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PubMed2005

Homo- and heteromeric assembly of TRP channel subunits.

Mammalian homologues of the Drosophila melanogaster transient receptor potential (TRP) channels are the second largest cation channel family within the superfamily of hexahelical cation channels. Most mammalian TRP channels function as homooligomers and mediate mono- or divalent cation entry upon activation by a variety of stimuli. Because native TRP channels may be multimeric proteins of possibly complex composition, it is difficult to compare cation conductances in native tissues to those of clearly defined homomeric TRP channel complexes in living cells. Therefore, the possibility of heteromeric TRP channel assembly has been investigated in recent years by several groups. As a major conclusion of these studies, most heteromeric TRP channel complexes appear to consist of subunit combinations only within relatively narrow confines of phylogenetic subfamilies. Although the general capability of heteromer formation between closely related TRP channel subunits is now clearly established, we are only beginning to understand whether these heteromeric complexes are of physiological significance. This review summarizes the current knowledge on the promiscuity and specificity of the assembly of channel complexes composed of TRPC-, TRPV- and TRPM-subunits of mammalian TRP channels.

Animals↗
PubMed2006

Role of Src in C3 transient receptor potential channel function and evidence for a heterogeneous makeup of receptor- and store-operated Ca2+ entry channels.

Receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) are known to be inhibited by tyrosine kinase inhibitors and 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 we now report that src phosphorylates TRPC3 at Y226 and that formation of phospho-Y226 is essential for TRPC3 activation. This requirement is unique for TRPC3 because (i) mutation of the cognate tyrosines of the closely related TRPC6 and TRPC7 had no effect; (ii) TRPC6 and TRPC7 were activated in src-, yes-, and fyn-deficient cells; and (iii) src, but not yes or fyn, rescued TRPC3 activation in src-, yes-, and fyn-deficient cells. The Src homology 2 domain of src was found to interact 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 ROCE in HEK cells mediated by TRPC3, TRPC6, TRPC7, and TRPC5 showed differences that argue for ROCE and SOCE channels to be heterogeneous.

Animals↗
PubMed2004

Invertebrate TRP proteins as functional models for mammalian channels.

Transient receptor potential (TRP) channels constitute a large and diverse family of channel proteins that are expressed in many tissues and cell types in both vertebrates and invertebrates. While the biophysical features of many of the mammalian TRP channels have been described, relatively little is known about their biological roles. Invertebrate TRPs offer valuable genetic handles for characterizing the functions of these cation channels in vivo. Importantly, studies in model organisms can help to identify fundamental mechanisms involved in normal cellular functions and human disease. In this review, we give an overview of the different TRP channels known in the two most utilized invertebrate models, the nematode Caenorhabditis elegans and the fruit-fly Drosophila melanogaster, and discuss briefly the heuristic impact of these invertebrate channels with respect to TRP function in mammals.

Animals↗
PubMedDate not supplied

TRPC1 store-operated cationic channel subunit.

TRPC1 is a membrane protein that is highly conserved in mammals, amphibians and birds. It is widely expressed in cells throughout the body including in the heart and nervous system. Amino acid sequence analysis and over-expression studies indicate it is an ion channel that allows the transmembrane flux of small cations including sodium and calcium. In some cell types it is apparent that at least a fraction of TRPC1 exists in the plasma membrane. Inhibition of TRPC1 expression or block by TRPC1-specific antibody leads to attenuation of the plasma membrane calcium influx that occurs in response to depletion of calcium levels in sarcoplasmic or endoplasmic reticulum. TRPC1 would, therefore, seem to be a key subunit of store-operated channels (SOCs). TRPC1 is, nevertheless, unlikely to act alone. There is good evidence that it can heteromultimerise with the related proteins TRPC4, TRPC5 and polycystin-2; a tetrameric arrangement is envisaged, but not demonstrated. Like its relative in Drosophila, TRPC1 looks likely to function in a signalplex, a protein complex including inositol 1,4,5-triphosphate (IP(3)) receptor, plasma membrane calcium-ATPase, caveolin-1 and calmodulin. Its localisation in membranes is punctate and associated with functionally discrete calcium signals. TRPC1's function may not only be linked to SOCs but also to other cellular events including the nuclear translocation of the NFAT transcription factor. There is still much to be learned about this fundamental protein.

Amino Acid Sequence↗
PubMed2005

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

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

Animals↗
PubMed2003

Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD.

An early key event in the activation of neutrophil granulocytes is Ca(2+) influx. Members of the transient receptor potential (TRP) channel family may be held responsible for this. The aim of the present study is to analyse the expression pattern of TRP mRNA and identify characteristic currents unambiguously attributable to particular TRP channels. mRNA was extracted from human neutrophils, isolated by gradient centrifugation and also by magnetically labelled CD15 antibodies. The presence of mRNA was demonstrated using reverse transcriptase-PCR in neutrophils (controlled to be CD5-negative) as well as in human leukaemic cell line 60 (HL-60) cells, for the following TRP species: the long TRPC2 (LTRPC2), the vanilloid receptor 1, the vanilloid receptor-like protein 1 and epithelial Ca(2+) channels 1 and 2. TRPC6 was specific for neutrophils, whereas only in HL-60 cells were TRPC1, TRPC2, TRPC3, melastatin 1 and melastatin-related 1 found. Patch-clamp measurements in neutrophils revealed non-selective cation currents evoked by intracellular ADP-ribose and by NAD(+). Both these modes of activation have been found to be characteristic of LTRPC2. Furthermore, single-channel activity was resolved in neutrophils and it was indistinguishable from that in LTRPC2-transfected HEK-293 cells. The results provide evidence that LTRPC2 in neutrophil granulocytes forms an entry pathway for Na(+) and Ca(2+), which is regulated by ADP-ribose and the redox state.

Adenosine Diphosphate Ribose↗
PubMed2003

Evidence that TRPC1 (transient receptor potential canonical 1) forms a Ca(2+)-permeable channel linked to the regulation of cell volume in liver cells obtained using small interfering RNA targeted against TRPC1.

The TRPC1 (transient receptor potential canonical 1) protein, which is thought to encode a non-selective cation channel activated by store depletion and/or an intracellular messenger, is expressed in a number of non-excitable cells. However, the physiological functions of TRPC1 are not well understood. The aim of these studies was to investigate the function of TRPC1 in liver cells using small interfering RNA (siRNA) to ablate the TRPC1 protein. Treatment of H4-IIE liver cells with siRNA targeted against TRPC1 caused an approx. 50% decrease in expression of the human TRPC1 protein in cells transfected with cDNA encoding human TRPC1, and a 50% decrease in expression of the endogenous TRPC1 protein (assessed by Western blot and immunofluorescence). The decrease in endogenous TRPC1 protein in cells transfected with TRPC1 siRNA was associated with a greater increase in cell volume (compared with the increase observed in control cells) immediately after cells were placed in a hypotonic medium, and an enhanced regulatory cell volume decrease after exposure to hypotonic medium. Treatment with siRNA targeted against TRPC1 also led to a 25% inhibition of thapsigargin-stimulated Ca(2+) inflow, a 40% inhibition of ATP and maitotoxin-stimulated Ca(2+) inflow, and a 50% inhibition of maitotoxin-stimulated Mn(2+) inflow. The idea that, in liver cells, TRPC1 encodes a non-selective cation channel involved directly or indirectly in the regulation of cell volume is consistent with the results obtained.

Animals↗
PubMed1998

Molecular cloning of a novel putative Ca2+ channel protein (TRPC7) highly expressed in brain.

We have isolated cDNA clones for a novel human protein, TRPC7 (transient receptor potential-related channels), which consists of 1503 amino acid residues from the fetal brain and caudate nucleus cDNA libraries. Northern blot analysis indicated that the TRPC7 gene is highly expressed as a 6.5-kb transcript in brain. The TRPC7 protein has significant homology with Caenorhabditis elegans hypothetical proteins T01H8.5, C05C12.3, and F54D1.5 and with Drosophila and human transient receptor potential (trp) proteins. The TRPC7 protein has seven putative transmembrane domains that probably constitute a Ca2+ channel as in the above-mentioned proteins. Genomic sequencing revealed that the TRPC7 gene consists of 32 exons spanning approximately 90 kb. The TRPC7 gene was mapped between D21S400 and D21S171 on human chromosome 21q22.3, 14 kb distal to a NotI site in D21S400. This novel TRPC7 gene could be a candidate gene for genetic disorders such as bipolar affective disorder, nonsyndromic hereditary deafness, Knobloch syndrome, and holoprosencephaly, which were mapped to this region.

Amino Acid Sequence↗
PubMed2004

TRPC3-like protein and vitamin D receptor mediate 1alpha,25(OH)2D3-induced SOC influx in muscle cells.

1alpha,25-Dihydroxy-Vitamin-D3 (1alpha,25(OH)2-Vitamin D3) stimulates in skeletal muscle cells Ca2+ release from inner stores and influx through both voltage-dependent and store-operated Ca2+ (SOC, CCE) channels. We investigated the involvement of TRPC proteins and Vitamin D receptor (VDR) in CCE induced by 1alpha,25(OH)2D3 in chick muscle cells. Two fragments were amplified by RT-PCR, exhibiting approximately 80% sequence homology with mammalian TRPC3/6/7. Northern and Western blots employing a TRPC3-probe and anti-TRPC3 antibodies, respectively, confirmed endogenous expression of a TRPC3-like protein of 140 kDa. Spectrofluorimetric measurements in Fura-2 loaded cells showed reduced CCE and Mn2+ entry in response to either thapsigargin or 1alpha,25(OH)2D3 upon transfection with anti-TRPC3/6/7 antisense oligodeoxynucleotides (ODNs). Transfection with anti-VDR antisense ODNs diminished 1alpha,25(OH)2D3-dependent Ca2+ and Mn2+ influx. Co-immunoprecipitation of TRPC3-like protein and VDR under non-denaturating conditions was observed. We propose that endogenous TRPC3-like proteins and the VDR participate in the modulation of CCE by 1alpha,25(OH)2D3 in muscle cells, which could be mediated by an interaction between these proteins.

Animals↗
PubMed2006

Pain signalling pathways: from cytokines to ion channels.

Pain is the major reason patients seek medical care. The treatment of pain, particularly chronic pain associated with cancer and damage to the nervous system, is at present inadequate. Lack of effective analgesics is partly due to the fact that pain signalling mechanisms are still not fully understood. Over the recent years, many channels, receptors, and regulatory proteins involved in pain pathways have bee identified, and novel pain signalling mechanisms and pathways at peripheral and spinal levels have been discovered. It is anticipated that increased understanding of the molecular mechanisms of pain would provide a hope for the future development of effective pain killers. This review examines the currently available information on the molecular aspects of pain signalling pathways, and discusses novel and promising therapeutic targets for the treatment of pain in humans.

Adenosine Triphosphate↗
PubMedDate not supplied

Making sense with TRP channels: store-operated calcium entry and the ion channel Trpm5 in taste receptor cells.

The sense of taste plays a critical role in the life and nutritional status of organisms. During the last decade, several molecules involved in taste detection and transduction have been identified, providing a better understanding of the molecular physiology of taste receptor cells. However, a comprehensive catalogue of the taste receptor cell signaling machinery is still unavailable. We have recently described the occurrence of calcium signaling mechanisms in taste receptor cells via apparent store-operated channels and identified Trpm5, a novel candidate taste transduction element belonging to the mammalian family of transient receptor potential channels. Trpm5 is expressed in a tissue-restricted manner, with high levels in gustatory tissue. In taste cells, Trpm5 is co-expressed with taste-signaling molecules such as alpha-gustducin, Ggamma(13), phospholipase C beta(2) and inositol 1,4,5-trisphosphate receptor type III. Biophysical studies of Trpm5 heterologously expressed in Xenopus oocytes and mammalian CHO-K1 cells indicate that it functions as a store-operated channel that mediates capacitative calcium entry. The role of store-operated channels and Trpm5 in capacitative calcium entry in taste receptor cells in response to bitter compounds is discussed.

Animals↗
PubMed2007

Assembly domains in TRP channels.

The large family of mammalian TRP (transient receptor potential) ion channels encompasses diverse sensory functions. TRP proteins consist of six transmembrane domains, with a pore-loop motif between the fifth and sixth domains and cytosolic N- and C-termini. The intracellular strands not only interact with various proteins and lipids, but also include essential multimerization regions. This review summarizes the current knowledge of the intrinsic assembly domains that assure tetrameric TRP channel formation.

Amino Acid Motifs↗
PubMed1999

TRP and calcium stores in Drosophila phototransduction.

Phototransduction in Drosophila is mediated by the ubiquitous phosphoinositide cascade, leading to opening of the TRP and TRPL channels, which are prototypical members of a novel class of membrane proteins. Drosophila mutants lacking the TRP protein display a response to light that declines to the dark level during illumination. It has recently been suggested that this response inactivation results from a negative feedback by calcium-calmodulin, leading to closure of the TRPL channels. It is also suggested that in contrast to other phosphoinositide-mediated systems, Ca2+ release from internal stores is neither involved in channel activation nor in phototransduction in general. We now show that inactivation of the light response in trp photoreceptors is enhanced upon reduction of the intracellular Ca2+ concentration. Furthermore, in Ca(2+)-free medium, when there is no Ca2+ influx into the photoreceptors, we demonstrate a significant elevation of intracellular Ca2+ upon illumination. This elevation correlates with ability of the cells to respond to light. Accordingly, malfunctioning of Ca2+ stores, either by Ca2+ deprivation or by application of the Ca2+ pump inhibitor, thapsigargin, confers a trp phenotype on wild type flies. The results indicate that the response inactivation in trp cells results from Ca2+ deficiency rather than from Ca(2+)-dependent negative feedback. The results also indicate that there is light-induced release of Ca2+ from intracellular stores. Furthermore, the response to light is correlated to Ca2+ release, and normal function of the stores is required for prolonged excitation. We suggest that phototransduction in Drosophila depends on Ca(2+)-release mediated signalling and that TRP is essential for the normal function of this process.

Animals↗
PubMed2000

Trp1, a candidate protein for the store-operated Ca(2+) influx mechanism in salivary gland cells.

The trp gene family has been proposed to encode the store-operated Ca(2+) influx (SOC) channel(s). This study examines the role of Trp1 in the SOC mechanism of salivary gland cells. htrp1, htrp3, and Trp1 were detected in the human submandibular gland cell line (HSG). HSG cells stably transfected with htrp1alpha cDNA displayed (i) a higher level of Trp1, (ii) a 3-5-fold increase in SOC (thapsigargin-stimulated Ca(2+) influx), determined by [Ca(2+)](i) and Ca(2+)-activated K(+) channel current measurements, and (iii) similar basal Ca(2+) permeability, and inhibition of SOC by Gd(3+) but not by Zn(2+), as compared with control cells. Importantly, (i) transfection of HSG cells with antisense trp1alpha cDNA decreased endogenous Trp1 level and significantly attenuated SOC, and (ii) transfection of HSG cells with htrp3 cDNA did not increase SOC. These data demonstrate an association between Trp1 and SOC and strongly suggest that Trp1 is involved in this mechanism in HSG cells. Consistent with this suggestion, Trp1 was detected in the plasma membrane region, the proposed site of SOC, of acinar and ductal cells in intact rat submandibular glands. Based on these aggregate data, we propose Trp1 as a candidate protein for the SOC mechanism in salivary gland cells.

Animals↗
PubMed2002

The role of endogenous human Trp4 in regulating carbachol-induced calcium oscillations in HEK-293 cells.

We utilized 2-aminoethyoxydiphenyl borane, an agent that blocks store-operated Ca(2+) entry, as well as an antisense approach to characterize endogenous Ca(2+) entry pathways in HEK-293 cells. The thapsigargin- and carbachol-induced, but not the 1-oleolyl-2-acytyl-sn-glycerol (OAG)-induced, entry was blocked by 2-aminoethyoxydiphenyl borane. Both reverse transcriptase-PCR and Western blot analyses demonstrated endogenous expression for HTRP1, HTRP3, and HTRP4 and specific suppression of mRNA levels and Trp protein levels in cells stably expressing antisense constructs. Expression of HTRP4 antisense inhibited 35% of the carbachol (CCh)-stimulated Ba(2+) entry and 46% of the OAG-stimulated Sr(2+) entry but in contrast had no effect on the thapsigargin-stimulated Ba(2+) or Sr(2+) entry. HTRP3 antisense reduced, while HTRP1 antisense had no effect on, OAG-induced Sr(2+) entry. Of greater importance, HTRP4 antisense expression, but not HTRP3 antisense expression, blocked the sustained Ca(2+) oscillations produced by low doses of CCh (15 microm), arguing that receptor-stimulated rather than store-operated channels are involved in these sustained oscillations. HTRP4 antisense also inhibited 75% of the arachidonic acid-induced Ca(2+) entry. In summary, these data suggest that HTRP4 proteins in HEK-293 cells, differing from HTRP3 and HTRP1 proteins, do not serve as functional subunits of store-operated channels but do function as subunits for CCh- and OAG-stimulated channels. Furthermore, evidence is provided for the first time for the involvement of a Trp isoform (HTRP4) in the formation of the channel responsible for both arachidonic acid-induced Ca(2+) entry and the Ca(2+) entry needed to sustain long term Ca(2+) oscillations induced by low doses of carbachol.

Arachidonic Acid↗
PubMed2002

Transient receptor potential 1 regulates capacitative Ca(2+) entry and Ca(2+) release from endoplasmic reticulum in B lymphocytes.

Capacitative Ca(2+) entry (CCE) activated by release/depletion of Ca(2+) from internal stores represents a major Ca(2+) influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca(2+) release-activated Ca(2+) currents and inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor-mediated Ca(2+) oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP(3) receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca(2+) signaling in B lymphocytes.

Animals↗
PubMed2005

Contribution of natural products to the discovery of the transient receptor potential (TRP) channels family and their functions.

Members of the transient receptor potential (TRP) family of nonselective cation channels are involved in several pathological and physiological conditions. The search for the molecular targets for naturally occurring substances, especially from plants, allowed the characterization of many TRP channels. In fact, attempts to understand the hot and painful action of the vanillyl group containing compounds capsaicin (from Capsicum sp.) and its ultrapotent analogue resiniferatoxin (RTX, from Euphorbia sp.) led to the cloning of the vanilloid receptor (TRPV1) 7 years ago. TRPV1 is found in sensory fibers and functions as a molecular integrator of several painful stimuli, being especially stimulated during inflammation. Since TRPV1 is involved in several pathological conditions, selective ligands or modulators of this channel are substances of potential interest to treat such diseases. Once again, natural products seem to be also interesting sources of compounds that might be prototype TRPV1 ligands. The cloning of TRPV1 also enabled the discovery of other members of the TRPV family of channels. Similar to TRPV1, these receptors function as molecular detectors of physical and chemical stimuli, such as innocuous and noxious heat, as well as mechanical force. Recently, novel TRP channels sensitive to low temperatures also have been cloned, namely, TRPM8 and TRPA1. Such channels are also activated by naturally occurring substances but knowledge of their involvement in health and disease is in its infancy. In the present review, we focused on the contribution of natural products to the discovery of TRP channels and to the development of novel drugs to treat pathological conditions in which these channels are involved.

Animals↗