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PubMed2005

TRP channels and mice deficient in TRP channels.

Transient receptor potential (TRP) channels are a superfamily of functionally versatile cation-permeant ion channels present in almost all mammalian cell types. Although they were initially proposed as store-operated calcium channels, recent progress shows that they exhibit a variety of regulatory and functional themes. Here, we summarize the most salient features of TRP channels, the approaches that are providing meaningful discoveries, and the challenges ahead. We primarily emphasize the understanding gleaned from mouse models engineered to be deficient in various members of TRP superfamily and from the human patients that suffer clinically due to defects in TRP channels.

Animals↗
PubMed2005

Molecular analysis of a store-operated and 2-acetyl-sn-glycerol-sensitive non-selective cation channel. Heteromeric assembly of TRPC1-TRPC3.

We have reported that internal Ca2+ store depletion in HSY cells stimulates a nonselective cation current which is distinct from I(CRAC) in RBL cells and TRPC1-dependent I(SOC) in HSG cells (Liu, X., Groschner, K., and Ambudkar, I. S. (2004) J. Membr. Biol. 200, 93-104). Here we have analyzed the molecular composition of this channel. Both thapsigargin (Tg) and 2-acetyl-sn-glycerol (OAG) stimulated similar non-selective cation currents and Ca2+ entry in HSY cells. The effects of Tg and OAG were not additive. HSY cells endogenously expressed TRPC1, TRPC3, and TRPC4 but not TRPC5 or TRPC6. Immunoprecipitation of TRPC1 pulled down TRPC3 but not TRPC4. Conversely, TRPC1 co-immunoprecipitated with TRPC3. Expression of antisense TRPC1 decreased (i) Tg- and OAG-stimulated currents and Ca2+ entry and (ii) the level of endogenous TRPC1 but not TRPC4. Antisense TRPC3 similarly reduced Ca2+ entry and endogenous TRPC3. Yeast two-hybrid analysis revealed an interaction between NTRPC1 and NTRPC3 (CTRPC1-CTRPC3, CTRPC3-CTRPC1, or CTRPC1-NTRPC3 did not interact), which was confirmed by glutathione S-transferase (GST) pull-down assays (GST-NTRPC3 pulled down TRPC1 and vice versa). Expression of NTRPC1 or NTRPC3 induced similar dominant suppression of Tg- and OAG-stimulated Ca2+ entry. NTRPC3 did not alter surface expression of TRPC1 or TRPC3 but disrupted TRPC1-TRPC3 association. In aggregate, our data demonstrate that TRPC1 and TRPC3 co-assemble, via N-terminal interactions, to form a heteromeric store-operated non-selective cation channel in HSY cells. Thus selective association between TRPCs generate distinct store-operated channels. Diversity of store-operated channels might be related to the physiology of the different cell types.

Biotinylation↗
PubMedDate not supplied

Role and regulation of TRP channels in neutrophil granulocytes.

Members of the transient receptor potential (TRP) family for which mRNA can be demonstrated in neutrophil granulocytes with RT-PCR include TRPC6 (as only "short" TRP), TRPM2, TRPV1, TRPV2, TRPV5 and TRPV6. When these are analyzed in heterologous overexpression experiments, TRPM2 is the only cation channel with characteristic properties that can be used as fingerprint to provide functional evidence for its expression in neutrophil granulocytes. As cells transfected with TRPM2, neutrophil granulocytes display non-selective cation currents and typical channel activity evoked by intracellular ADP-ribose and NAD. Thus, stimulation of TRPM2 is likely to occur after activation of CD38 (producing ADP-ribose) and during the oxidative burst (enhancing the NAD concentration). This novel mode of cation entry regulation may be of particular importance for the response of granulocytes to chemoattractants. TRPV6 is a likely but not exclusive candidate as subunit of the channels mediating store-operated Ca2+ entry (SOCE). Evidence for SOCE in granulocytes has been presented with the fura-2 technique but not with electrophysiological methods although Ca2+-selective store-operated currents can be demonstrated in HL-60 cells, a cell culture model of neutrophil granulocytes.

Adenosine Diphosphate Ribose↗
PubMed2005

Expression and functional evaluation of transient receptor potential channel 4 in bovine corneal endothelial cells.

We previously found that activation of purinergic receptors mobilizes Ca2+ and enhances bicarbonate transport in bovine corneal endothelial cells (BCEC). Since transient receptor potential channel 4 (TRPC) has been reported to be a candidate for capacitative calcium entry (CCE) and receptor operated calcium entry (ROC), we examined the expression of TRPC4 and evaluated the potential involvement of TRPC4 in CCE or ROC in BCEC. The C-terminus of TRPC4 was fused into the glutathione S-transferase (GST) expression vector. The fusion protein GST-TRPC4c was induced in bacteria and purified by affinity chromatography. An antibody was raised in rabbit by using the purified GST-TRPC4c antigen. In Western blotting, the TRPC4 antibody recognized the fusion protein while the pre-immune IgG did not. The TRPC4 antibody recognized a band at around 80 kD for membrane proteins from both the fresh and cultured BCEC. The pre-immune IgG could not detect bands at the same size. Incubation with the TRPC4c antigen abolished the 80 kD band. Immunofluorescence using the TRPC4 antibody stained both fresh and cultured BCEC, while pre-immune IgG did not. RNAi knocked down the expression of TRPC4 in cultured BCEC. Ca2+ entry induced by the purinergic receptor agonist ATP, was increased in TRPC4-siRNA transfected cells compared with the scrambled siRNA control, while Ca2+ entry induced by store depletion through blocking the endoplasmic reticulum Ca2+ pump, did not differ between the siRNA and scrambled siRNA-treated cells. Taken together, these results show that TRPC4 protein is expressed in the bovine corneal endothelial cells and may be a negative regulator in ROC stimulated by purinergic activation, but not by store depletion itself.

Animals↗
PubMed1998

Functional interaction between InsP3 receptors and store-operated Htrp3 channels.

Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current (I(crac)). A critical question in cell signalling is how SOC and I(crac) sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion; in an alternative model, InsP3 receptors in the stores are coupled to SOC and I(crac). The mammalian Htrp3 protein forms a well defined store-operated channel and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and I(crac). We show here that Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. Our results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate I(crac).

Animals↗
PubMed2004

Chronic lithium treatment of B lymphoblasts from bipolar disorder patients reduces transient receptor potential channel 3 levels.

Chronic lithium treatment of B-lymphoblast cell lines (BLCLs) from bipolar-I disorder (BD-I) patients and healthy subjects ex vivo attenuates agonist- and thapsigargin-stimulated intracellular calcium (Ca(2+)) responses. As these findings suggest that chronic lithium treatment modifies receptor (ROCE) and/or store-operated Ca(2+) entry (SOCE) mechanisms, we determined whether chronic lithium treatment of BLCLs modified the expression of two members of the transient receptor potential channels (TRPC1 & 3), which participate in ROCE/SOCE. Chronic lithium treatment significantly reduced BLCL TRPC3 immunoreactivity (repeated-measures ANOVA, P=0.00005), with interaction effects of diagnosis (P=0.037) and sex (P=0.040). The lithium-induced decrease was greatest in BLCLs from female BD-I patients compared with those from healthy females (-27%) and with vehicle-treated BLCLs from female BD-I patients (-33%). However, lithium treatment did not affect TRPC1 and 3 mRNA levels, and TRPC1 immunoreactivity. Downregulation of TRPC3 may be an important mechanism by which lithium ameliorates pathophysiological Ca(2+) disturbances as observed in BD.

Adult↗
PubMed2005

Endogenous TRPC1, TRPC3, and TRPC7 proteins combine to form native store-operated channels in HEK-293 cells.

Endogenously expressed canonical transient receptor potential (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 indicated that the individual suppression of TRPC1, TRPC3, or TRPC7 protein levels, by small interfering RNA (siRNA) techniques, dramatically inhibited (52-68%) store-operated calcium entry (SOCE), whereas suppression of TRPC4 or TRPC6 had no effect. Combined suppression of TRPC1-TRPC3, TRPC1-TRPC7, TRPC3-TRPC7, or TRPC1-TRPC3-TRPC7 gave only slightly more inhibition of SOCE (74-78%) 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, induced a high Ba(2+) leak flux that was 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, on the other hand, could 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.

Barium↗
PubMed2005

Phospholipase cgamma1 is required for activation of store-operated channels in human keratinocytes.

Store-operated calcium entry depicts the movement of extracellular Ca2+ into cells through plasma membrane Ca2+ channels activated by depletion of intracellular Ca2+ stores. The members of the canonical subfamily of transient receptor potential channels (TRPC) have been implicated as the molecular bases for store-operated channels (SOC). Here we investigate the role of phospholipase C (PLC) in regulation of native SOC and the expression of endogenous TRPC in human epidermal keratinocytes. Calcium entry in response to store depletion with thapsigargin was reversibly blocked by 2-aminoethoxydiphenyl borane, an effective SOC inhibitor, and suppressed by the diacylglycerol analoge, 1-oleoyl-2-acetyl-sn-glycerol. Inhibition of PLC with U73122 or transfection of a PLCgamma1 antisense cDNA construct completely blocked SOC activity, indicating a requirement for PLC, especially PLCgamma1, in the activation of SOC. RT-PCR and immunoblotting analyses showed that TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 are expressed in keratinocytes. Knockdown of the level of endogenous TRPC1 or TRPC4 inhibited store-operated calcium entry, indicating they are part of the native SOC. Co-immunoprecipitation studies demonstrated that TRPC1, but not TRPC4, interacts with PLCgamma1 and the inositol 1,4,5-trisphosphate receptor (IP3R). The association of TRPC1 with PLCgamma1 and IP3R decreased in keratinocytes with higher intracellular Ca2+, coinciding with a downregulation in SOC activity. Our results indicate that the activation of SOC in keratinocytes depends, at least partly, on the interaction of TRPC with PLCgamma1 and IP3R.

Calcium↗
PubMed1997

[New aspects of the mechanism of Ca2+ entry in non-excitable cells].

The capacitative Ca2+ entry has been accepted to play a crucial role in Ca2+ signaling in non-excitable cells, and the mechanisms linking the depletion of intracellular Ca2+ stores to the activation of Ca2+ entry are presently the subject of intensive investigation. There are two hypotheses to explain the molecular mechanism of capacitative Ca2+ entry. One is that a diffusible second messenger released from intracellular Ca2+ stores may activate a Ca2+ channel at the plasma membrane. Numerous molecules, including CIF, cGMP, arachidonic acid, and a small G-protein, are proposed as the candidates for the diffusible messenger. In addition, the capacitative Ca2+ entry has been suggested to be modulated by protein phosphorylation/dephosphorylation. Another hypothesis is termed the "conformational coupling model". This model suggests that information between the IP3 receptor and a plasma membrane Ca2+ channel may be transferred directly through conformational protein-protein interactions. The plasma membrane Ca2+ channel involved in the capacitative Ca2+ entry is still neither purified nor cloned, but recent studies suggest that the mammalian homologue of the Drosophila protein Trp may function as a capacitative Ca2+ entry channel.

Animals↗
PubMed2003

[Progress in research on the functions of calcium and chloride channels in vascular endothelial cells].

Endothelial cells (EC) lining the inner lumen of the blood vessels are rich in ion channels that constitute the basis of a unique signal transduction function of the cells. The abundance of calcium and chloride channels in the plasma membrane of these nonexcitable cells has raised questions in regard to their functional roles. This review presents evidence for the involvement of these channels in the endothelial cell functions, in an attempt to further explore the relationship between cardiovascular diseases and functional abnormalities of the ion channels.

Animals↗
PubMed2006

TRPC-like conductance mediates restoration of intracellular Ca2+ in cochlear outer hair cells in the guinea pig and rat.

Ca2+ signalling is central to cochlear sensory hair cell physiology through its influence on sound transduction, membrane filter properties and neurotransmission. However, the mechanism for establishing Ca2+ homeostasis in these cells remains unresolved. Canonical transient receptor potential (TRPC) Ca2+ entry channels provide an important pathway for maintaining intracellular Ca2+ levels. TRPC3 subunit expression was detected in guinea pig and rat organ of Corti by RT-PCR, and localized to the sensory and neural poles of the inner and outer hair cells (OHCs) by confocal immunofluorescence imaging. A cation entry current with a TRPC-like phenotype was identified in guinea pig and rat OHCs by whole-cell voltage clamp. This slowly activating current was induced by the lowering of cytosolic Ca2+ levels ([Ca2+]i) following a period in nominally Ca2+-free solution. Activation was dependent upon the [Ca2+]o and was sustained until [Ca(2+)]i was restored. Ca2+ entry was confirmed by confocal fluorescence imaging, and rapidly recruited secondary charybdotoxin- and apamin-sensitive K(Ca) currents. Dual activation by the G protein-coupled receptor (GPCR)-phospholipase C-diacylglycerol (DAG) second messenger pathway was confirmed using the analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Ion substitution experiments showed that the putative TRPC Ca2+ entry current was selective for Na+ > K+ with a ratio of 1: 0.6. The Ca2+ entry current was inhibited by the TRPC channel blocker 2-aminoethyl diphenylborate (2APB) and the tyrosine kinase inhibitor, erbstatin analogue. We conclude that TRPC Ca2+ entry channels, most likely incorporating TRPC3 subunits, support cochlear hair cell Ca2+ homeostasis and GPCR signalling.

Animals↗
PubMed1998

TRP trapped in fly signaling web.

In the Drosophila eye, photoactivation of rhodopsin leads to the opening of the light-sensitive cation influx channels TRP and TRPL. This response is extremely rapid and results in depolarization of the photoreceptor cells followed by Ca(2+)-mediated feedback regulation of the visual signaling cascade. The mechanisms that facilitate the rapid kinetics of activation and feedback regulation are poorly understood. However, the recent discovery that most of the proteins that function in fly phototransduction associate into a supramolecular complex permits a re-evaluation of the mechanisms underlying the activation and regulation of the cascade. The central player in the signaling complex is INAD, a protein with five protein-interaction motifs known as PDZ domains. The INAD complex does not appear to be a particle, but a massive signaling web composed of an INAD polymer with which some of the target proteins associate through complex multivalent interactions.

Animals↗
PubMed2005

Physiological mechanisms of TRPC activation.

TRPC (canonical transient receptor potential) channels are vertebrate homologs of the Drosophila photoreceptor channel, TRP. Considerable research has been brought to bear on the seven members of this family, especially with regard to their possible role in calcium entry. Unfortunately, the current literature presents a confusing picture, with different laboratories producing widely differing results and interpretations. It appears that ectopically expressed TRPC channels can be activated by phospholipase C products (generally, diacylglycerols), by stimulation of trafficking to the plasma membrane, or by depletion of intracellular Ca(2+) stores. Here, I discuss the possibility that these diverse experimental findings arise because TRPC channels can, under both experimental as well as physiological conditions, be activated in three distinct ways, possibly depending on their subunit composition and/or signaling complex environment. The TRPCs may be unique among ion-channel subunit families in being able to participate in the assembly and function of multiple types of physiologically important ion channels.

Animals↗
PubMed2006

Mechanism and functional significance of TRPC channel multimerization.

Ca(2+) signaling regulates many important physiological events within a diverse set of living organisms. In particular, sustained Ca(2+) signals play an important role in controlling cell proliferation, cell differentiation and the activation of immune cells. Two key elements for the generation of sustained Ca(2+) signals are store-operated and receptor-operated Ca(2+) channels that are activated downstream of phospholipase C (PLC) stimulation, in response to G-protein-coupled receptor or growth factor receptor stimulation. One goal of this review is to help clarify the role of canonical transient receptor potential (TRPC) proteins in the formation of native store-operated and native receptor-operated channels. Toward that end, data from studies of endogenous TRPC proteins will be reviewed in detail to highlight the strong case for the involvement of certain TRPC proteins in the formation of one subtype of store-operated channel, which exhibits a low Ca(2+)-selectivity, in contrast to the high Ca(2+)-selectivity exhibited by the CRAC subtype of store-operated channel. A second goal of this review is to highlight the growing body of evidence indicating that native store-operated and native receptor-operated channels are formed by the heteromultimerization of TRPC subunits. Furthermore, evidence will be provided to argue that some TRPC proteins are able to form multiple channel types.

Animals↗
PubMed2006

A review of TRP channels splicing.

Ion channel functional diversity can be achieved at the structural level by means of three main mechanisms: (1) transcriptional regulation and processing of mRNA, (2) heteromerization of different pore-forming channel subunits and (3) incorporation of regulatory subunits to the functional channel complex. In this review article we will focus on one of these mechanisms, alternative pre-mRNA splicing, in the context of the TRP superfamily of cation channels. For this purpose, the basic principles governing pre-mRNA splicing will be introduced and comprehensive tables classifying only published spliced-variants of TRP channels will be presented.

Alternative Splicing↗
PubMed1999

Properties of heterologously expressed hTRP3 channels in bovine pulmonary artery endothelial cells.

1. We combined patch clamp and fura-2 fluorescence methods to characterize human TRP3 (hTRP3) channels heterologously expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which do not express the bovine trp3 isoform (btrp3) but express btrp1 and btrp4. 2. ATP, bradykinin and intracellular InsP3 activated a non-selective cation current (IhTRP3) in htrp3-transfected CPAE cells but not in non-transfected wild-type cells. During agonist stimulation, the sustained rise in [Ca2+]i was significantly higher in htrp3-transfected cells than in control CPAE cells. 3. The permeability for monovalent cations was PNa > PCs approximately PK >> PNMDG and the ratio PCa/PNa was 1.62 +/- 0.27 (n = 11). Removal of extracellular Ca2+ enhanced the amplitude of the agonist-activated IhTRP3 as well as that of the basal current The trivalent cations La3+ and Gd3+ were potent blockers of IhTRP3 (the IC50 for La3+ was 24.4 +/- 0.7 microM). 4. The single-channel conductance of the channels activated by ATP, assessed by noise analysis, was 23 pS. 5. Thapsigargin and 2,5-di-tert-butyl-1, 4-benzohydroquinone (BHQ), inhibitors of the organellar Ca2+-ATPase, failed to activate IhTRP3. U-73122, a phospholipase C blocker, inhibited IhTRP3 that had been activated by ATP and bradykinin. Thimerosal, an InsP3 receptor-sensitizing compound, enhanced IhTRP3, but calmidazolium, a calmodulin antagonist, did not affect IhTRP3. 6. It is concluded that hTRP3 forms non-selective plasmalemmal cation channels that function as a pathway for agonist-induced Ca2+ influx.

Adenosine Triphosphate↗
PubMed2004

Pharmacological properties and functional role of a TRP-related ion channel in lobster olfactory receptor neurons.

Odors activate lobster olfactory receptor neurons (ORNs) through phosphoinositide signaling that appears to target a Na(+)-gated nonselective cation channel. The Na(+)-gated channel is a potential member of the growing family of transient receptor potential (TRP) channels. Here, we test the effect of potential antagonists on the channel in cell-free patches from cultured lobster ORNs. We show that the channel is antagonized by H+ and the TRP channel blockers 2-aminoethoxydiphenyl borate, SKF96365, ruthenium red, Al3+, Gd3+, and La3+. We then use this enhanced antagonist profile together with the agonists Na+ and Ca2+ to implicate the channel in signal amplification in the cells.

Animals↗