Ca(2+)-calmodulin regulates receptor-operated Ca(2+) entry activity of TRPC6 in HEK-293 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.     

Calcium store contents control the expression of TRPC1, TRPC3 and TRPV6 proteins in LNCaP prostate cancer cell line

The mammalian homologues of the Drosophila transient receptor potential (TRP) represent a superfamily of ion channels involved in Ca(2+) homeostasis. Several members of this family are activated either by a depletion of the internal stores of Ca(2+) or by stimulation of G protein-coupled receptors. In androgen responsive prostate cancer cell line LNCaP, TRPC1, TRPC4 and/or TRPV6 have been reported to function as store-operated channels (SOCs) while TRPC3 might be involved in the response to agonist stimulation, possibly through the induction of diacylglycerol production by phospholipase C. However, the control of expression of these TRP proteins is largely unknown. In the present study, we have investigated if the expression of the TRP proteins possibly involved in the capacitative influx of calcium is influenced by the contents of Ca(2+) in the endoplasmic reticulum. Using real-time PCR and Western blot techniques, we show that the expression of TRPC1, TRPC3 and TRPV6 proteins increases after a prolonged (24-48 h) depletion of the stores with thapsigargin. The upregulation of TRPC1 and TRPC3 depends on the store contents level and involves the activation of the Ca(2+)/calmodulin/calcineurin/NFAT pathway. Functionally, cells overexpressing TRPC1, TRPC3 and TRPV6 channels after a prolonged depletion of the stores showed an increased [Ca(2+)](i) response to alpha-adrenergic stimulation. However, the store-operated entry of calcium was unchanged. The isolated overexpression of TRPV6 (without overexpression of TRPC1 and TRPC3) did not produce this increased response to agonists, therefore suggesting that TRPC1 and/or TRPC3 proteins are responsible for the response to alpha-adrenergic stimulation but that TRPC1, TPRC3 and TRPV6 proteins, expressed alone or concomitantly, are not sufficient for SOC formation.     

A functional link between store-operated and TRPC channels revealed by the 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2

The coupling between receptor-mediated Ca2+ store release and the activation of "store-operated" Ca2+ entry channels is an important but so far poorly understood mechanism. The transient receptor potential (TRP) superfamily of channels contains several members that may serve the function of store-operated channels (SOCs). The 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2, is a recently described inhibitor of SOC activity in T-lymphocytes. We compared its action on SOC activation in a number of cell types and evaluated its modification of three specific TRP channels, canonical transient receptor potential 3 (TRPC3), TRPC5, and TRPV6, to throw light on any link between SOC and TRP channel function. Using HEK293 cells, DT40 B cells, and A7r5 smooth muscle cells, BTP2 blocked store-operated Ca2+ entry within 10 min with an IC50 of 0.1-0.3 microM. Store-operated Ca2+ entry induced by Ca2+ pump blockade or in response to muscarinic or B cell receptor activation was similarly sensitive to BTP2. Using the T3-65 clonal HEK293 cell line stably expressing TRPC3 channels, TRPC3-mediated Sr2+ entry activated by muscarinic receptors was also blocked by BTP2 with an IC50 of <0.3 microM. Importantly, direct activation of TRPC3 channels by diacylglycerol was also blocked by BTP2 (IC50 approximately 0.3 microM). BTP2 still blocked TRPC3 in medium with N-methyl-D-glucamine-chloride replacing Na+, indicating BTP2 did not block divalent cation entry by depolarization induced by activating monovalent cation entry channels. Whereas whole-cell carbachol-induced TRPC3 current was blocked by 3 microM BTP2, single TRPC3 channel recordings revealed persistent short openings suggesting BTP2 reduces the open probability of the channel rather than its pore properties. TRPC5 channels transiently expressed in HEK293 cells were blocked by BTP2 in the same range as TRPC3. However, function of the highly Ca(2+)-selective TRPV6 channel, with many channel properties akin to SOCs, was entirely unaffected by BTP2. The results indicate a strong functional link between the operation of expressed TRPC channels and endogenous SOC activity.     

Protein kinase C-dependent phosphorylation of transient receptor potential canonical 6 (TRPC6) on serine 448 causes channel inhibition

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry following the stimulation of a G(q)-protein coupled or tyrosine kinase receptor. A dysregulation of TRPC6 activity causes abnormal proliferation of smooth muscle cells and glomerulosclerosis. In the present study, we investigated the regulation of TRPC6 activity by protein kinase C (PKC). We showed that inhibiting PKC with GF1 or activating it with phorbol 12-myristate 13-acetate potentiated and inhibited agonist-induced Ca(2+) entry, respectively, into cells expressing TRPC6. Similar results were obtained when TRPC6 was directly activated with 1-oleyl-2-acetyl-sn-glycerol. Activation of the cells with carbachol increased the phosphorylation of TRPC6, an effect that was prevented by the inhibition of PKC. The target residue of PKC was identified by an alanine screen of all canonical PKC sites on TRPC6. Unexpectedly, all the mutants, including TRPC6(S768A) (a residue previously proposed to be a target for PKC), displayed PKC-dependent inhibition of channel activity. Phosphorylation prediction software suggested that Ser(448), in a non-canonical PKC consensus sequence, was a potential target for PKCδ. Ba(2+) and Ca(2+) entry experiments revealed that GF1 did not potentiate TRPC6(S448A) activity. Moreover, activation of PKC did not enhance the phosphorylation state of TRPC6(S448A). Using A7r5 vascular smooth muscle cells, which endogenously express TRPC6, we observed that a novel PKC isoform is involved in the inhibition of the vasopressin-induced Ca(2+) entry. Furthermore, knocking down PKCδ in A7r5 cells potentiated vasopressin-induced Ca(2+) entry. In summary, we provide evidence that PKCδ exerts a negative feedback effect on TRPC6 through the phosphorylation of Ser(448).     

TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating the interaction between inositol 1,4,5-trisphosphate receptor and RACK1

Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-mediated intracellular Ca(2+) release and associated with phosphatidylinositol 4,5-bisphosphate hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with receptor for activated C-kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also the interaction of IP(3)R with RACK1 and IP(3)-dependent intracellular Ca(2+) release. We show that TRPC3 interacts with RACK1 via N-terminal residues Glu-232, Asp-233, Glu-240, and Glu-244. Carbachol (CCh) stimulation of HEK293 cells expressing wild type TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP(3)R complex and increased surface expression of TRPC3 and Ca(2+) entry. Mutation of the putative RACK1 binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP(3)R complex as well as increased surface expression of TRPC3 and receptor-operated Ca(2+) entry were also attenuated. Importantly, CCh-induced intracellular Ca(2+) release was significantly reduced as was RACK1-IP(3)R association without any change in thapsigargin-stimulated Ca(2+) release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP(3)R association and decreased CCh-stimulated Ca(2+) entry. Furthermore, an oscillatory pattern of CCh-stimulated intracellular Ca(2+) release was seen in these cells compared with the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca(2+) release was seen after CCh stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP(3)R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca(2+) release by mediating interaction between IP(3)R and RACK1.     

Involvement of phosphoinositide 3-kinase and PTEN protein in mechanism of activation of TRPC6 protein in vascular smooth muscle cells

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry after the stimulation of a G(q)-protein-coupled or tyrosine-kinase receptor. TRPC6 translocates to the plasma membrane upon stimulation and remains there as long as the stimulus is present. However, the mechanism that regulates the trafficking and activation of TRPC6 are unclear. In this study we showed phosphoinositide 3-kinase and its antagonistic phosphatase, PTEN, are involved in the activation of TRPC6. The inhibition of PI3K by PIK-93, LY294002, or wortmannin decreased carbachol-induced translocation of TRPC6 to the plasma membrane and carbachol-induced net Ca(2+) entry into T6.11 cells. Conversely, a reduction of PTEN expression did not affect carbachol-induced externalization of TRPC6 but increased Ca(2+) entry through TRPC6 in T6.11 cells. We also showed that the PI3K/PTEN pathway regulates vasopressin-induced translocation of TRPC6 to the plasma membrane and vasopressin-induced Ca(2+) entry into A7r5 cells, which endogenously express TRPC6. In summary, we provided evidence that the PI3K/PTEN pathway plays an important role in the translocation of TRPC6 to the plasma membrane and may thus have a significant impact on Ca(2+) signaling in cells that endogenously express TRPC6.     

TRPC4 can be activated by G-protein-coupled receptors and provides sufficient Ca(2+) to trigger exocytosis in neuroendocrine cells

Transient receptor potential (TRP) channels form a large family of plasma membrane cation channels. Mammalian members of the "short" TRP family (TRP channel (TRPC) 1-7 are Ca(2+)-permeant, non-selective cation channels that are widely expressed in various cell types, including neurons. TRPC activity is linked through unknown mechanisms to G-protein-coupled receptors or receptor tyrosine kinases that activate phospholipase C. To investigate the properties and function of TRPC4 in neuronally derived cells, we transiently expressed mouse TRPC4 and histamine H(1) receptor in mouse adrenal chromaffin cells and PC12 cells. Histamine, but not thapsigargin, stimulated Mn(2+) influx in transfected cells. In the whole-cell patch clamp mode, histamine triggered a transient current in TRPC4-expressing cells. No current was evoked by perfusion with inositol 1,4,5-trisphosphate. When exocytosis was monitored with the capacitance detection technique, the magnitude of the membrane capacitance increase (Delta C(m)) on application of histamine in H(1) receptor/TRPC4-expressing chromaffin cells was comparable with that triggered by a train of depolarizing pulses. Our results indicate that TRPC4 channels behave as receptor, but not store-operated, channels in neuronally derived cells. TRPC4 channels can provide sufficient Ca(2+) influx to trigger a robust secretory response in voltage-clamped neurosecretory cells. Similar mechanisms may modulate exocytosis in other neuronal systems.     

The overexpression of presenilin2 and Alzheimer's-disease-linked presenilin2 variants influences TRPC6-enhanced Ca2+ entry into HEK293 cells

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.     

Nitric oxide activates TRP channels by cysteine S-nitrosylation

Transient receptor potential (TRP) proteins form plasma-membrane cation channels that act as sensors for diverse cellular stimuli. Here, we report a novel activation mechanism mediated by cysteine S-nitrosylation in TRP channels. Recombinant TRPC1, TRPC4, TRPC5, TRPV1, TRPV3 and TRPV4 of the TRPC and TRPV families, which are commonly classified as receptor-activated channels and thermosensor channels, induce entry of Ca(2+) into cells in response to nitric oxide (NO). Labeling and functional assays using cysteine mutants, together with membrane sidedness in activating reactive disulfides, show that cytoplasmically accessible Cys553 and nearby Cys558 are nitrosylation sites mediating NO sensitivity in TRPC5. The responsive TRP proteins have conserved cysteines on the same N-terminal side of the pore region. Notably, nitrosylation of native TRPC5 upon G protein-coupled ATP receptor stimulation elicits entry of Ca(2+) into endothelial cells. These findings reveal the structural motif for the NO-sensitive activation gate in TRP channels and indicate that NO sensors are a new functional category of cellular receptors extending over different TRP families.     

The Na+/Ca2+ exchange inhibitor KB-R7943 potently blocks TRPC channels

Na(+)/Ca(2+) exchangers (NCXs) and members of the canonical transient receptor potential (TRPC) channels play an important role in Ca(2+) homeostasis in heart and brain. With respect to their overlapping expression and their role as physiological Ca(2+) influx pathways a functional discrimination of both mechanisms seems to be necessary. Here, the effect of the reverse-mode NCX inhibitor KB-R7943 was investigated on different TRPC channels heterologously expressed in HEK293 cells. In patch-clamp recordings KB-R7943 potently blocked currents through TRPC3 (IC(50)=0.46 microM), TRPC6 (IC(50)=0.71 microM), and TRPC5 (IC(50)=1.38 microM). 1-Oleoyl-2-acetyl-sn-glycerol-induced Ca(2+) entry was nearly completely suppressed by 10 microM KB-R7943 in TRPC6-transfected cells. Thus, KB-R7943 is able to block receptor-operated TRP channels at concentrations which are equal or below those required to inhibit reverse-mode NCX activity. These data further suggest that the protective effects of KB-R7943 in ischemic tissue may, at least partly, be due to inhibition of TRPC channels.     

The canonical transient receptor potential 6 channel as a putative phosphatidylinositol 3,4,5-trisphosphate-sensitive calcium entry system

We previously reported that phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), a lipid product of phosphoinositide 3-kinase (PI3K), induced Ca(2+) influx via a noncapacitative pathway in platelets, Jurkat T cells, and RBL-2H3 mast cells. The identity of this Ca(2+) influx system, however, remains unclear. Here, we investigate a potential link between PIP(3)-sensitive Ca(2+) entry and the canonical transient receptor potential (TRPC) channels by developing stable human embryonic kidney (HEK) 293 cell lines expressing TRPC1, TRPC3, TRPC5, and TRPC6. Two lines of evidence support TRPC6 as a putative target by which PIP(3) induces Ca(2+) influx. First, Fura-2 fluorometric Ca(2+) analysis shows the ability of PIP(3) to selectively stimulate [Ca(2+)](i) increase in TRPC6-expressing cells. Second, pull-down analysis indicates specific interactions between biotin-PIP(3) and TRPC6 protein. Our data indicate that PIP(3) activates store-independent Ca(2+) entry in TRPC6 cells via a nonselective cation channel. Although the activating effect of PIP(3) on TRPC6 is reminiscent to that of 1-oleoyl-2-acetyl-sn-glycerol, this activation is not attributable to the diacylglycerol substructure of PIP(3) since other phosphoinositides failed to trigger Ca(2+) responses. The PIP(3)-activated Ca(2+) entry is inhibited by known TRPC6 inhibitors such as Gd(3+) and SKF96365 and is independent of IP(3) production. Furthermore, we demonstrated that TRPC6 overexpression or antisense downregulation significantly alters the amplitude of PIP(3)- and anti-CD3-activated Ca(2+) responses in Jurkat T cells. Consequently, the link between TRPC6 and PIP(3)-mediated Ca(2+) entry provides a framework to account for an intimate relationship between PI3K and PLCgamma in initiating Ca(2+) response to agonist stimulation in T lymphocytes.     

Activation of Transient Receptor Potential Canonical 3 (TRPC3)-mediated Ca2+ Entry by A1 Adenosine Receptor in Cardiomyocytes Disturbs Atrioventricular Conduction

Although the activation of the A(1)-subtype of the adenosine receptors (A(1)AR) is arrhythmogenic in the developing heart, little is known about the underlying downstream mechanisms. The aim of this study was to determine to what extent the transient receptor potential canonical (TRPC) channel 3, functioning as receptor-operated channel (ROC), contributes to the A(1)AR-induced conduction disturbances. Using embryonic atrial and ventricular myocytes obtained from 4-day-old chick embryos, we found that the specific activation of A(1)AR by CCPA induced sarcolemmal Ca(2+) entry. However, A(1)AR stimulation did not induce Ca(2+) release from the sarcoplasmic reticulum. Specific blockade of TRPC3 activity by Pyr3, by a dominant negative of TRPC3 construct, or inhibition of phospholipase Cs and PKCs strongly inhibited the A(1)AR-enhanced Ca(2+) entry. Ca(2+) entry through TRPC3 was activated by the 1,2-diacylglycerol (DAG) analog OAG via PKC-independent and -dependent mechanisms in atrial and ventricular myocytes, respectively. In parallel, inhibition of the atypical PKCζ by myristoylated PKCζ pseudosubstrate inhibitor significantly decreased the A(1)AR-enhanced Ca(2+) entry in both types of myocytes. Additionally, electrocardiography showed that inhibition of TRPC3 channel suppressed transient A(1)AR-induced conduction disturbances in the embryonic heart. Our data showing that A(1)AR activation subtly mediates a proarrhythmic Ca(2+) entry through TRPC3-encoded ROC by stimulating the phospholipase C/DAG/PKC cascade provide evidence for a novel pathway whereby Ca(2+) entry and cardiac function are altered. Thus, the A(1)AR-TRPC3 axis may represent a potential therapeutic target.     

Homer 1 mediates store- and inositol 1,4,5-trisphosphate receptor-dependent translocation and retrieval of TRPC3 to the plasma membrane

Store-operated Ca(2+) channels (SOCs) mediate receptor-stimulated Ca(2+) influx. Accumulating evidence indicates that members of the transient receptor potential (TRP) channel family are components of SOCs in mammalian cells. Agonist stimulation activates SOCs and TRP channels directly and by inducing translocation of channels in intracellular vesicles to the plasma membrane (PM). The mechanism of TRP channel translocation in response to store depletion and agonist stimulation is not known. Here we use TRPC3 as a model to show that IP(3) and the scaffold Homer 1 (H1) regulate the rate of translocation and retrieval of TRPC3 from the PM. In resting cells, TRPC3 exists in TRPC3-H1b/c-IP(3)Rs complexes that are located in part at the PM and in part in intracellular vesicles. Binding of IP(3) to the IP(3)Rs dissociates the interaction between IP(3)Rs and H1 but not between H1 and TRPC3 to form IP(3)Rs-TRPC3-H1b/c. TIRFM and biotinylation assays show robust receptor- and store-dependent translocation of the TRPC3 to the PM and their retrieval upon termination of cell stimulation. The translocation requires depletion of stored Ca(2+) and is prevented by inhibition of the IP(3)Rs. In HEK293, dissociating the H1b/c-IP(3)R complex with H1a results in TRPC3 translocation to the PM, where it is spontaneously active. The TRPC3-H1b/c-IP(3)Rs complex is reconstituted by infusing H1c into these cells. Reconstitution is inhibited by IP(3). Deletion of H1 in mice markedly reduces the rates of translocation and retrieval of TRPC3. Conversely, infusion of H1c into H1(-/-) cells eliminates spontaneous channel activity and increases the rate of channel activation by agonist stimulation. The effects of H1c are inhibited by IP(3). These findings together with our earlier studies demonstrating gating of TRPC3 by IP(3)Rs were used to develop a model in which assembly of the TRPC3-H1b/c-IP(3)Rs complexes by H1b/c mediates both the translocation of TRPC3-containing vesicles to the PM and gating of TRPC3 by IP(3)Rs.     

The HECT ubiquitin ligase AIP4 regulates the cell surface expression of select TRP channels

TRPV4 is a widely expressed member of the transient receptor potential (TRP) family that facilitates Ca(2+) entry into nonexcitable cells. TRPV4 is activated by several stimuli, but it is largely unknown how the activity of this channel is terminated. Here, we show that ubiquitination represents an important mechanism to control the presence of TRPV4 at the plasma membrane. Ubiquitination of TRPV4 is dramatically increased by the HECT (homologous to E6-AP carboxyl terminus)-family ubiquitin ligase AIP4 without inducing degradation of this channel. Instead, AIP4 promotes the endocytosis of TRPV4 and decreases its amount at the plasma membrane. Consequently, the basal activity of TRPV4 is reduced despite an overall increase in TRPV4 levels. This mode of regulation is not limited to TRPV4. TRPC4, another member of the TRP channel family, is also strongly ubiquitinated in the presence of AIP4, leading to the increased intracellular localization of TRPC4 and the reduction of its basal activity. However, ubiquitination of several other TRP channels is not affected by AIP4, demonstrating that AIP4-mediated regulation is a unique property of select TRP channels.