionally, chronic application of the TRPC inhibitor SKF96365 caused near complete growth arrest. A detailed analysis, by fluorescence-activated cell sorting and time-lapse microscopy, showed that growth inhibition occurred at the G₂+ M phase of the cell cycle with cytokinesis defects. Cells underwent incomplete cell divisions and became multinucleate, enlarged cells. Conclusions : Nuclear atypia and enlarged cells are histopathological hallmarks for glioblastoma multiforme , the highest grade glioma, suggesting that a defect in TRPC channel function may contribute to cellular abnormalities in these tumours.     

Rapid vesicular translocation and insertion of TRP channels

The broadly expressed transient receptor potential (TRP) family of ion channels are permeant to cations, most resulting in increased intracellular calcium. However, their regulation and gating is not well understood. Here, we report that growth factor stimulation initiates the rapid translocation of the transient receptor potential ion channel, TRPC5, from vesicles held in reserve just under the plasma membrane. This process, which we term 'rapid vesicular insertion of TRP' (RiVIT), dramatically increases membrane-associated TRPC5 channels and functional TRPC5 current, resulting in tight spatial-temporal control of these Ca(2+)-permeant nonselective channels. Epidermal growth factor (EGF)-induced incorporation of functional TRP channels requires phosphatidylinositide 3-kinase (PI(3)K), the Rho GTPase Rac1 and phosphatidylinositol 4-phosphate 5-kinase (PIP(5)K alpha). The increase in TRPC5 availability affects neurite extension rates in cultured hippocampal neurons, and may be a general mechanism for initiating Ca(2+) influx and cell morphological changes in response to stimuli.     

Involvement of calmodulin and myosin light chain kinase in activation of mTRPC5 expressed in HEK cells

The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channels in mammalian cells. Because TRPC channels have calmodulin (CaM) binding sites at their COOH termini, we investigated the effect of CaM on mTRPC5. TRPC5 was initially activated by muscarinic stimulation with 50 microM carbachol and then decayed rapidly even in the presence of carbachol. Intracellular CaM (150 microg/ml) increased the amplitude of mTRPC5 current activated by muscarinic stimulation. CaM antagonists (W-7 and calmidazolium) inhibited mTRPC5 currents when they were applied during the activation of mTRPC5. Pretreatment of W-7 and calmidazolium also inhibited the activation of mTRPC5 current. Inhibitors of myosin light chain kinase (MLCK) inhibited the activation of mTRPC5 currents, whereas inhibitors of CaM-dependent protein kinase II did not. Small interfering RNA against cardiac type MLCK also inhibited the activation of mTRPC5 currents. However, inhibitors of CaM or MLCK did not show any effect on GTPgammaS-induced currents. Application of both Rho kinase inhibitor and MLCK inhibitor inhibited GTPgammaS-induced currents. We conclude that CaM and MLCK modulates the activation process of mTRPC5.     

Stimulation of TRPC5 cationic channels by low micromolar concentrations of lead ions (Pb)

Lead toxicity is long-recognised but continues to be a major public health problem. Its effects are wide-ranging and include induction of hyper-anxiety states. In general it is thought to act by interfering with Ca²⁺ signalling but specific targets are not clearly identified. Transient receptor potential canonical 5 (TRPC5) is a Ca²⁺-permeable ion channel that is linked positively to innate fear responses and unusual amongst ion channels in being stimulated by trivalent lanthanides, which include gadolinium. Here we show investigation of the effect of lead, which is a divalent ion (Pb²⁺). Intracellular Ca²⁺ and whole-cell patch-clamp recordings were performed on HEK 293 cells conditionally over-expressing TRPC5 or other TRP channels. Extracellular application of Pb²⁺ stimulated TRPC5 at concentrations greater than 1 μM. Control cells without TRPC5 showed little or no response to Pb²⁺ and expression of other TRP channels (TRPM2 or TRPM3) revealed partial inhibition by 10 μM Pb²⁺. The stimulatory effect on TRPC5 depended on an extracellular residue (E543) near the ion pore: similar to gadolinium action, E543Q TRPC5 was resistant to Pb²⁺ but showed normal stimulation by the receptor agonist sphingosine-1-phosphate. The study shows that Pb²⁺ is a relatively potent stimulator of the TRPC5 channel, generating the hypothesis that a function of the channel is to sense metal ion poisoning.     

Co-expression of non-selective cation channels of the transient receptor potential canonical family in central aminergic neurones

The mammalian transient receptor potential canonical (TRPC) group of channels is a family of Ca2+-permeable cation channels that are activated following receptor-mediated stimulation of different isoforms of phospholipase C. In vitro TRPC proteins can form hetero- or homo-oligomeric channels. We performed single-cell RT-PCR analysis to reveal the co-expression of seven TRPC channels in identified rat aminergic neurones. All serotonergic neurones of the dorsal raphe (DR), the majority of histaminergic (tuberomamillary nucleus; TMN) and dopaminergic cells of the ventral tegmental area (VTA), as well as some GABAergic neurones from the VTA, expressed at least one variant of TRPC channels. No TRPC channel expression was found in the locus coeruleus. In raphe neurones TRPC6 and TRPC5 mRNAs occurred most frequently. In VTA and TMN co-expression of TRPC4 with TRPC5 and TRPC6 with TRPC7 was not found in individual neurones (in contrast to the whole-brain regions). Their co-expression in non-neuronal cells could not be excluded. The neonatal TRPC3 subunit was rarely seen. In DR, but not in the other nuclei studied, the expression of orexin receptors correlated with the expression of TRPC channels. We conclude that several TRPC channel populations exist in individual neurones and that their subunit co-expression pattern is region and cell-type specific.     

A benzothiadiazine derivative and methylprednisolone are novel and selective activators of transient receptor potential canonical 5 (TRPC5) channels

The transient receptor potential canonical channel 5 (TRPC5) is a Ca²⁺-permeable ion channel, which is predominantly expressed in the brain. TRPC5-deficient mice exhibit a reduced innate fear response and impaired motor control. In addition, outgrowth of hippocampal and cerebellar neurons is retarded by TRPC5. However, pharmacological evidence of TRPC5 function on cellular or organismic levels is sparse. Thus, there is still a need for identifying novel and efficient TRPC5 channel modulators.We, therefore, screened compound libraries and identified the glucocorticoid methylprednisolone and N-[3-(adamantan-2-yloxy)propyl]-3-(6-methyl-1,1-dioxo-2H-1λ⁶,2,4-benzothiadiazin-3-yl)propanamide (BTD) as novel TRPC5 activators. Comparisons with closely related chemical structures from the same libraries indicate important substructures for compound efficacy. Methylprednisolone activates TRPC5 heterologously expressed in HEK293 cells with an EC₅₀ of 12 μM, while BTD-induced half-maximal activation is achieved with 5-fold lower concentrations, both in Ca²⁺ assays (EC₅₀ = 1.4 μM) and in electrophysiological whole cell patch clamp recordings (EC₅₀ = 1.3 μM). The activation resulting from both compounds is long lasting, reversible and sensitive to clemizole, a recently established TRPC5 inhibitor. No influence of BTD on homotetrameric members of the remaining TRPC family was observed. On the main sensory TRP channels (TRPA1, TRPV1, TRPM3, TRPM8) BTD exerts only minor activity. Furthermore, BTD can activate heteromeric channel complexes consisting of TRPC5 and its closest relatives TRPC1 or TRPC4, suggesting a high selectivity of BTD for channel complexes bearing at least one TRPC5 subunit.     

Protein kinase Calpha phosphorylates the TRPC1 channel and regulates store-operated Ca2+ entry in endothelial cells

The TRPC1 (transient receptor potential canonical-1) channel is a constituent of the nonselective cation channel that mediates Ca2+ entry through store-operated channels (SOCs) in human endothelial cells. We investigated the role of protein kinase Calpha (PKCalpha) phosphorylation of TRPC1 in regulating the opening of SOCs. Thrombin or thapsigargin added to the external medium activated Ca2+ entry after Ca2+ store depletion, which we monitored by changes in cellular Fura 2 fluorescence. Internal application of the metabolism-resistant analog of inositol 1,4,5-trisphosphate (IP3) activated an inward cationic current within 1 min, which we recorded using the whole cell patch clamp technique. La3+ or Gd3+ abolished the current, consistent with the known properties of SOCs. Pharmacological (G?6976) or genetic (kinase-defective mutant) inhibition of PKCalpha markedly inhibited IP3-induced activation of the current. Thrombin or thapsigargin also activated La3+-sensitive Ca2+ entry in a PKCalpha-dependent manner. We determined the effects of a specific antibody directed against an extracellular epitope of TRPC1 to address the functional importance of TRPC1. External application of the antibody blocked thrombin- or IP3-induced Ca2+ entry. In addition, we showed that addithrombin or thapsigargin induced phosphorylation of TRPC1 within 1 min. Thrombin failed to induce TRPC1 phosphorylation in the absence of PKCalpha activation. Phosphorylation of TRPC1 and the resulting Ca2+ entry were essential for the increase in permeability induced by thrombin in confluent endothelial monolayers. These results demonstrate that PKCalpha phosphorylation of TRPC1 is an important determinant of Ca2+ entry in human endothelial cells.