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PubMedDate not supplied

The venerable inveterate invertebrate TRP channels.

The transient receptor potential (TRP) superfamily is subdivided into four main classes of cation channels, TRPC, TRPV, TRPM and TRPN, each of which includes members in worms, flies, mice and humans. While the biophysical features of many of the mammalian channels have been described, relatively little is known concerning the biological roles of these channels. Forward genetic screens in Drosophila melanogaster and Caenorhabditis elegans have led to the identification of the founding members of each of these four subfamilies. Moreover, phenotypic analyses of invertebrate mutants have contributed greatly to our understanding of the roles of TRP proteins. A recurring theme is that many of these proteins function in sensory signaling processes ranging from vision to olfaction, osmosensation, light touch, social feeding, and temperature- and mechanically-induced nociception. In addition, at least one invertebrate TRP protein is required for cell division. As many of these functions may be conserved among the mammalian TRPs, the invertebrate TRPs offer valuable genetic handles for characterizing the functions of these cation channels in vivo.

Animals↗
PubMed2003

ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures.

Mammals detect temperature with specialized neurons in the peripheral nervous system. Four TRPV-class channels have been implicated in sensing heat, and one TRPM-class channel in sensing cold. The combined range of temperatures that activate these channels covers a majority of the relevant physiological spectrum sensed by most mammals, with a significant gap in the noxious cold range. Here, we describe the characterization of ANKTM1, a cold-activated channel with a lower activation temperature compared to the cold and menthol receptor, TRPM8. ANKTM1 is a distant family member of TRP channels with very little amino acid similarity to TRPM8. It is found in a subset of nociceptive sensory neurons where it is coexpressed with TRPV1/VR1 (the capsaicin/heat receptor) but not TRPM8. Consistent with the expression of ANKTM1, we identify noxious cold-sensitive sensory neurons that also respond to capsaicin but not to menthol.

Amino Acid Sequence↗
PubMed2003

N-linked protein glycosylation is a major determinant for basal TRPC3 and TRPC6 channel activity.

The TRPC family of receptor-activated cation channels (TRPC channels) can be subdivided into four subfamilies based on sequence homology as well as functional similarities. Members of the TRPC3/6/7 subfamily share common biophysical characteristics and are activated by diacylglycerol in a membrane-delimited manner. At present, it is only poorly understood whether members of the TRPC3/6/7 subfamily are functionally redundant or whether they serve distinct cellular roles. By electrophysiological and fluorescence imaging strategies we show that TRPC3 displays considerable constitutive activity, while TRPC6 is a tightly regulated channel. To identify potential molecular correlates accounting for the functional difference, we analyzed the glycosylation pattern of TRPC6 compared with TRPC3. Two NX(S/T) motifs in TRPC6 were mutated (Asn to Gln) by in vitro mutagenesis to delete one or both extracellular N-linked glycosylation sites. Immunoblotting analysis of HEK 293 cell lysates expressing TRPC6 wild type and mutants favors a model of TRPC6 that is dually glycosylated within the first (e1) and second extracellular loop (e2) as opposed to the monoglycosylated TRPC3 channel (Vannier, B., Zhu, X., Brown, D., and Birnbaumer, L. (1998) J. Biol. Chem. 273, 8675-8679). Elimination of the e2 glycosylation site, missing in the monoglycosylated TRPC3, was sufficient to convert the tightly receptor-regulated TRPC6 into a constitutively active channel, displaying functional characteristics of TRPC3. Reciprocally, engineering of an additional second glycosylated site in TRPC3 to mimic the glycosylation status in TRPC6 markedly reduced TRPC3 basal activity. We conclude that the glycosylation pattern plays a pivotal role for the tight regulation of TRPC6 through phospholipase C-activating receptors.

Amino Acid Motifs↗
PubMed2002

Subunit composition of mammalian transient receptor potential channels in living cells.

Hormones, neurotransmitters, and growth factors give rise to calcium entry via receptor-activated cation channels that are activated downstream of phospholipase C activity. Members of the transient receptor potential channel (TRPC) family have been characterized as molecular substrates mediating receptor-activated cation influx. TRPC channels are assumed to be composed of multiple TRPC proteins. However, the cellular principles governing the assembly of TRPC proteins into homo- or heteromeric ion channels still remain elusive. By pursuing four independent experimental approaches--i.e., subcellular cotrafficking of TRPC subunits, differential functional suppression by dominant-negative subunits, fluorescence resonance energy transfer between labeled TRPC subunits, and coimmunoprecipitation--we investigate the combinatorial rules of TRPC assembly. Our data show that (i) TRPC2 does not interact with any known TRPC protein and (ii) TRPC1 has the ability to form channel complexes together with TRPC4 and TRPC5. (iii) All other TRPCs exclusively assemble into homo- or heterotetramers within the confines of TRPC subfamilies--e.g., TRPC4/5 or TRPC3/6/7. The principles of TRPC channel formation offer the conceptual framework to assess the physiological role of distinct TRPC proteins in living cells.

Calcium Channels↗
PubMed2003

Substance P evokes cation currents through TRP channels in HEK293 cells.

Activation of any of the three known tachykinin receptors (NK1R, -2R, or -3R) can cause a rise in [Ca2+]i via a pertussis toxin-insensitive heterotrimeric G protein, Gq/G11, activation of phospholipase C (PLC), and a membrane depolarization. Tachykinins can depolarize neurons by two distinct mechanisms: 1) they reduce a resting K+ current in many neurons or 2) in parasympathetic and vagal primary sensory neurons, they activate a nonspecific cation current (Icat). Transient receptor potential channels (TRPC) are nonspecific cation channels that can be activated by a rise in [Ca2+]i in a PLC-dependent manner. The present work tests whether NK2R can signal TRPC. We applied standard whole cell patch-clamp recordings to HEK293 cells stably transfected with the human TRP3 channels (TRP3C), and transiently transfected with a functional NK2R-EGFP. Bath applied Substance P (SP, 1 microM) induced an Icat in the cells expressing both TRP3C and NK2R. Icat reached its peak value in approximately 3 min (195 +/- 120.0 s, mean +/- SE, n = 20), had a peak density of 11.3 +/- 3.48 pA/pF (n = 24), and was blocked by an NK2R-specific antagonist (SR48968, 100 nM). The Erev value for the SP current was 6.8 +/- 7.66 mV (n = 6), suggestive of a nonspecific cation channel. Icat was not measurable in TRP3C-expressing HEK293 cells without NK2R expression (n = 6) or in wild-type HEK293 cells with NK2R expression (n = 12). These data indicate that NK2R can be functionally coupled to TRP channels in HEK293 cells and suggest that SP-induced cation currents in vagal primary sensory neurons might be mediated by TRPC.

Calcium Channels↗
PubMed2001

Expression of ryanodine receptor type 3 and TRP channels in endothelial cells: comparison of in situ and cultured human endothelial cells.

OBJECTIVE: Ca(2+) mobilization plays an important role in endothelial function by stimulating Ca(2+)-dependent synthesis of vasodilating factors. In addition to inositol-1,4,5-trisphosphate (InsP(3)) mediated Ca(2+) mobilization, Ca(2+) release from ryanodine-sensitive pools and Ca(2+)-influx through TRP channels have been suggested to be important in endothelial Ca(2+)-signaling. However, the function and molecular identity of TRP channels and ryanodine receptors in human endothelium in situ are still elusive. We hypothesized that expression of ryanodine-receptors (RyR) and TRP channels differs between human endothelium in situ and in cultured cells. METHODS: By combining single-cell RT-PCR and patch-clamp techniques, expression of RyR and TRP channels was determined in situ in endothelial cells of human mesenteric artery (HMAECs) obtained from patients undergoing bowel resection and in the endothelial cell line EA.hy926. RESULTS: At the single cell level, expression of RyR 3 was detected in 25 and 5% of HMAECs and EA.hy926 samples, respectively. Expression of the RyR 1 and 2 was not detected in either HMAECs or EA.hy926. In patch-clamp experiments in HMAECs, applications of caffeine (0.5 mM) induced sustained hyperpolarization mediated by activation of Ca(2+)-activated K channels. In EA.hy926, caffeine-induced hyperpolarization was not detected. Single HMAECs expressed the TRP genes, TRP1 and TRP3, but not TRP 4 and 6. The TRP1 was the predominantly expressed TRP gene in HMAECs in situ whereas TRP3 expression was rarely detected. EA.hy926 expressed only TRP1. In patch clamp experiments in HMAECs, Ca(2+)-store depletion activated non-selective cation currents leading to Ca(2+) entry. CONCLUSIONS: Our findings suggest that, in addition to InsP(3) mediated Ca(2+) release, Ca(2+) release from ryanodine-sensitive stores mediated by RyR3 and Ca(2+) entry through TRP1 might represent important components of endothelial Ca(2+) signaling in situ and thereby of endothelial function in intact human blood vessels.

Calcium↗
PubMed2000

Gating of store-operated channels by conformational coupling to ryanodine receptors.

We report here that RyRs interact with and gate the store-operated hTrp3 and Icrac channels. This gating contributes to activation of hTrp3 and Icrac by agonists. Coupling of hTrp3 to IP3Rs or RyRs in the same cells was found to be mutually exclusive. Biochemical and functional evidence suggest that mutually exclusive coupling reflects clustering and segregation of hTrp3-IP3R and hTrp3-RyR complexes in plasma membrane microdomains. Gating of CCE by RyRs indicates that gating by conformational coupling is not unique to skeletal muscle but is a general mechanism for communication between events in the plasma and endoplasmic reticulum membranes.

Barium↗
PubMed2000

Store depletion by caffeine/ryanodine activates capacitative Ca(2+) entry in nonexcitable A549 cells.

Capacitative Ca(2+) entry is essential for refilling intracellular Ca(2+) stores and is thought to be regulated primarily by inositol 1, 4,5-trisphosphate (IP(3))-sensitive stores in nonexcitable cells. In nonexcitable A549 cells, the application of caffeine or ryanodine induces Ca(2+) release in the absence of extracellular Ca(2+) similar to that induced by thapsigargin (Tg), and Ca(2+) entry occurs upon the readdition of extracellular Ca(2+). The channels thus activated are also permeable to Mn(2+). The channels responsible for this effect appear to be activated by the depletion of caffeine/ryanodine-sensitive stores per se, as evidenced by the activation even in the absence of increased intracellular Ca(2+) concentration. Tg pretreatment abrogates the response to caffeine/ryanodine, whereas Tg application subsequent to caffeine/ryanodine treatment induces further Ca(2+) release. The response to caffeine/ryanodine is also abolished by initial ATP application, whereas ATP added subsequent to caffeine/ryanodine induces additional Ca(2+) release. RT-PCR analyses showed the expression of a type 1 ryanodine receptor, two human homologues of transient receptor potential protein (hTrp1 and hTrp6), as well as all three types of the IP(3) receptor. These results suggest that in A549 cells, (i) capacitative Ca(2+) entry can also be regulated by caffeine/ryanodine-sensitive stores, and (ii) the RyR-gated stores interact functionally with those sensitive to IP(3), probably via Ca(2+)-induced Ca(2+) release.

Caffeine↗
PubMed2003

Ion channel gene expression in human lung, skin, and cord blood-derived mast cells.

Immunoglobulin E (IgE)-dependent activation of human mast cells (HMC) is characterized by an influx of extracellular calcium (Ca(2+)), which is essential for subsequent release of preformed (granule-derived) mediators and newly generated autacoids and cytokines. In addition, flow of ions such as K(+) and Cl(-) is likely to play an important role in mast cell activation, proliferation, and chemotaxis through their effect on membrane potential and thus Ca(2+) influx. It is therefore important to identify these critical molecular effectors of HMC function. In this study, we have used high-density oligonucleotide probe arrays to characterize for the first time the profile of ion channel gene expression in human lung, skin, and cord blood-derived mast cells. These cells express mRNA for inwardly rectifying and Ca(2+)-activated K(+) channels, voltage-dependent Na(+) and Ca(2+) channels, purinergic P2X channels, transient receptor potential channels, and voltage-dependent and intracellular Cl(-) channels. IgE-dependent activation had little effect on ion channel expression, but distinct differences for some channels were observed between the different mast cell phenotypes, which may contribute to the mechanism of functional mast cell heterogeneity.

Adult↗
PubMedDate not supplied

TRPC4 and TRPC5: receptor-operated Ca2+-permeable nonselective cation channels.

The seven mammalian channels from the classical (TRPC) subfamily of transient receptor potential (TRP) channels are thought to be receptor-operated cation channels activated in a phospholipase C (PLC)-dependent manner. Based on sequence similarity, TRPC channels can be divided into four subgroups. Group 4 comprises TRPC4 and TRPC5, and is most closely related to group 1 (TRPC1). The functional properties observed following heterologous expression of TRPC4 or TRPC5 in mammalian cells are contradictory and, therefore, controversial. In our hands, and in several independent studies, both channels, probably as homotetramers, form receptor-operated, Ca2+-permeable, nonselective cation channels activated independently of inositol 1,4,5-trisphosphate (InsP(3)) receptor activation or Ca2+ store-depletion. As heteromultimers with TRPC1, TRPC4 and TRPC5 form receptor-operated, Ca2+-permeable, nonselective cation channels with biophysical properties distinct from homomeric TRPC4 or TRPC5. In other studies, TRPC4 and TRPC5 have been shown to be store-operated channels, with moderate to high Ca2+ permeabilities. At present there is no clear explanation for these major differences in functional properties. To date, little is known as to which native cation channels are formed by TRPC4 and TRPC5. Endothelial cells from TRPC4(-/-) mice lack a highly Ca2+-permeable, store-dependent current, and data support a role for TRPC4 in endothelium-mediated vasorelaxation. A similar current in adrenal cortical cells is reduced by TRPC4 antisense. From similarities in the properties of the currents and expression of appropriate isoforms in the tissues, it is likely that heteromultimers of TRPC1 and TRPC4 or TRPC5 form receptor-operated nonselective cation channels in central neurones, and that TRPC4 contributes to nonselective cation channels in intestinal smooth muscle.

Animals↗
PubMed2003

Transient receptor potential channels in endothelium: solving the calcium entry puzzle?

Many endothelial cell (EC) functions depend on influx of extracellular Ca2+, which is triggered by a variety of mechanical and chemical signals. Here, we discuss possible pathways for this Ca2+ entry. The superfamily of cation channels derived from the "transient receptor potential" (TRP) channels is introduced. Several members of this family are expressed in ECs, and they provide pathways for Ca2+ entry. All TRP subfamilies may contribute to the Ca2+ entry channels or to the regulation of Ca2+ entry in EC. Members of Ca2+ entry channels in endothelium probably belong to the canonical TRP subfamily, TRPC. All TRPC1-6 have been discussed as Ca2+ entry channels that might be store-operated and/or receptor-operated. More importantly, knockout models of TRPC4 have proven that this channel is functionally involved in the regulation of endothelial-dependent vasorelaxation and in the control of EC barrier function. TRPC1 might be an important candidate for involvement of endothelial growth factors. TRPC3 is unequivocally important for a sustained EC Ca2+ entry. ECs express different patterns of TRPCs, which may increase the variability of TRPC channel function by formation of different multiheteromers. Among the two other TRP subfamilies, TRPMV and TRPM, at least TRPV4 and TRPM4 are EC channels. TRPV4 is a Ca2+ entry channel that is activated by an increase in cell volume, which might be involved in mechano-sensing, by an increase in temperature, and perhaps by ligand-activation. TRPM4 is a nonselective cation channel, which is not Ca2+ permeable. It is probably modulated by NO and might be essential for regulating the inward driving force for Ca2+ entry. Possible modes of TRP channel regulation are described, involving (a) activation via the phospholipase (PL)Cbeta and PLC-gamma pathways; (b) activation by lipids (diacylglycerol [DAG], arachidonic acid); (c) Ca2+ depletion of Ca2+ stores in the endoplasmic reticulum; (d) shear stress; and (e) radicals.

Animals↗
PubMed1999

Activation of a TRPC3-dependent cation current through the neurotrophin BDNF.

Nonvoltage-gated cation currents, which are activated following stimulation of phospholipase C (PLC), appear to be major modes for Ca2+ and Na+ entry in mammalian cells. The TRPC channels may mediate some of these conductances since their expression in vitro leads to PLC-dependent cation influx. We found that the TRPC3 protein was highly enriched in neurons of the central nervous system (CNS). The temporal and spatial distribution of TRPC3 paralleled that of the neurotrophin receptor TrkB. Activation of TrkB by brain-derived nerve growth factor (BDNF) led to production of a PLC-dependent, nonselective cation conductance in pontine neurons. Evidence is provided that TRPC3 contributes to this current in vivo. Thus, activation of TrkB and PLC leads to a TRPC3-dependent cation influx in CNS neurons.

Animals↗
PubMed2001

Increased inwardly rectifying potassium currents in HEK-293 cells expressing murine transient receptor potential 4.

Drosophila transient receptor potential (Trp) and its mammalian homologues are postulated to form capacitative Ca2+ entry or store-operated channels. Here we show that expression of murine Trp4 in HEK 293 cells also leads to an increase in inwardly rectifying K+ currents. No similar increase was found in cell lines expressing Trp1, Trp3 or Trp6. Consistent with typical characteristics of inward rectifiers, the K+ currents in Trp4-expressing cells were blocked by low millimolar concentrations of Cs+ and Ba2+, but not by 1.2 mM Ca2+, and were only slightly inhibited by 5 mM tetraethylammonium. Single channel recordings of excised inside-out patches revealed the presence of two conducting states of 51 pS and 94 pS in Trp4-expressing cells. The outward current in the excised patches was blocked by 1 mM spermine, but not by 1 mM Mg2+. How Trp4 expression causes the increase in the K+ currents is not known. We propose that Trp4 either participates in the formation of a novel K+ channel or up-regulates the expression or activity of endogenous inwardly rectifying K+ channels.

Animals↗
PubMed2002

TRP genes: candidates for nonselective cation channels and store-operated channels in insulin-secreting cells.

Nonselective cation channels may play a role in insulin secretion by regulating pancreatic beta-cell plasma membrane potential, Ca(2+) homeostasis, and thereby glucose signaling. Transient receptor potential channel (TRPC)-related genes encode nonselective cation channels, some of which are similar to those described for beta-cells. Some TRPC-like channels are activated via G-protein--coupled mechanisms, some have been reported to be calcium-store-operated channels (SOC), and others are activated by novel signaling molecules or are sensitive to pressure and osmotic strength. Here we report the cloning and expression of mSTRPC4 from a mouse insulinoma cDNA library. mSTRPC4 encoded a protein of 97 kd, expressed in both endocrine cells and the brain. Stable cell lines expressing mSTRPC4 showed abundant mSTRPC4 protein, but no reproducible currents could be detected. mSTRPC4 therefore probably functions as a heteromultimer. We also report that LTRPC2, a G-protein and adenosine 5'-diphosphoribose (ADPR)-activated nonselective cation channel, is also expressed in human islets. TRPC-like channels may provide a pathway for depolarization or Ca(2+) entry in beta-cells and may be interesting targets for manipulating beta-cell function.

Animals↗
PubMed2003

Formation of novel TRPC channels by complex subunit interactions in embryonic brain.

Mammalian short TRP channels (TRPCs) are putative receptor- and store-operated cation channels that play a fundamental role in the regulation of cellular Ca2+ homeostasis. Assembly of the seven TRPC homologs (TRPC1-7) into homo- and heteromers can create a large variety of different channels. However, the compositions as well as the functional properties of native TRPC complexes are largely undefined. We performed a systematic biochemical study of TRPC interactions in mammalian brain and identified previously unrecognized channel heteromers composed of TRPC1, TRPC4, or TRPC5 and the diacylglycerol-activated TRPC3 or TRPC6 subunits. The novel TRPC heteromers were found exclusively in embryonic brain. In heterologous systems, we demonstrated that assembly of these novel heteromers required the combination of TRPC1 plus TRPC4 or TRPC5 subunits along with diacylglycerol-sensitive subunits in the channel complexes. Functional interaction of the TRPC subunits was verified using a dominant negative TRPC5 mutant (TRPC5DN). Co-expression of TRPC5DN suppressed currents through TRPC5- and TRPC4-containing complexes; TRPC3-associated currents were unaffected by TRPC5DN unless TRPC1 was also co-expressed. This complex assembly mechanism increases the diversity of TRPC channels in mammalian brain and may generate novel heteromers that have specific roles in the developing brain.

Animals↗