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PubMed2004

Regulation of TRPV2 by axotomy in sympathetic, but not sensory neurons.

Neuropathic pain results from traumatic or disease-related insults to the nervous system. Mechanisms that have been postulated to underlie peripheral neuropathy commonly implicate afferent neurons that have been damaged but still project centrally to the spinal cord, and/or intact neurons that interact with degenerating distal portions of the injured neurons. One pain state that is observed following peripheral nerve injury in the rat is thermal hyperalgesia. The noxious heat-gated ion channel TRPV1 may be responsible for this increased sensitivity, as it is up-regulated in L4 dorsal root ganglion (DRG) neurons following L5 spinal nerve lesion (SpNL). The TRPV1 homologue TRPV2 (or VRL-1) is another member of the TRPV subfamily of TRP ion channels. TRPV2 is a nonselective cation channel activated by high noxious temperatures (>52 degrees C) and is present in a subset of medium- to large-diameter DRG neurons. To establish whether TRPV2 is endogenous to the spinal cord, we examined its expression in the dorsal horn following rhizotomy. We found no significant decrease in TRPV2 immunoreactivity, suggesting that TRPV2 is endogenous to the spinal cord. In order to determine whether TRPV2, like TRPV1, is regulated by peripheral axotomy, we performed L5 SpNL and characterized TRPV2 distribution in the DRG, spinal cord, brainstem, and sympathetic ganglia. Our results show that peripheral axotomy did not regulate TRPV2 in the DRG, spinal cord, or brainstem; however, TRPV2 was up-regulated in sympathetic postganglionic neurons following injury, suggesting a potential role for TRPV2 in sympathetically mediated neuropathic pain.

Animals↗
PubMed2005

MxA, a member of the dynamin superfamily, interacts with the ankyrin-like repeat domain of TRPC.

Mammalian transient receptor potential canonical channels have been proposed as the molecular entities associated with calcium entry activity in nonexcitable cells. Amino acid sequence analyses of TRPCs revealed the presence of ankyrin-like repeat domains, one of the most common protein-protein interaction motifs. Using a yeast two-hybrid interaction assay, we found that the second ankyrin-like repeat domain of TRPC6 interacted with MxA, a member of the dynamin superfamily. Using a GST pull-down and co-immunoprecipitation assay, we showed that MxA interacted with TRPC1, -3, -4, -5, -6, and -7. Overexpression of MxA in HEK293T cells slightly increased endogenous calcium entry subsequent to stimulation of G(q) protein-coupled receptors or store depletion by thapsigargin. Co-expression of MxA with TRPC6 enhanced agonist-induced or OAG-induced calcium entry activity. GTP binding-defective MxA mutants had only a minor potentiating effect on OAG-induced TRPC6 activity. However, a MxA mutant that could bind GTP but that lacked GTPase activity produced the same effect as MxA on OAG-induced TRPC6 activity. These results indicated that MxA interacted specifically with the second ankyrin-like repeat domain of TRPCs and suggested that monomeric MxA regulated the activity of TRPC6 by a mechanism requiring GTP binding. Additional results showed that an increase in the endogenous expression of MxA, induced by a treatment with interferon alpha, regulated the activity of TRPC6. The study clearly identified MxA as a new regulatory protein involved in Ca2+ signaling.

Amino Acid Motifs↗
PubMed2006

Regulation of TRP channels by N-linked glycosylation.

A subset of TRP channel proteins undergoes regulatory N-linked glycosylation. A glycosylation site in the first extracellular loop of TRPV5 is enzymatically cleaved by a secreted glucuronidase, indirectly regulating channel function. Members of the TRPC family share a similar site, although details about a regulatory role are lacking. A second conserved TRP channel glycosylation site is found immediately adjacent to the channel pore-forming loop; both TRPV1 and TRPV4--and perhaps other TRPV family members--are influenced by glycosylation at this site. N-linked glycosylation, and the dynamic regulation of this process, substantially impacts function and targeting of TRP channels.

Amino Acid Sequence↗
PubMedDate not supplied

Store-operated Ca2+ channels in prostate cancer epithelial cells: function, regulation, and role in carcinogenesis.

Ca2+ homeostasis mechanisms, in which the Ca2+ entry pathways play a key role, are critically involved in both normal function and cancerous transformation of prostate epithelial cells. Here, using the lymph node carcinoma of the prostate (LNCaP) cell line as a major experimental model, we characterize prostate-specific store-operated Ca2+ channels (SOCs)--a primary Ca2+ entry pathway for non-excitable cells--for the first time. We show that prostate-specific SOCs share major store-dependent, kinetic, permeation, inwardly rectifying, and pharmacological (including dual, potentiation/inhibition concentration-dependent sensitivity to 2-APB) properties with "classical" Ca2+ release-activated Ca2+ channels (CRAC), but have a higher single channel conductance (3.2 and 12pS in Ca2+- and Na+-permeable modes, respectively). They are subject to feedback inhibition via Ca2+-dependent PKC, CaMK-II and CaM regulatory pathways and are functionally dependent on caveolae integrity. Caveolae also provide a scaffold for spatial co-localization of SOCs with volume-regulated anion channels (VRAC) and their Ca2+-mediated interaction. The TRPC1 and TRPV6 members of the transient receptor potential (TRP) channel family are the most likely molecular candidates for the formation of prostate-specific endogenous SOCs. Differentiation of LNCaP cells to an androgen-insensitive, apoptotic-resistant neuroendocrine phenotype downregulates SOC current. We conclude that prostate-specific SOCs are important determinants in the transition to androgen-independent prostate cancer.

Biomarkers↗
PubMed1998

TRP, TRPL and trouble in photoreceptor cells.

Calcium influx through the TRP and TRPL light-activated channels triggers a complex regulatory hierarchy resulting in positive and negative feedback regulation of the phototransduction cascade. Recent studies have begun to elucidate the function of TRP and TRPL in vivo, and to examine their relationship to intracellular calcium changes during the light response.

Animals↗
PubMed1999

TRP2: a candidate transduction channel for mammalian pheromone sensory signaling.

The vomeronasal organ (VNO) of terrestrial vertebrates plays a key role in the detection of pheromones, chemicals released by animals that elicit stereotyped sexual and aggressive behaviors among conspecifics. Sensory transduction in the VNO appears unrelated to that in the vertebrate olfactory and visual systems: the putative pheromone receptors of the VNO are evolutionarily independent from the odorant receptors and, in contrast to vertebrate visual and olfactory transduction, vomeronasal transduction is unlikely to be mediated by cyclic-nucleotide-gated channels. We hypothesized that sensory transduction in the VNO might instead involve an ion channel of the transient receptor potential (TRP) family, members of which mediate cyclic-nucleotide-independent sensory responses in Drosophila melanogaster and Caenorhabditis elegans and play unknown functions in mammals. We have isolated a cDNA (rTRP2) from rat VNO encoding a protein of 885 amino acids that is equally distant from vertebrate and invertebrate TRP channels (10-30% amino acid identity). rTRP2 mRNA is exclusively expressed in VNO neurons, and the protein is highly localized to VNO sensory microvilli, the proposed site of pheromone sensory transduction. The absence of Ca2+ stores in sensory microvilli suggests that, in contrast to a proposed mechanism of activation of mammalian TRP channels, but in accord with analysis of TRP function in Drosophila phototransduction, the gating of TRP2 is independent from the depletion of internal Ca2+ stores. Thus, TRP2 is likely to participate in vomeronasal sensory transduction, which may share additional similarities with light-induced signaling in the Drosophila eye.

Amino Acid Sequence↗
PubMed1998

The membrane topology of human transient receptor potential 3 as inferred from glycosylation-scanning mutagenesis and epitope immunocytochemistry.

Transient receptor potential (Trp) proteins form ion channels implicated in the calcium entry observed after stimulation of the phospholipase C pathway. Kyte-Doolittle analysis of the amino acid sequence of Trp proteins identifies seven hydrophobic regions (H1-H7) with potential of forming transmembrane segments. A limited sequence similarity to voltage-gated calcium channel alpha1 subunits lead to the prediction of six transmembrane (TM) segments flanked by intracellular N and C termini and a putative pore region between TM5 and TM6. However, experimental evidence supporting this model is missing. Using human Trp 3 to test Trp topology, we now confirm the intracellular nature of the termini by immunocytochemistry. We also demonstrate presence of a unique glycosylation site in position 418, which defines one extracellular loop between H2 and H3. After removal of this site and insertion of ten separate glycosylation sites, we defined two additional extracellular loops between H4 and H5, and H6 and H7. This demonstrated the existence of six transmembrane segments formed of H2-H7. Thus, the first hydrophobic region of Trp rather than being a transmembrane segment is intracellular and available for protein-protein interactions. A site placed in the center of the putative pore region was glycosylated, suggesting that this region may have been luminal and was reinserted into the membrane at a late stage of channel assembly.

Animals↗
PubMed2000

Ca2+ entry mediated by store depletion, S-nitrosylation, and TRP3 channels. Comparison of coupling and function.

The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. SOC-mediated Ca(2+) entry is complex and may reflect more than one type of channel and coupling mechanism. To assess such possible divergence the function and coupling of SOCs was compared with two other distinct yet related Ca(2+) entry mechanisms. SOC coupling in DDT(1)MF-2 smooth muscle cells was prevented by the permeant inositol 1,4,5-trisphosphate (InsP(3)) receptor blockers, 2-aminoethoxydiphenyl borate (2-APB) and xestospongin C. In contrast, Ca(2+) entry induced by S-nitrosylation and potentiated by store depletion (Ma, H-T., Favre, C. J., Patterson, R. L., Stone, M. R., and Gill, D. L. (1999) J. Biol. Chem. 274, 35318-35324) was unaffected by 2-APB, suggesting that this entry mechanism is independent of InsP(3) receptors. The cycloalkyl lactamimide, MDL-12, 330A (MDL), prevented SOC activation (IC(50) 10 micrometer) and similarly completely blocked S-nitrosylation-mediated Ca(2+) entry. Ca(2+) entry mediated by the TRP3 channel stably expressed in HEK293 cells was activated by phospholipase C-coupled receptors but independent of Ca(2+) store depletion (Ma, H.-T., Patterson, R. L., van Rossum, D. B., Birnbaumer, L., Mikoshiba, K., and Gill, D. L. (2000) Science 287, 1647-1651). Receptor-induced TRP3 activation was 2-APB-sensitive and fully blocked by MDL. Direct stimulation of TRP3 channels by the permeant diacylglycerol derivative, 1-oleoyl-2-acetyl-sn-glycerol, was not blocked by 2-APB, but was again prevented by MDL. The results indicate that although the activation and coupling processes for each of the three entry mechanisms are distinct, sensitivity to MDL is a feature shared by all three mechanisms, suggesting there may be a common structural feature in the channels themselves or an associated regulatory component.

Animals↗
PubMed2003

Expression level of the canonical transient receptor potential 3 (TRPC3) channel determines its mechanism of activation.

Studies on the mechanism of activation of canonical transient receptor potential (TRPC) channels have often yielded conflicting results. In the current study, we have investigated the influence of expression level on the mode of regulation of TRPC3 channels. At relatively low levels of expression in DT40 chicken B-lymphocytes, TRPC3 was activated by the depletion of Ca2+ stores. Expression was increased by either transfecting with a 10-fold greater concentration of plasmid or transfecting with TRPC3 under control of a more efficient avian beta-actin promoter. At higher levels of expression, TRPC3 was no longer store-operated but could be activated through receptor-coupled phospholipase C. Under these expression conditions, TRPC3 was efficiently activated in DT40 cells lacking inositol 1,4,5-trisphosphate receptors. The Ca2+ store-operated channels formed upon expression of TRPC3 at limited levels were blocked by gadolinium; the receptor-activated channels formed upon expression of higher levels of TRPC3 were insensitive to gadolinium. These findings indicate that a single ion channel protein can form or contribute to the formation of channels regulated in two very distinct ways, i.e. either by phospholipase C-derived messengers or Ca2+ store-depletion. The mechanism of regulation of the channels depends on their level of expression.

Animals↗
PubMed2005

Compartmentalization of calcium entry pathways in mouse rods.

Photoreceptor metabolism, gene expression and synaptic transmission take place in a highly polarized structure consisting of the ellipsoid, subellipsoid, cell body and synaptic terminal regions. Although calcium, a key second messenger, regulates cellular functions throughout the photoreceptor, the molecular mechanisms underlying local region-specific action of Ca2+ in photoreceptors are poorly understood. I have investigated the compartmentalization of voltage-dependent and independent Ca2+ channels in mouse photoreceptors. Transient receptor potential channels isoform 6 (TRPC6), a putative store-operated Ca2+ channel, was selectively localized to the cell body of rods. By contrast, voltage-operated Ca2+ channels were expressed in the synaptic terminal and in the ellipsoid/subellipsoid regions. Likewise, Ca2+ store transporters and channels were strongly associated with the subellipsoid region. A moderate TRPC6 signal was observed in cell bodies of bipolar, amacrine and ganglion cells, but was absent from both plexiform layers. These results suggest that Ca2+ entry mechanisms in mammalian photoreceptors and bipolar cells are highly compartmentalized, consistent with local, region-specific activation of Ca2+-dependent processes.

Animals↗
PubMed2006

Calcium-sensing mechanism in TRPC5 channels contributing to retardation of neurite outgrowth.

The calcium- and sodium-permeable transient receptor potential channel TRPC5 has an inhibitory role in neuronal outgrowth but the mechanisms governing its activity are poorly understood. Here we propose a mechanism involving the neuronal calcium sensor-1 (NCS-1) protein. Inhibitory mutants of TRPC5 and NCS-1 enhance neurite outgrowth similarly. Mutant NCS-1 does not inhibit surface-expression of TRPC5 but generally suppresses channel activity, irrespective of whether it is evoked by carbachol, store depletion, lanthanides or elevated intracellular calcium. NCS-1 and TRPC5 are in the same protein complex in rat brain and NCS-1 directly binds to the TRPC5 C-terminus. The data suggest protein-protein interaction between NCS-1 and TRPC5, and involvement of this protein complex in retardation of neurite outgrowth.

Animals↗
PubMed2004

TRPC4 and TRPC4-deficient mice.

TRP proteins, in most cases, provide localized Ca2+ increases for spatially defined signal transduction processes. They are activated by as yet unclear mechanisms, many involving the complex phospholipase C and phosphatidylinositol pathways. In mouse endothelial cells at least seven TRPs are expressed, including TRPC1, TRPC2, TRPC3, TRPC4, TRPC6, TRPV4 and TRPM4. As shown previously, TRPC4 is an indispensable component of agonist-induced Ca2+ entry channels in native endothelial cells which essentially contributes to agonist-induced vessel relaxation and microvascular endothelial permeability, although, it is still open, whether TRPC4 acts as channel-forming subunit and/or essential constituent for channel activation. Utilizing the mouse model is one way to address this question and to provide novel insights for the biological functions of TRPC4. Here we review recent results on heterologously expressed TRPC4 and summarize what is known on the phenotype of the TRPC4-/- mice generated in our laboratory.

Amino Acid Sequence↗
PubMed2002

Molecular candidates for capacitative and non-capacitative Ca2+ entry in smooth muscle.

Recent investigations have revealed that mammalian homologues of transient receptor potential (TRP) protein (TRP1-7) are promising candidates for Ca2+ entry mechanisms (or channels) associated with various metabotropic G protein-coupled receptors (GPCRs) in smooth muscle, stimulation of which generates lipid second messengers and depletes internal stores. RT-PCR and immunocytochemical experiments have demonstrated that although the level of expression varies depending on tissues, the major TRP isoforms expressed in smooth muscle are TRP4, 6 and 7. In some vascular preparations, the significant expression of TRP1 mRNA and protein is also detected. Consistent with these findings, recent functional studies using TRP6- and TRP1-specific antisense oligonucleotides and antibodies have suggested that TRP6 is the essential component of alpha1-adrenoceptor activated, store depletion-independent Ca2+ entry channels, while TRP1 is partly involved in Ca2+ entry associated with store depletion or capacitative Ca2+ entry. In addition, coexpression of different TRP isoforms results in the appearance of cation channels showing novel properties reminiscent of some native GPCR-activated Ca2+-permeable non-selective cation channels. Thus, at present, TRP proteins may be the most important clues for elucidating the molecular entities of receptor- and store-operated Ca2+ entry mechanisms in smooth muscle and their roles in smooth muscle functions.

Animals↗
PubMed2000

Coassembly of Trp1 and Trp3 proteins generates diacylglycerol- and Ca2+-sensitive cation channels.

To analyze the functional consequences of coassembly of transient receptor potential 1 (Trp1) and Trp3 channel proteins, we characterized membrane conductances and divalent cation entry derived by separate overexpression and by coexpression of both Trp isoforms. Trp1 expression generated a 1-oleoyl-2-acetyl-sn-glycerol (OAG)-activated conductance that was detectable only in Ca(2+)-free extracellular solution. Trp3 expression gave rise to an OAG-activated conductance that was suppressed but clearly detectable at physiological Ca(2+) concentrations. Coexpression of both species resulted in a constitutively active, OAG-sensitive conductance, which exhibited distinctive cation selectivity and high sensitivity to inhibition by intracellular Ca(2+). Trp1-expressing cells displayed only modest carbachol-induced Ca(2+) entry and lacked OAG-induced Sr(2+) entry, whereas Trp3-expressing cells responded to both agents with a substantial divalent cation entry. Coexpression of Trp1 plus Trp3 suppressed carbachol-induced Ca(2+) entry compared with Trp3 expression and abolished OAG-induced Sr(2+) entry signals. We concluded that coassembly of Trp1 and Trp3 resulted in the formation of oligomeric Trp channels that are subject to regulation by phospholipase C and Ca(2+). The distinguished Ca(2+) sensitivity of these Trp1/Trp3 hetero-oligomers appeared to limit Trp-mediated Ca(2+) signals and may be of importance for negative feedback control of Trp function in mammalian cells.

Animals↗
PubMed2002

The PDZ scaffold protein INAD abolishes apparent store-dependent regulation of the light-activated cation channel TRP.

In fly photoreceptor cells, light initiates a G protein-coupled phospholipase Cb-dependent signaling cascade that results in the depolarization of the cell membrane, which is mediated by the cation channels TRP and TRPL. Together with phospholipase Cb and an eye-specific protein kinase C, TRP is tethered to the scaffolding protein INAD, which forms a multimolecular signaling complex. Divergent data from expressed TRP and studies from photoreceptor cells have brought up a controversy whether or not a capacitative calcium entry (CCE) mechanism is involved in the Drosophila phototransduction pathway. Our initial characterization of TRP from photoreceptors of Calliphora vicina supported the hypothesis of a CCE mechanism, as heterologously expressed TRP was stimulated after application of thapsigargin. The situation changed when the PDZ domain protein INAD was coexpressed with TRP. In cells coexpressing TRP and INAD, no calcium entry was detectable on application of store depletion protocols. Suppression of CCE by INAD was not observed when the described interaction was disrupted by mutations in TRP and INAD. Our data show that apparent activation of TRP by CCE is abolished by INAD. Within the complex, the proteins necessary for phototransduction mutually influence their activities. The results support the hypothesis of a store-independent activation of TRP.

Animals↗