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PubMed2004

Evidence that TRPC3 is a molecular component of the 1alpha,25(OH)2D3-activated capacitative calcium entry (CCE) in muscle and osteoblast cells.

In chick skeletal muscle and in rat osteoblast-like cells (ROS 17/2.8), 1alpha,25-dihydroxy-Vitamin-D(3) [1alpha,25(OH)(2)D(3)] stimulates release of Ca(2+) from inner stores and extracellular cation influx through both voltage-dependent and capacitative Ca(2+) entry (CCE) channels. We investigated the involvement of TRPC proteins in CCE induced by 1alpha,25(OH)(2)D(3). Two fragments were amplified by RT-PCR, exhibiting >85% sequence homology with human TRPC3. Northern and Western blots employing TRPC3-probes and anti-TRPC3 antibodies, respectively, confirmed endogenous expression of a TRPC3-like protein. Both cell types transfected with anti-TRPC3 antisense oligodeoxynucleotides showed reduced CCE and Mn(2+) entry induced by either thapsigargin or 1alpha,25(OH)(2)D(3). In muscle cells, anti-VDR antisense inhibited steroid-induced Ca(2+) and Mn(2+) influx and co-immunoprecipitation of TRPC3 and VDR was observed, suggesting an association between both proteins and a functional role of the receptor in 1alpha,25(OH)(2)D(3) activation of CCE. In osteoblasts, two PCR fragments showing high homology with human INAD-like sequences were obtained. Northern blot and antisense functional assays suggested the involvement of the INAD-like protein in CCE regulation by the hormone. Therefore, we propose that an endogenous TRPC3 protein mediates 1alpha,25(OH)(2)D(3) modulation of CCE in muscle and osteoblastic cells, which seems to implicate VDR-TRPC3 association and the participation of a INAD-like scaffold protein.

Animals↗
PubMed2004

Role of store-dependent influx of Ca2+ and efflux of K+ in apoptosis of CHO cells.

Agents mobilising Ca(2+) from the endoplasmic reticulum are known to activate apoptosis. Whatever means are used, the release of Ca(2+) is often followed by a store-dependent entry of Ca(2+). Whether apoptosis is triggered by the depletion of the stores or by the subsequent store-dependent entry of Ca(2+) is still a matter of controversy. Here we studied apoptosis in CHO cells transfected with the rat neurotensin (NT) receptor, in which the store-dependent entry of Ca(2+) is abolished by repressing the transient receptor potential channel 2 (TRPC2) by an antisense oligonucleotide strategy (TRPC2(-) cells) [Cell Calcium 30 (2001) 157]. When stimulated with thapsigargin (TG), apoptosis occurred in both TRPC2(+) and TRPC2(-) cells but 12h earlier in TRPC2(+) cells, suggesting that store-dependent entry of Ca(2+) can accelerate the process. The expression and localisation of caspase-12, an enzyme that has been involved in the apoptosis triggered by a stress on the endoplasmic reticulum, was not different in TRPC2(+) and TRPC2(-) cells. On the contrary, the expression of GADD153 (Growth Arrest and DNA Damage inducible gene 153) triggered by TG treatment depended on external Ca(2+) and occurred earlier in TRPC2(+) than in TRPC2(-) cells. In these cells, we also noted the presence of K(+) channels activated by Ca(2+) (K(Ca) channels). Stimulation of TRPC2(+) cells with TG or with NT triggered a long sustained K(+) current, parallel to [Ca(2+)](i) transients, and resulting in a sustained hyperpolarisation of the cell membrane. K(+) current and hyperpolarisation were transient and not sustained in TRPC2(-) cells. Inhibition of K(Ca) channels with charybdotoxin dramatically reduced the K(+) current and also significantly brought down the level of apoptosis, suggesting that a prolonged efflux of K(+) could be involved in the apoptosis process. We conclude that in CHO cells, store-dependent entry of Ca(2+) can accelerate apoptosis by accelerating the expression of GADD153 and by inducing a prolonged efflux of K(+) out of the cell.

Animals↗
PubMed2002

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↗
PubMed2004

Canonical transient receptor potential TRPC7 can function as both a receptor- and store-operated channel in HEK-293 cells.

Previous studies on the activation mechanism of canonical transient receptor potential (TRPC) channels have often produced conflicting conclusions. All seven have been shown to be activated by phospholipase C (PLC)-coupled receptors, but TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, and TRPC7 have also been proposed to function as store-operated channels.(1)1Although PLC activation inevitably leads to activation of store-operated channels, in this report when we refer to PLC-activated channels, we mean those channels that are specifically activated by PLC independently of store depletion. In the case of TRPC3, the expression environment and the expression level appear to determine the mode of regulation. Evidence of a close structural relative of TRPC3, TRPC7, has been presented that this channel is activated by receptor activation or by store depletion. On the basis of previous findings for TRPC3, we reasoned that subtle differences in structure or expression conditions might account for the apparent distinct gating mechanisms of TRPC7. To reexamine the mode of activation of TRPC7, we stably and transiently transfected human embryonic kidney (HEK)-293 cells with cDNA encoding for human TRPC7. We examined the ability of a PLC-activating agonist and an intracellular Ca(2+) store-depleting agent to activate these channels. Our findings demonstrate that when transiently expressed in HEK-293 cells, TRPC7 forms channels that are activated by PLC-stimulating agonists, but not by Ca(2+) store depletion. However, when stably expressed in HEK-293 cells, TRPC7 can be activated by either Ca(2+) store depletion or PLC activation. To our knowledge, this is the first demonstration of a channel protein that can be activated by both receptor- and store-operated modes in the same cell. In addition, the results reconcile the apparently conflicting findings of other laboratories regarding TRPC7 regulation.

Barium↗
PubMed2006

Chemosensory properties of murine nasal and cutaneous trigeminal neurons identified by viral tracing.

BACKGROUND: Somatosensation of the mammalian head is mainly mediated by the trigeminal nerve that provides innervation of diverse tissues like the face skin, the conjunctiva of the eyes, blood vessels and the mucouse membranes of the oral and nasal cavities. Trigeminal perception encompasses thermosensation, touch, and pain. Trigeminal chemosensation from the nasal epithelia mainly evokes stinging, burning, or pungent sensations. In vitro characterization of trigeminal primary sensory neurons derives largely from analysis of complete neuronal populations prepared from sensory ganglia. Thus, functional properties of primary trigeminal afferents depending on the area of innervation remain largely unclear. RESULTS: We established a PrV based tracing technique to identify nasal and cutaneous trigeminal neurons in vitro. This approach allowed analysis and comparison of identified primary afferents by means of electrophysiological and imaging measurement techniques. Neurons were challenged with several agonists that were reported to exhibit specificity for known receptors, including TRP channels and purinergic receptors. In addition, TTX sensitivity of sodium currents and IB4 binding was investigated. Compared with cutaneous neurons, a larger fraction of nasal trigeminal neurons showed sensitivity for menthol and capsaicin. These findings pointed to TRPM8 and TRPV1 receptor protein expression largely in nasal neurons whereas for cutaneous neurons these receptors are present only in a smaller fraction. The majority of nasal neurons lacked P2X3 receptor-mediated currents but showed P2X2-mediated responses when stimulated with ATP. Interestingly, cutaneous neurons revealed largely TTX resistant sodium currents. A significantly higher fraction of nasal and cutaneous afferents showed IB4 binding when compared to randomly chosen trigeminal neurons. CONCLUSION: In conclusion, the usability of PrV mediated tracing of primary afferents was demonstrated. Using this technique it could be shown that compared with neurons innervating the skin nasal trigeminal neurons reveal pronounced chemosensitivity for TRPM8 and TRPV1 channel agonists and only partially meet properties typical for nociceptors. In contrast to P2X3 receptors, TRPM8 and TRPV1 receptors seem to be of pronounced physiological relevance for intranasal trigeminal sensation.

Afferent Pathways↗
PubMed2005

TRP channels in Drosophila photoreceptor cells.

TRP cation channels are conserved throughout animal phylogeny and include many members that function in sensory physiology. The founding TRP is required for Drosophila phototransduction and has served as a paradigm for unravelling the roles and macromolecular organizations of TRP channels in native tissues. Two other TRPC channels, TRPL and TRPgamma, are expressed in photoreceptor cells and form heteromultimers with TRP and with each other. TRP is a member of a supramolecular signalling complex, the signalplex, which includes the PDZ scaffold protein, INAD, and two other core members that remain bound and depend on INAD for localization. Other INAD binding proteins are proposed to interact dynamically with INAD, one of which, TRPL, undergoes light-dependent translocation in photoreceptor cells. Surprisingly, TRP has non-channel functions, including an anchoring role necessary for retaining INAD in the rhabdomeres. Loss of TRP function or constitutive TRP activity results in retinal degeneration, which can be suppressed by disruption or overexpression of the Na+/Ca2+ exchanger, CalX, respectively. Given that hypoxia-induced constitutive activity of some mammalian TRPs leads to neuronal cell death, interventions that increase Na+/Ca2+ exchanger or decrease TRP function have the potential to reduce the severity of cell death due to ischaemia.

Animals↗
PubMed2004

Diversity of TRP channel activation.

Calcium entry controls a plethora of short- and long-term cell functions. With respect to the activation mechanisms of Ca2+ entry channels they are subdivided in the thoroughly characterized voltage-gated Ca2+ channels and the non-voltage gated Ca2+ channels. The latter group includes cation channels of the 'transient receptor potential' (TRP) superfamily, which consists of three subfamilies (TRPC, TRPV, TRPM). Activation of TRP channels is not yet completely understood, but examples of activation mechanisms of TRP channels from all three subfamilies will be discussed. The main focus is on the members of the TRPV subfamily, among which the TRPV4 channel shows a surprising gating promiscuity. It can be activated by cell swelling, heat and phorbol esters. Endogenous activators of the channel have not yet been described. It will be shown that lipid messengers related to arachidonic acid are endogenous TRPV4 activators. For TRPV5 and 6, the only highly Ca2+ selective channels within the TRP super-family, a voltage-dependent gating mechanism will be discussed, which includes an open pore block by Mg2+ and a highly Ca2+-sensitive mechanism of inactivation. Regulation of channel availability by interaction with a protein bound to a site at the C-terminus of both channels will be demonstrated. Functional consequences of these different mechanisms of gating will be discussed.

Animals↗
PubMed2006

An introduction to TRP channels.

The aim of this review is to provide a basic framework for understanding the function of mammalian transient receptor potential (TRP) channels, particularly as they have been elucidated in heterologous expression systems. Mammalian TRP channel proteins form six-transmembrane (6-TM) cation-permeable channels that may be grouped into six subfamilies on the basis of amino acid sequence homology (TRPC, TRPV, TRPM, TRPA, TRPP, and TRPML). Selected functional properties of TRP channels from each subfamily are summarized in this review. Although a single defining characteristic of TRP channel function has not yet emerged, TRP channels may be generally described as calcium-permeable cation channels with polymodal activation properties. By integrating multiple concomitant stimuli and coupling their activity to downstream cellular signal amplification via calcium permeation and membrane depolarization, TRP channels appear well adapted to function in cellular sensation. Our review of recent literature implicating TRP channels in neuronal growth cone steering suggests that TRPs may function more widely in cellular guidance and chemotaxis. The TRP channel gene family and its nomenclature, the encoded proteins and alternatively spliced variants, and the rapidly expanding pharmacology of TRP channels are summarized in online supplemental material.

Amino Acid Sequence↗
PubMed2006

TRPC3 and TRPC4 associate to form a redox-sensitive cation channel. Evidence for expression of native TRPC3-TRPC4 heteromeric channels in endothelial cells.

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 we have previously suggested TRPC proteins as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), we set out to analyze the TRPC subunit composition of endogenous endothelial TRPC channels and report here on a redox-sensitive TRPC3-TRPC4 channel complex. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex was 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. We propose TRPC3 and TRPC4 as subunits of native endothelial cation channels that are governed by the cellular redox state.

Animals↗
PubMed1996

The roles of trp and calcium in regulating photoreceptor function in Drosophila.

Invertebrate photoreceptors use the ubiquitous inositol-lipid signaling pathway for phototransduction. This pathway depends on Ca2+ release from internal stores and on Ca2+ entry via light-activated channels to replenish the loss of Ca2+ in those stores. The Drosophila transient receptor potential (TRP) protein is essential for the high Ca2+ permeability and other biophysical properties of these light-activated channels, which affect both excitation and adaptation in photoreceptor cells. Physiological and heterologous expression studies indicate that TRP is a putative subunit of a surface membrane channel that can be activated by depletion of internal Ca2+ stores. Furthermore, trp is an archetypal member of a multigene family whose products share a structure that is highly conserved throughout evolution, from worms to humans.

Adaptation, Ocular↗
PubMed2003

Nonselective cation channels activated by the stimulation of muscarinic receptors in mammalian gastric smooth muscle.

Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptors were present in circular smooth muscle cells of the guinea-pig gastric antrum. Nonselective cation channels (NSCC) activated by ACh or CCh are coupled to pertussis toxin (PTX)-sensitive Go protein through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach. CCh-activated currents (I(CCh)), especially the steady-state I-V relationship of I(CCh) showed a chracteristic U-shaped curve; reversal potential of around 0 mV and inward rectification at around +15 mV and a negative slope conductance at negative potential range. Under physiological conditions, the measured single channel conductance of NSCC was approximately 25 pS. The single channel conductance was modulated by external monovalent and divalent cations including Na+, Cs+, Li+, and Ca2+ through changing both the open probability and unitary conductance. Through the NSCC, Ca2+ can move into the cell from extracellular solution as well as Na+. Calculated fractional Ca2+ current of I(CCh) (f(Ca)) was around 1% at the 2 mM [Ca2+]o and at the 4 mM [Ca2+]o, f(Ca) was 2.3%. Quinidine blocked I(CCh) potently in a reversible manner; IC50 was 0.25 microM. There were two kinds of I(CCh) modulations through Ca(2+)-dependent pathways in guinea-pig gastric smooth muscle cells; 1) Facilitation of I(CCh) via Ca2+/CaM-dependent MLCK pathway, 2) Desensitization of I(CCh) via Ca(2+)-dependent PKC pathway. In the mouse stomach, all seven types of TRPC mRNA were detected with RT-PCR. On the basis of electrophysiological, pharmacological, and molecular biological experiments, we reported the mTRPC5 as a candidate for the NSCC activated by muscarinic stimulation in mouse stomach.

Animals↗
PubMed1998

Ionic currents underlying HTRP3 mediated agonist-dependent Ca2+ influx in stably transfected HEK293 cells.

hTrp3 is a human homologue of the Drosophila gene responsible for a transient receptor potential (trp) mutation. When stably expressed in HEK293 cells, hTrp3 formed ion channels that were active under resting conditions but could be further stimulated by carbachol or ATP via endogenous muscarinic or purinergic receptors, respectively. Agonist evoked currents reversed polarity near 0 mV in physiological ionic conditions and were associated with a significant increase in the current variance. These results suggest the involvement of a non-selective cation channel with relatively large unitary amplitude. Consistent with this, resolved unitary events had a conductance of approximately 60 pS in the negative voltage range and an extrapolated reversal potential near 0 mV.

Adenosine Triphosphate↗
PubMed1998

Functional expression of TrpC1: a human homologue of the Drosophila Trp channel.

TrpC1 appears to be a store-operated channel (SOC) when expressed in mammalian cells. In the present study, TrpC1 was expressed in Sf9 insect cells using the baculovirus expression system. Expression of TrpC1 caused an increase in basal cytosolic free Ca2+ concentration ([Ca2+]i) as a function of post-infection time. Basal Ba2+ influx, an index of plasmalemmal Ca2+ permeability, was also increased and was blocked by La3+. Although the thapsigargin-induced change in [Ca2+]i was greater in TrpC1-expressing cells than controls, Ba2+ influx was unaffected by thapsigargin. Whole-cell membrane currents recorded in TrpC1-expressing cells increased as a function of post-infection time and were (1) inwardly rectifying in symmetrical sodium gluconate solutions, (2) non-selective with respect to Na+, Ca2+ and Ba2+, and (3) blocked by La3+. Furthermore TrpC1 currents were unaffected by (1) thapsigargin, (2) dialysis of the cell with Ins(1,4,5)P3 or (3) dialysis of the cell with solutions containing high concentrations of the Ca2+ chelator, EGTA. These results suggest that TrpC1 forms non-selective cation channels that are constitutively active when expressed in Sf9 cells, but insensitive to depletion of the internal Ca2+ stores. Thus TrpC1 may be a subunit of a SOC which alone can form functional channels in Sf9 cells, but which requires additional subunits or cytoplasmic factors present in mammalian cells for expression of SOC activity.

Animals↗
PubMed2002

Vanilloid and TRP channels: a family of lipid-gated cation channels.

The emergence of the TRP (C) and vanilloid (TRPV) receptor family of Ca(2+) permeable channels has started to provide molecular focus to a linked group of ion channels whose common feature is activation primarily by intracellular ligands. These channels have a central role in Ca(2+) homeostasis in virtually all cells and in particular those that lack voltage-gated Ca(2+) channels. We will discuss recent work that is more precisely defining both molecular form and physiological function of this important group of Ca(2+) permeable channels with particular focus on the intracellular ligands that gate and modulate channel activity.

Animals↗
PubMed2005

TRPC channel expression during calcium-induced differentiation of human gingival keratinocytes.

BACKGROUND: Extracellular calcium is an important regulator of keratinocyte differentiation. An increase in intracellular calcium ion concentration is required for activation of calcium-induced keratinocyte differentiation. The signaling elements in this differentiation response include the calcium sensing receptor, phospholipase C, release of calcium ions from intracellular stores, and store-operated calcium channels. Nothing is currently known about the calcium-entry channels activated by the increase in external calcium. However, canonical transient receptor potential (TRPC) channels have been identified as store-operated calcium channels in several tissues. OBJECTIVE: To examine the expression of TRPC channels in human gingival keratinocytes (HGKs) in primary culture under both low calcium (basal) and high calcium (differentiating) conditions, and in gingival tissue. METHODS: TRPC channel expression was evaluated via RT-PCR, Western blots, and immunohistology. RESULTS: TRPC1, TRPC5, TRPC6 and TRPC7 mRNAs were detected in undifferentiated keratinocytes. Their levels initially increased, then decreased during calcium-induced differentiation. TRPC1 and TRPC6 protein expression reflected these changes. CONCLUSION: TRPC channels are present in both proliferating and differentiating keratinocytes in primary culture and in gingival tissue. The above expression patterns suggest that these channels may be involved in calcium-induced differentiation of keratinocytes.

Calcium↗
PubMed2003

Capacitative cation entry in human myometrial cells and augmentation by hTrpC3 overexpression.

Transient receptor potential (Trp) channels have been implicated in mediating store- and receptor-activated Ca2+ influx. Different properties of this influx in various cell types may stem from the assembly of these Trp proteins into homo- or heterotetramers or association with other regulatory proteins. We examined the properties of endogenous capacitative Ca2+ entry in PHM1 immortalized human myometrial cells that express endogenous hTrpCs 1, 3, 4, 6, and 7 mRNA and in primary human myocytes. In PHM1 cells, activation of the oxytocin receptor or depletion of intracellular Ca2+ stores with the endoplasmic reticulum calcium pump-inhibitor thapsigargin induced capacitative Ca2+ entry, which was inhibited both by SKF 96365 and gadolinium (Gd3+). Whereas unstimulated cells did not exhibit Sr2+ entry, oxytocin and thapsigargin enhanced Sr2+ entry that was also inhibited by SKF 96365 and Gd3+. In contrast, Ba2+, a poor substrate for Ca2+ pumps, accumulated in these cells in the absence of the capacitative entry stimulus and also after oxytocin and thapsigargin treatment. Both types of entry were markedly decreased by SKF 96365 and Gd3+. The membrane-permeant derivative of diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), elicited oscillatory increases in PHM1 intracellular Ca2+ that were dependent on extracellular Ca2+. These properties were also observed in primary human myocytes. Overexpression of hTrpC3 in PHM1 cells enhanced thapsigargin-, oxytocin-, and OAG-induced Ca2+ entry. These data are consistent with the expression of endogenous hTrpC activity in myometrium. Capacitative Ca2+ entry can potentially contribute to Ca2+ dynamics controlling uterine smooth muscle contractile activity.

Adenoviridae↗
PubMed2005

The non-selective cation-permeable channel TRPC3 is a tetrahedron with a cap on the large cytoplasmic end.

TRPC3 plays important roles in neuronal differentiation and immune cell maturation by mediating the cationic current in response to phospholipase C activation, Ca2+ depletion, and diacylglycerol stimulation. Here, we purified the TRPC3 channel using a glycosylated tetramer and observed the structure using electron microscopy. Negatively stained specimens demonstrate homogeneous protein particles containing an internal cavity-like structure. These particle images were picked up by automated pick-up programs, aligned, and classified by the growing neural gas network method. Similarly oriented projections were averaged to decrease the signal-to-noise ratio. The averaged images progress from the top view to the side views, which are representative of their raw images. The top view confirmed the hypothesis of a four-domain structure, and the side view demonstrates a large cytoplasmic domain with a capped structure at the bottom, which is near a predicted locus of ion release. The total image of the protein is a blunt-edged trapezoid of 200 x 200 x 235 A. This large dimension of TRPC3 is also supported by the Stokes radius (92 A) obtained from gel filtration chromatography.

Chromatography, Gel↗