Cellular calcium homeostasis is regulated by hormones and neurotransmitters, resulting in the activation of a variety of proteins, in particular, channel proteins of the plasma membrane and of intracellular compartments. Such channels are, for example, TRP channels of the TRPC protein family that are activated by various mediators from receptor-stimulated signaling cascades. In Drosophila, two TRPC channels, TRP and TRPL, are involved in phototransduction. In addition, a third Drosophila TRPC channel, TRPgamma, has been identified and described as an auxiliary subunit of TRPL. The current data show that heterologously expressed TRPgamma forms a receptor-activated, outwardly rectifying cation channel independent from TRPL co-expression. Analysis of the activation mechanism revealed that TRPgamma is activated by various polyunsaturated fatty acids generated in a phospholipase C- and phospholipase A(2)-dependent manner. The most potent activator of TRPgamma, the stable analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid, induced currents in single channel recordings. Upon heterologous expression TRPgamma forms a homomeric channel complex that is activated by polyunsaturated fatty acids as mediators of receptor-dependent signaling pathways. Reverse transcription PCR analysis showed that TRPgamma is expressed in Drosophila heads and bodies. Its body-wide expression pattern and its activation mechanism suggest that TRPgamma forms a fly cation channel responsible for the regulation of intracellular calcium in a variety of hormonal signaling cascades (Jors, 2006).
Transient receptor potential (TRP) channels mediate light-induced Ca(2+) entry and the electrical response in Drosophila photoreceptors. The role of TRP channels in other invertebrate photoreceptors is unknown, particularly those, exemplified by Limulus ventral eye photoreceptors, in which calcium release from intracellular stores is prominent. cDNA encoding three variants of a Limulus TRP channel were amplified. LptrpA and LptrpB encode proteins of 896 and 923 amino acids, that differ by a 27 amino acid insert within the C-terminus. LptrpC encodes an alternative 63 amino acid sequence in the pore domain compared with LptrpB. LptrpB and LptrpC are present in ventral eye mRNA, while LptrpA is only present in brain mRNA. In situ hybridization indicates the presence of Lptrp in photoreceptors of the Limulus ventral eye. Some canonical TRP channels can be activated by diacylglycerol analogs. Injection of a diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), into Limulus photoreceptors can activate an inward current with electrical characteristics similar to the light-activated current. However, simultaneous elevation of cytosolic calcium concentration appears to be necessary. Illumination attenuates the response to OAG injections and vice versa. These results provide molecular and pharmacological evidence for a TRP channel in Limulus ventral eye that may contribute to the light-sensitive conductance (Bandyopadhyay, 2004).
Ca(2+) influx via plasma membrane Trp3 channels is proposed to be regulated by a reversible interaction with inositol trisphosphate receptor [IP(3)R] in the endoplasmic reticulum. Condensation of the cortical actin layer has been suggested to physically disrupt this interaction and inhibit Trp3-mediated Ca(2+) influx. This study examines the effect of cytoskeletal reorganization on the localization and function of Trp3 and key Ca(2+) signaling proteins. Calyculin-A treatment resulted in formation of condensed actin layer at the plasma membrane; internalization of Trp3, Galpha(q/11), phospholipase Cbeta, and caveolin-1, and attenuation of 1-oleoyl-2-acetyl-sn-glycerol- and ATP-stimulated Sr(2+) influx. Importantly, Trp3 and IP(3)R-3 remained co-localized inside the cell and were co-immunoprecipitated. Jasplakinolide also induced internalization of Trp3 and caveolin-1. Pretreatment of cells with cytochalasin D or staurosporine did not affect Trp3 but prevented calyculin-A-induced effects. Based on these data, it is suggest that Trp3 is assembled in a caveolar Ca(2+) signaling complex with IP(3)R, SERCA, Galpha(q/11), phospholipase Cbeta, caveolin-1, and ezrin. Furthermore, the data demonstrate that conditions that stabilize cortical actin induce loss of Trp3 activity due to internalization of the Trp3-signaling complex, not disruption of IP(3)R-Trp3 interaction. This suggests that localization of the Trp3-associated signaling complex, rather than Trp3-IP(3)R coupling, depends on the status of the actin cytoskeleton (Lockwich, 2001).
Receptor-coupled [Ca2+]i increase is initiated in the apical region of epithelial cells and has been associated with apically localized Ca2+-signaling proteins. However, localization of Ca2+ channels that are regulated by such Ca2+-signaling events has not yet been established. This study examines the localization of TRPC channels in polarized epithelial cells and demonstrates a role for TRPC3 in apical Ca2+ uptake. Endogenously and exogenously expressed TRPC3 localizes apically in polarized Madin-Darby canine kidney cells (MDCK) and salivary gland epithelial cells. In contrast, TRPC1 localizes basolaterally, whereas TRPC6 is detected in both locations. Localization of Galpha(q/11), inositol 1,4,5-trisphosphate receptor-3, and phospholipase Cbeta1 and -beta2 is also predominantly apical. TRPC3 co-immunoprecipitates with endogenous TRPC6, phospholipase Cbetas, Galpha(q/11), inositol 1,4,5-trisphosphate receptor-3, and syntaxin 3 but not with TRPC1. Furthermore, 1-oleoyl-2-acetyl-sn-glycerol (OAG)-stimulated apical 45Ca2+ uptake is higher in TRPC3-MDCK cells compared with control (MDCK) cells. Bradykinin-stimulated apical 45Ca2+ uptake and transepithelial 45Ca2+ flux are also higher in TRPC3-expressing cells. Consistent with this, OAG induces [Ca2+]i increase in the apical, but not basal, region of TRPC3-MDCK cells that is blocked by EGTA addition to the apical medium. Most importantly, (1) TRPC3 is detected in the apical region of rat submandibular gland ducts, whereas TRPC6 is present in apical as well as basolateral regions of ducts and acini; and (2) OAG stimulates Ca2+ influx into dispersed ductal cells. These data demonstrate functional localization of TRPC3/TRPC6 channels in the apical region of polarized epithelial cells. In salivary gland ducts this could contribute to the regulation of salivary [Ca2+] and secretion (Bandyopadhyay, 2005).
Mammalian homologues of Drosophila Trp have been implicated to form channels that are activated following the depletion of Ca(2+) from internal stores. Recent studies indicate that actin redistribution is required for the activation of these channels. Murine Trp4 and Trp5, as well as phospholipase C beta1 and beta2 interact with the first PDZ domain of NHERF, regulatory factor of the Na+/H+ exchanger. The association of Trp4 and phospholipase C-beta1 with NHERF in vivo in an HEK293 cell line expressing Trp4 and in adult mouse brain has been demonstrated by immuno-coprecipitation. NHERF is a two PDZ domain-containing protein that associates with the actin cytoskeleton via interactions with members of ezrin/radixin/moesin family. Thus, store-operated channels involving Trp4 and Trp5 can form signaling complexes with phospholipase C isozymes via interactions with NHERF, thereby linking the lipase and the channels to the actin cytoskeleton. The interaction with the PDZ protein may constitute an important mechanism for distribution and regulation of store-operated channels (Tang, 2000).
Mammalian homologs of the Drosophila TRP protein have been shown to form cation-permeable channels in the plasma membrane but very little is known about the mechanisms that control their cell surface localization. Recently it has been demonstrated that the last three C-terminal amino acids (TRL) of TRPC4 comprise a PDZ-interacting domain that binds to the scaffold protein EBP50 [ezrin/moesin/radixin-binding phosphoprotein 50]. In this report, the influence of the TRL motif on the subcellular distribution of TRPC4 was examined in human embryonic kidney (HEK) 293 cells. The consequences of the interaction between EBP50 and the membrane-cytoskeletal adaptors of the ezrin/radixin/moesin (ERM) family was examined for the cell surface expression of TRPC4. Using immunofluorescence microscopy, it was found that the mutant lacking the TRL motif accumulated into cell outgrowths and exhibited a punctate distribution pattern whereas the wild-type channel was evenly distributed on the cell surface. Deletion of the PDZ-interacting domain also decreased the expression of TRPC4 in the plasma membrane by 2.4-fold, as assessed by cell surface biotinylation experiments. Finally, in a large percentage of cells co-expressing TRPC4 and an EBP50 mutant lacking the ERM-binding site, TRPC4 was not present in the plasma membrane but co-localized with the truncated scaffold in a perinuclear compartment (most probably representing the Golgi apparatus) and in vesicles associated with actin filaments. The data demonstrate that the PDZ-interacting domain of TRPC4 controls its localization and surface expression in transfected HEK293 cells. They also point to a yet unexplored role of the EBP50-ERM complex in the regulation of protein insertion into the plasma membrane (Merv, 2002).
TRPC1-7 proteins are members of a family of mammalian non-specific cation channels that mediate receptor-operated, phospholipase Cbeta/Cgamma dependent Ca(2+) influx in various cell types. TRPC4 and TRPC5 form a subfamily within TRPCs. Uniquely in the TRPC family, these channels possess a C-terminal 'VTTRL' motif that binds to PDZ-domains of the scaffolding protein, EBP50 (NHERF1). The functional effects of EBP50 on TRPC4/5 activity have not been investigated. Rat TRPC5 (rTRPC5) was cloned, functionally expressed in HEK293 cell, and channel regulation was studied with patch-clamp techniques. Both rTRPC5 and its VTTRL deletion mutant (r5dV) localize to the plasma membrane. rTRPC5 does not display any significant basal activity in unstimulated HEK293 cells. In cells co-expressing rTRPC5 and H1 histamine receptor, rTRPC5 current evoked by GTPgammaS or histamine develops in two phases: a slowly developing, small inward current followed by a rapidly developing, transient, large inward current. Each phase has a characteristic non-linear current-voltage (I-V) relationship. Deletion of the VTTRL motif has no detectable effect on the biophysical properties of the channel. Co-expression of EBP50 with rTRPC5 causes a significant delay in the time-to-peak of the histamine-evoked, transient large inward current. EBP50 does not modify the activation kinetics of the VTTRL-deletion mutant. It is concluded that the VTTRL motif is not necessary for activation of TRPC5, but may mediate the modulatory effect of EBP50 on TRPC5 activation kinetics (Obukhov, 2004).
Various members of the canonical family of transient receptor potential channels (TRPCs) exhibit increased cation influx following receptor stimulation or Ca(2+) store depletion. Tyrosine phosphorylation of TRP family members also results in increased channel activity; however, the link between the two events is unclear. Two tyrosine residues in the C terminus of human TRPC4 (hTRPC4), Tyr-959 and Tyr-972, are phosphorylated following epidermal growth factor (EGF) receptor stimulation of COS-7 cells. This phosphorylation is mediated by Src family tyrosine kinases (STKs), with Fyn appearing to be the dominant kinase. In addition, EGF receptor stimulation induces the exocytotic insertion of hTRPC4 into the plasma membrane dependent on the activity of STKs and is accompanied by a phosphorylation-dependent increase in the association of hTRPC4 with Na+/H+ exchanger regulatory factor. Furthermore, this translocation and association is defective upon mutation of Tyr-959 and Tyr-972 to phenylalanine. Significantly, inhibition of STKs is concomitant with a reduction in Ca(2+) influx in both native COS-7 cells and hTRPC4-expressing HEK293 cells, with cells expressing the Y959F/Y972F mutant exhibiting a reduced EGF response. These findings represent the first demonstration of a mechanism for phosphorylation to modulate TRPC channel function (Odell, 2005).
Mammalian TRPC channels have been proposed as the molecular entities associated with calcium entry activity in nonexcitable cells. Amino acid sequence analyses of TRPCs reveals the presence of ankyrin-like repeat domains, one of the most common protein-protein interaction motifs. Using a yeast two-hybrid interaction assay, it was found that the second ankyrin-like repeat domain of TRPC6 interacts with MxA, a member of the dynamin superfamily. Using a GST pull-down and co-immunoprecipitation assay, it was shown that MxA interacts with TRPC1, -3, -4, -5, -6, and -7. Overexpression of MxA in HEK293T cells slightly increased endogenous calcium entry subsequent to stimulation of G(q) protein-coupled receptors or store depletion by thapsigargin. Co-expression of MxA with TRPC6 enhanced either agonist-induced or OAG-induced calcium entry activity. GTP binding-defective MxA mutants had only a minor potentiating effect on OAG-induced TRPC6 activity. However, a MxA mutant that could bind GTP but that lacked GTPase activity produced the same effect as MxA on OAG-induced TRPC6 activity. These results indicated that MxA interacts specifically with the second ankyrin-like repeat domain of TRPCs and suggests that monomeric MxA regulates the activity of TRPC6 by a mechanism requiring GTP binding. Additional results showed that an increase in the endogenous expression of MxA, induced by a treatment with interferon alpha, regulates the activity of TRPC6. The study clearly identified MxA as a new regulatory protein involved in Ca2+ signaling (Lussier, 2005).
The acrosome reaction, the first step of the fertilization, is induced by calcium influx through TRPC channels. The molecular nature of TRPC involved is still a debated question. In mouse, TRPC2 plays the most important role and is responsible for the calcium plateau. However, TRPC1 and TRPC5 are also localized in the acrosomal crescent of the sperm head and may participate in calcium signaling, especially in TRPC2-deficient mice. Activation of TRPC channels is an unresolved question in germ and somatic cells as well. In particular, in sperm, little is known concerning the molecular events leading to TRPC2 activation. From the discovery of IP3R binding domains on TRPC2, it has been suggested that TRPC channel activation may be due to a conformational coupling between IP3R and TRPC channels. Moreover, recent data demonstrate that junctate, an IP3R associated protein, participates also in the gating of some TRPC. This study shows that junctate is expressed in sperm and co-localizes with the IP3R in the acrosomal crescent of the anterior head of rodent sperm. Consistent with its specific localization, pull-down experiments show that junctate interacts with TRPC2 and TRPC5 but not with TRPC1. Focus was placed on the interaction between TRPC2 and junctate; the N-terminus of junctate interacts with the C-terminus of TRPC2, both in vitro and in a heterologous expression system. Junctate binds to TRPC2 independently of the calcium concentration and the junctate binding site does not overlap with the common IP3R/calmodulin binding sites. TRPC2 gating is downstream phospholipase C activation, which is a key and necessary step during the acrosome reaction. TRPC2 may then be activated directly by diacylglycerol (DAG), as in neurons of the vomeronasal organ. The present study investigated whether DAG could promote the acrosome reaction. It was found that 100 microM OAG, a permeant DAG analogue, was unable to trigger the acrosome reaction. Altogether, these results provide a new hypothesis concerning sperm TRPC2 gating: TRPC2 activation may be due to modifications of its interaction with both junctate and IP3R, induced by depletion of calcium from the acrosomal vesicle (Stamboulian, 2005).
Mammalian TRPC genes encode a family of nonselective cation channels that are activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. In Drosophila photoreceptor cells, TRP channels are found in large, multimolecular signaling complexes in association with the PDZ-containing scaffolding protein, INAD. A similar mammalian TRPC 'signalplex' has been proposed, but has yet to be defined. In the present study, affinity-purified polyclonal antibodies against TRPC5 and TRPC6 were used to immunoprecipitate signalplex components from rat brain lysates. Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis, digested with trypsin, and sequenced by mass spectrometry. Proteins identified in the immunoprecipitates included cytoskeletal proteins spectrin, myosin, actin, drebrin, tubulin, and neurabin; endocytic vesicle-associated proteins clathrin, dynamin and AP-2, and the plasmalemmal Na(+)/K(+)-ATPase (NKA) pump. Several of these interactions were confirmed by reciprocal immunoprecipitation followed by Western blot analysis. In lysates from rat kidney, TRPC6, but not TRPC3, was found to coimmunoprecipitate with the NKA pump. Likewise, TRPC6, stably expressed in human embryonic kidney (HEK) cells, coimmunoprecipitated with endogenous NKA and colocalized with the pump to the plasmalemma when examined by immunofluorescence microscopy. Cell surface biotinylation experiments, in intact HEK cells, confirms that both the Na(+) pump and TRPC6 are present in the surface membrane and appear to interact. Lastly, TRPC6 coimmunoprecipitated with the NKA pump when the proteins were coexpressed in Spodoptera frugiperda insect cells using recombinant baculoviruses. These observations suggest that TRPC6 and the Na(+) pump are part of a functional complex that may be involved in ion transport and homeostasis in both the brain and kidney (Goel, 2005).
TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. The activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade was studied along with the role of calmodulin (CaM) on the activation. Exogenous application of 10 microM CaM through patch pipette accelerates the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 to 1.56 min. A novel CaM-binding site was identified located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuates the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 renders the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, high intracellular free Ca2+ inhibits the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrate that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation (Ordaz, 2005).
Receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) are known to be inhibited by tyrosine kinase inhibitors. Activation of C-type transient receptor potential channel (TRPC) isoform 3 (TRPC3), a cation channel thought to be involved in SOCE and/or ROCE, was recently shown to depend on src tyrosine kinase activity. What is not known is the step at which src acts on TRPC3 and whether the role for tyrosine kinases in ROCE or SOCE is a general phenomenon. Using in vitro and in cell protein-protein interaction assays it is now reported that src phosphorylates TRPC3 at Y226 and that formation of phospho-Y226 is essential for TRPC3 activation. This requirement is unique for TRPC3 because (1) mutation of the cognate tyrosines of the closely related TRPC6 and TRPC7 have no effect; (2) TRPC6 and TRPC7 are activated in src-, yes-, and fyn-deficient cells; and (3) src, but not yes or fyn, rescue TRPC3 activation in src-, yes-, and fyn-deficient cells. The Src homology 2 domain of src interacts with either the N or the C termini of all TRPCs, suggesting that other tyrosine kinases may play a role in ion fluxes mediated by TRPCs other than TRPC3. A side-by-side comparison of the effects of genistein (a general tyrosine kinase inhibitor) on endogenous ROCE and SOCE in mouse fibroblasts, HEK and COS-7 cells, and on ROCE in HEK cells mediated by TRPC3, TRPC6, TRPC7, and TRPC5 showed differences that argue for ROCE and SOCE channels to be heterogeneous (Kawasaki, 2006).
Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor-activated Ca(2+) influx in vertebrate cells. Intracellular Ca(2+) is involved in the receptor-mediated activation of mammalian TRPC5 channels. This study investigated the role of calmodulin, an important sensor of changes in intracellular Ca(2+), and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca(2+) entry through TRPC5 channels, induced upon stimulation of the G-protein-coupled ATP receptor, is abolished by treatment with W-13, an inhibitor of calmodulin. ML-9 and wortmannin, inhibitors of Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK), and the expression of a dominant-negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML-9 impairs the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole-cell patch-clamp technique is inhibited by ML-9, whereas TRPC5 channel activity observed in the cell-excised, inside-out patch is unaffected by ML-9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions is reduced by ML-9 in HEK293 cells. These findings strongly suggest that intracellular Ca(2+)-calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC (Shimizu, 2006).
Endogenously expressed TRPC homologs were investigated for their role in forming store-operated, 1-oleoyl-2-acetyl-sn-glycerol-stimulated, or carbachol (CCh)-stimulated calcium entry pathways in HEK-293 cells. Measurement of thapsigargin-stimulated Ba(2+) entry indicate that the individual suppression of TRPC1, TRPC3, or TRPC7 protein levels, by siRNA techniques, dramatically inhibits store-operated calcium entry (SOCE), whereas suppression of TRPC4 or TRPC6 has no effect. Combined suppression of TRPC1-TRPC3, TRPC1-TRPC7, TRPC3-TRPC7, or TRPC1-TRPC3-TRPC7 gives only slightly more inhibition of SOCE than seen with suppression of TRPC1 alone (68%), suggesting that these three TRPC homologs work in tandem to mediate a large component of SOCE. Evidence from co-immunoprecipitation experiments indicates that a TRPC1-TRPC3-TRPC7 complex, predicted from siRNA results, does exist. The suppression of either TRPC3 or TRPC7, but not TRPC1, induces a high Ba(2+) leak flux that is inhibited by 2-APB and SKF96365, suggesting that the influx is via leaky store-operated channels. The high Ba(2+) leak flux is eliminated by co-suppression of TRPC1-TRPC3 or TRPC1-TRPC7. For 1-oleoyl-2-acetyl-sn-glycerol-stimulated cells, siRNA data indicate that TRPC1 plays no role in mediating Ba(2+) entry, which appears to be mediated by the participation of TRPC3, TRPC4, TRPC6, and TRPC7. CCh-stimulated Ba(2+) entry, in contrast, can be inhibited by suppression of any of the five endogenously expressed TRPC homologs, with the degree of inhibition being consistent with CCh stimulation of both store-operated and receptor-operated channels. In summary, endogenous TRPC1, TRPC3, and TRPC7 participate in forming heteromeric store-operated channels, whereas TRPC3 and TRPC7 can also participate in forming heteromeric receptor-operated channels (Zagranichnaya, 2005).
Canonical transient receptor potential proteins (TRPC) have been proposed to form homo- or heteromeric cation channels in a variety of tissues, including the vascular endothelium. Assembly of TRPC multimers is incompletely understood. In particular, heteromeric assembly of distantly related TRPC isoforms is still a controversial issue. Because TRPC proteins have been proposed as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), the TRPC subunit composition of endogenous endothelial TRPC channels was studied. A redox-sensitive TRPC3-TRPC4 channel complex is described. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex is supported by four lines of evidence as follows: (1) Co-immunoprecipitation experiments in PAECs and in HEK293 cells demonstrated the association of TRPC3 and TRPC4 in the same complex. (2) Fluorescence resonance energy transfer analysis demonstrated TRPC3-TRPC4 association, involving close proximity between the N terminus of TRPC4 and the C terminus of TRPC3 subunits. (3) Co-expression of TRPC3 and TRPC4 in HEK293 cells generated a channel that displayed distinct biophysical and regulatory properties. (4) Expression of dominant-negative TRPC4 proteins suppressed TRPC3-related channel activity in the HEK293 expression system and in native endothelial cells. Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti-HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. TRPC3 and TRPC4 are proposed as subunits of native endothelial cation channels that are governed by the cellular redox state (Poteser, 2006).
Many ion channels are regulated by lipids, but prominent motifs for lipid binding have not been identified in most ion channels. Phospholipase Cgamma1 (PLC-gamma1) binds to and regulates TRPC3 channels, components of agonist-induced Ca2+ entry into cells. This interaction requires a domain in PLC-gamma1 that includes a partial pleckstrin homology (PH) domain that functions as consensus lipid-binding and protein-binding sequence. A gestalt algorithm was developed to detect hitherto 'invisible' PH and PH-like domains, and the partial PH domain of PLC-gamma1 interacts with a complementary partial PH-like domain in TRPC3 to elicit lipid binding and cell-surface expression of TRPC3. These findings imply a far greater abundance of PH domains than previously appreciated, and suggest that intermolecular PH-like domains represent a widespread signalling mode (van Rossum, 2005).
TRPC3 has been suggested to be a component of cation channel complexes that are targeted to cholesterol-rich lipid membrane microdomains. This study investigates the potential role of membrane cholesterol as a regulator of cellular TRPC3 conductances. Functional experiments have demonstrated that cholesterol loading activates a non-selective cation conductance and a Ca2+ entry pathway in TRPC3-overexpressing cells but not in wild-type HEK-293 (human embryonic kidney 293) cells. The cholesterol-induced membrane conductance exhibits a current-to-voltage relationship similar to that observed upon PLC (phospholipase C)-dependent activation of TRPC3 channels. Nonetheless, the cholesterol-activated conductance lacked negative modulation by extracellular Ca2+, a typical feature of agonist-activated TRPC3 currents. Involvement of TRPC3 in the cholesterol-dependent membrane conductance was further corroborated by a novel dominant-negative strategy for selective blockade of TRPC3 channel activity. Expression of a TRPC3 mutant, which contains a haemagglutinin epitope tag in the second extracellular loop, confers antibody sensitivity to both the classical PLC-activated as well as the cholesterol-activated conductance in TRPC3-expressing cells. Moreover, cholesterol loading as well as PLC stimulation increased surface expression of TRPC3. Promotion of TRPC3 membrane expression by cholesterol was persistent over 30 min, while PLC-mediated enhancement of plasma membrane expression of TRPC3 was transient in nature. It is suggested the cholesterol content of the plasma membrane is a determinant of cellular TRPC3 activity and evidence is provided for cholesterol dependence of TRPC3 surface expression (Grazini, 2006).
TRPC calcium channels are emerging as a ubiquitous feature of vertebrate cells, but understanding of them is hampered by limited knowledge of the mechanisms of activation and identity of endogenous regulators. One of the TRPC channels, TRPC5, is strongly activated by common endogenous lysophospholipids including lysophosphatidylcholine (LPC) but, by contrast, not arachidonic acid. Although TRPC5 was stimulated by agonists at G-protein-coupled receptors, TRPC5 activation by LPC occurs downstream and independently of G-protein signaling. The effect is not due to the generation of reactive oxygen species or because of a detergent effect of LPC. LPC activates TRPC5 when applied to excised membrane patches and thus has a relatively direct action on the channel structure, either because of a phospholipid binding site on the channel or because of sensitivity of the channel to perturbation of the bilayer by certain lipids. Activation shows dependence on side-chain length and the chemical head-group. The data reveal a previously unrecognized lysophospholipid-sensing capability of TRPC5 that confers the property of a lipid ionotropic receptor (Fleming, 2006).
Members of the TRP cation channel family control a wide variety of cellular functions by regulating calcium influx. In neurons, TRP channels may also modulate cell excitability. TRPC5 is a neuronal TRP channel that plays a role in controlling neurite extension in the hippocampus. Transiently expressed TRPC5 exhibits a doubly rectifying current-voltage relationship characterized by relatively large inward currents and a unique outwardly rectifying current with a 'flat' segment between +10 and +40 mV that may be attributable to Mg2+ block. Intracellular Mg2+ blocks the outward current through TRPC5 with an IC50 of 457 microM. The block is mediated by a cytosolic aspartate residue, D633, situated between the termination of the sixth transmembrane domain and the 'TRP box.' The substitution of noncharged or positively charged residues for the negatively charged D633 resulted in a channel with markedly reduced inward currents, in addition to decreased Mg2+ block. This suggests that electrostatic attraction of cations by D633 may contribute to inward current amplitude in TRPC5. It is proposed that cytosolic negatively charged residues can modulate the conductivity of TRP cation channels (Obukhov, 2005).
A full-length RIKEN mouse cDNA has been identified that encodes a putative variant of the C3-type TRPC that differs from the previously cloned murine TRPC3 cDNA in that it has a 5' extension stemming from inclusion of an additional exon (exon 0). The extended cDNA adds 62 aa to the sequence of the murine TRPC3. A cDNA encoding the human homologue of this extended TRPC3 has a highly homologous 73-aa N-terminal extension, referred to as hTRPC3a. A query of the GenBank genomic database predicts the existence of a similar gene product also found in rats. Transient expression of the longer TRPC3a in human embryonic kidney (HEK) cells showed that it mediates Ca2+ entry in response to stimulation of the Gq-phospholipase C beta pathway, which is similar to that mediated by the shorter hTRPC3. However, after isolation of HEK cells expressing hTRPC3 in stable form, TRPC3a gave rise to Ca2+-entry channels that are not only activated by the Gq-phospholipase C beta pathway (receptor-activated Ca entry) but also by thapsigargin triggered store depletion. In conjunction with findings that TRPC1, TRPC2, TRPC4, TRPC5, and TRPC7, can each mediate store-depletion-activated Ca2+ entry in mammalian cells, these findings with hTRC3a support the proposal that TRPCs form capacitative Ca-entry channels (Yildirim, 2005).
Progressive kidney failure is a genetically and clinically heterogeneous group of disorders. Podocyte foot processes and the interposed glomerular slit diaphragm are essential components of the permeability barrier in the kidney. Mutations in genes encoding structural proteins of the podocyte lead to the development of proteinuria, resulting in progressive kidney failure and focal segmental glomerulosclerosis. The TRPC6 ion channel is expressed in podocytes and is a component of the glomerular slit diaphragm. Five families were identified with autosomal dominant focal segmental glomerulosclerosis in which disease segregated with mutations in the gene TRPC6 on chromosome 11q. Two of the TRPC6 mutants had increased current amplitudes. These data show that TRPC6 channel activity at the slit diaphragm is essential for proper regulation of podocyte structure and function (Reiser, 2005).
Among the TRPC subfamily of TRP channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, a TRPC6-deficient mouse model was developed. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, is completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6-/- smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells results in increased basal activity, while TRPC6 expression in TRPC6-/- smooth muscle cells reduces basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone (Dietrich, 2005).
Focal and segmental glomerulosclerosis (FSGS) is a kidney disorder of unknown etiology, and up to 20% of patients on dialysis have been diagnosed with it. A large family with hereditary FSGS carries a missense mutation in the TRPC6 gene on chromosome 11q, encoding the ion-channel protein TRPC6. The proline-to-glutamine substitution at position 112, which occurs in a highly conserved region of the protein, enhances TRPC6-mediated calcium signals in response to agonists such as angiotensin II and appears to alter the intracellular distribution of TRPC6 protein. Previous work has emphasized the importance of cytoskeletal and structural proteins in proteinuric kidney diseases. These findings suggest an alternative mechanism for the pathogenesis of glomerular disease (Winn, 2005).
Epidermal growth factor (EGF) in corneal epithelial cells stimulates proliferation by inducing capacitative calcium entry (CCE). However, neither the identity nor the mechanism of activation of the plasma membrane influx pathway that mediates CCE is known. Accordingly, it was determined, in human corneal epithelial cells, whether or not (1) CCE is dependent upon stimulation of storeoperated channel (SOC) activity; (2) whether the canonical TRP protein isoform TRPC4 is a component of such channels, and (3) whether suppression of TRPC4 protein expression decreases EGF-induced stimulation of SOC activity and proliferation. The whole cell patch-clamp technique was used to monitor TRPC4-mediated stimulation of SOC activity following intracellular calcium store depletion and induction of CCE. TRPC4 small interfering RNA transfection suppresses TRPC4 protein expression. Reverse transcription-PCR and Western blot analysis were used to assess knockdown efficiency of mRNA and protein expression. [(3)H]Thymidine incorporation was used to evaluate EGF-induced mitogenesis. Ca(2+) transients were measured by single-cell fluorescence imaging. TRPC4 knockdown decreased mRNA and protein expression by 89% and 87%, respectively. In these cells, EGF-induced SOC activation elicited by intracellular calcium store depletion was obviated; EGF-induced CCE fell by 76%; EGF-induced stimulation of SOC activity was eliminated, and EGF-induced increases in proliferation fell by 54%. Thus, TRPC4 is a component of SOC in human corneal epithelial cells whose activation by EGF is requisite for an optimum mitogenic response to this growth factor (Yang, 2005).
TRPC3 is sharply up-regulated during the early part of myotube differentiation and remains elevated in mature myotubes compared with myoblasts. To examine its functional roles in muscle, TRPC3 was 'knocked down' in mouse primary skeletal myoblasts using retroviral-delivered small interference RNAs and single cell cloning. TRPC3 knockdown myoblasts were differentiated into myotubes (TRPC3 KD) and subjected to functional and biochemical assays. By measuring rates of Mn(2+) influx with Fura-2 and Ca(2+) transients with Fluo-4, it was found that neither excitation-coupled Ca(2+) entry nor thapsigargin-induced store-operated Ca(2+) entry was significantly altered in TRPC3 KD, indicating that expression of TRPC3 is not required for engaging either Ca(2+) entry mechanism. In Ca(2+) imaging experiments, the gain of excitation-contraction coupling and the amplitude of the Ca(2+) release seen after direct RyR1 activation with caffeine was significantly reduced in TRPC3 KD. The decreased gain appears to be due to a decrease in RyR1 Ca(2+) release channel activity, because sarcoplasmic reticulum (SR) Ca(2+) content was not different between TRPC3 KD and wild-type myotubes. Immunoblot analysis demonstrated that TRPC1, calsequestrin, triadin, and junctophilin 1 were up-regulated in TRPC3 KD. Based on these data, it is concluded that expression of TRPC3 is tightly regulated during muscle cell differentiation and it is proposed that functional interaction between TRPC3 and RyR1 may regulate the gain of SR Ca(2+) release independent of SR Ca(2+) load (Lee, 2006).
The ubiquitously expressed TRPC ion channels are considered important in Ca2+ signal generation, but their mechanisms of activation and roles remain elusive. Whereas most studies have examined overexpressed TRPC channels, molecular, biochemical, and electrophysiological approaches were used to assess the expression and function of endogenous TRPC channels in A7r5 smooth muscle cells. Real time PCR and Western analyses reveal TRPC6 as the only member of the diacylglycerol-responsive TRPC3/6/7 subfamily of channels expressed at significant levels in A7r5 cells. TRPC1, TRPC4, and TRPC5 were also abundant. An outwardly rectifying, nonselective cation current was activated by phospholipase C-coupled vasopressin receptor activation or by the diacylglycerol analogue, oleoyl-2-acetyl-sn-glycerol (OAG). Introduction of TRPC6 small interfering RNA sequences into A7r5 cells by electroporation led to 90% reduction of TRPC6 transcript and 80% reduction of TRPC6 protein without any detectable compensatory changes in the expression of other TRPC channels. The OAG-activated nonselective cation current was similarly reduced by TRPC6 RNA interference. Intracellular Ca2+ measurements using fura-2 revealed that thapsigargin-induced store-operated Ca2+ entry is unaffected by TRPC6 knockdown, whereas vasopressin-induced Ca2+ entry was suppressed by more than 50%. In contrast, OAG-induced Ca2+ transients were unaffected by TRPC6 knockdown. Nevertheless, OAG-induced Ca2+ entry bears the hallmarks of TRPC6 function; it is inhibited by protein kinase C and blocked by the Src-kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Importantly, OAG-induced Ca2+ entry is blocked by the potent L-type Ca2+ channel inhibitor, *nimodipine. Thus, TRPC6 activation probably results primarily in Na ion entry and depolarization, leading to activation of L-type channels as the mediators of Ca2+ entry. Calculations reveal that even 90% reduction of TRPC6 channels allows depolarization sufficient to activate L-type channels. This tight coupling between TRPC6 and L-type channels is probably important in mediating smooth muscle cell membrane potential and muscle contraction (Soboloff, 2005).
LTRPC2 is a cation channel recently reported to be activated by adenosine diphosphate-ribose (ADP-ribose) and NAD. Since ADP-ribose can be formed from NAD and NAD is elevated during oxidative stress, whole cell currents and increases in the intercellular free calcium concentration [Ca(2+)](i) were studed in LTRPC2-transfected HEK 293 cells after stimulation with hydrogen peroxide (H2O2). Cation currents carried by monovalent cations and Ca(2+) are induced by H2O2 as well as by intracellular ADP-ribose but not by NAD. H2O2-induced currents develop slowly after a characteristic delay of 3-6 min and recedes after wash-out of H2O2. [Ca(2+)](i) is rapidly increased by H2O2 in LTRPC2-transfected cells as well as in control cells; however, in LTRPC2-transfected cells, H2O2 evokes a second delayed rise in [Ca(2+)](i). A splice variant of LTRPC2 with a deletion in the C terminus (amino acids 1292-1325) was identified in neutrophil granulocytes. This variant is stimulated by H2O2 as the wild type. However, it does not respond to ADP-ribose. It is concluded that activation of LTRPC2 by H2O2 is independent of ADP-ribose and that LTRPC2 may mediate the influx of Na(+) and Ca(2+) during oxidative stress, such as the respiratory burst in granulocytes (Wehage, 2002).
Ion channels formed by the TRP superfamily of proteins act as sensors for temperature, osmolarity, mechanical stress and taste. The growth cones of developing axons are responsible for sensing extracellular guidance factors, many of which trigger Ca2+ influx at the growth cone; however, the identity of the ion channels involved remains to be clarified. TRP-like channel activity exists in the growth cones of cultured Xenopus neurons and can be modulated by exposure to netrin-1 and brain-derived neurotrophic factor, two chemoattractants for axon guidance. Whole-cell recording from growth cones showed that netrin-1 induces a membrane depolarization, part of which remains after all major voltage-dependent channels are blocked. Furthermore, the membrane depolarization is sensitive to blockers of TRP channels. Pharmacological blockade of putative TRP currents or downregulation of Xenopus TRP-1 (xTRPC1) expression with a specific morpholino oligonucleotide abolishes the growth-cone turning and Ca2+ elevation induced by a netrin-1 gradient. Thus, TRPC currents reflect early events in the growth cone's detection of some extracellular guidance signals, resulting in membrane depolarization and cytoplasmic Ca2+ elevation that mediates the turning of growth cones (G. X. Wang, 2005).
Brain-derived neurotrophic factor (BDNF) is known to promote neuronal survival and differentiation and to guide axon extension both in vitro and in vivo. The BDNF-induced chemo-attraction of axonal growth cones requires Ca2+ signalling, but how Ca2+ is regulated by BDNF at the growth cone remains largely unclear. Extracellular application of BDNF triggers membrane currents resembling those through TRPC channels in rat pontine neurons and in Xenopus spinal neurons. In cultured cerebellar granule cells, TRPC channels contribute to the BDNF-induced elevation of Ca2+ at the growth cone and are required for BDNF-induced chemo-attractive turning. Several members of the TRPC family are highly expressed in these neurons, and both Ca2+ elevation and growth-cone turning induced by BDNF are abolished by pharmacological inhibition of TRPC channels, overexpression of a dominant-negative form of TRPC3 or TRPC6, or downregulation of TRPC3 expression via short interfering RNA. Thus, TRPC channel activity is essential for nerve-growth-cone guidance by BDNF (Li, 2005).
The calcium- and sodium-permeable transient receptor potential channel TRPC5 has an inhibitory role in neuronal outgrowth but the mechanisms governing its activity are poorly understood. A mechanism is proposed involving the neuronal calcium sensor-1 (NCS-1) protein. Inhibitory mutants of TRPC5 and NCS-1 enhance neurite outgrowth similarly. Mutant NCS-1 does not inhibit surface-expression of TRPC5 but generally suppresses channel activity, irrespective of whether it is evoked by carbachol, store depletion, lanthanides or elevated intracellular calcium. NCS-1 and TRPC5 are in the same protein complex in rat brain and NCS-1 directly binds to the TRPC5 C-terminus. The data suggest protein-protein interaction between NCS-1 and TRPC5, and the involvement of this protein complex in retardation of neurite outgrowth (Hui, 2006).
Excitotoxicity in brain ischemia triggers neuronal death and neurological disability, and yet these are not prevented by antiexcitotoxic therapy (AET) in humans. In neurons subjected to prolonged oxygen glucose deprivation (OGD), AET unmasks a dominant death mechanism perpetuated by a Ca2+-permeable nonselective cation conductance (IOGD). IOGD is activated by reactive oxygen/nitrogen species (ROS), and permits neuronal Ca2+ overload and further ROS production despite AET. IOGD currents corresponded to those evoked in HEK-293 cells expressing the nonselective cation conductance TRPM7. In cortical neurons, blocking IOGD or suppressing TRPM7 expression blocked TRPM7 currents, anoxic 45Ca2+ uptake, ROS production, and anoxic death. TRPM7 suppression eliminates the need for AET to rescue anoxic neurons and permits the survival of neurons previously destined to die from prolonged anoxia. Thus, excitotoxicity is a subset of a greater overall anoxic cell death mechanism, in which TRPM7 channels play a key role (Aarts, 2003).
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