TRP Domain Research Articles

Capsaicin, transient receptor potential (TRP) protein subfamilies and the particular relationship between capsaicin receptors and small primary sensory neurons

A number of subfamilies of the capsaicin receptor, collectively called TRP, have been reported since the discovery of vanilloid receptor 1 (VR1). The term 'TRP' is derived from 'transient receptor potential', which means the transient and rapid defect of reaction following long stimulation with light in the photoreceptor cells of mutant Drosophila. The common features of TRP family members are the centrally situated six transmembrane domain, in which an ion channel is located, three to four ankirin repeats at the N-terminus and a TRP domain comprising 25 amino acids at the C-terminus. The TRP family members are present in animals, including invertebrates and vertebrates, and in the cells in various tissues in individual animals. During evolution, the original TRP seems to have acquired a wide variety of functions related to sensing the inner or outer environment (e.g. to sensing light (Drosophila), osmolarity, protons, temperature, ligands and mechanical force). In mammals, the TRPV subfamily is exclusively expressed in small- to medium-sized primary sensory neurons that also co-express some chemical markers (i.e. isolectin B4 (IB4), fluoride-resistant acid phosphatase (FRAP), the P2X3 purinoceptor (a receptor provoked by ATP-induced nociception) and Ret, a glial cell line-derived neurotrophic factor receptor). There is a paradox in that regardless of the marked or complete loss of noxious, small sensory neurons (polymodal nociceptors) in mice treated with capsaicin during the neonatal period, as well as in VR1 (TRPV1)-deficient knock-out mice, the responses to noxious heat are normal. Regarding the paradox in mice treated with capsaicin as neonates, our explanation is that although capsaicin probably reduces the number of a subgroup of small neurons (IB4-, VR1+), the remaining IB4+ (VR1-) neurons can sense noxious heat normally. One working hypothesis is that mice lacking TRPV1/2 can sense noxious heat under normal conditions, presumably via another still unknown pathway, and TRPV1 has been suggested to be involved in noxious heat transduction under pathological conditions, such as inflammation and tissue injury. Further studies will be required to clarify these complexities. Mice treated with capsaicin as neonates would provide a model to investigate the above paradoxes, as would TRPV1-knock-out mice, although different mechanisms may be operating in the two models.

High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol

Menthol is a cooling compound derived from mint leaves and is extensively used as a flavoring chemical. Menthol activates transient receptor potential melastatin 8 (TRPM8), an ion channel also activated by cold, voltage and phosphatidylinositol-4,5-bisphosphate (PIP2). Here we investigated the mechanism by which menthol activates mouse TRPM8. Using a new high-throughput approach, we screened a random mutant library consisting of approximately 14,000 individual TRPM8 mutants for clones that are affected in their response to menthol while retaining channel function. We identified determinants of menthol sensitivity in two regions: putative transmembrane segment 2 (S2) and the C-terminal TRP domain. Analysis of these mutants indicated that activation by menthol involves a gating mechanism distinct and separable from gating by cold, voltage or PIP2. Notably, TRP domain mutations mainly attenuated menthol efficacy, suggesting that this domain influences events downstream of initial binding. In contrast, S2 mutations strongly shifted the concentration dependence of menthol activation, raising the possibility that S2 influences menthol binding.

The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate

Transient receptor potential (TRP) channel, melastatin subfamily (TRPM)4 is a Ca2+-activated monovalent cation channel that depolarizes the plasma membrane and thereby modulates Ca2+ influx through Ca2+-permeable pathways. A typical feature of TRPM4 is its rapid desensitization to intracellular Ca2+ ([Ca2+]i). Here we show that phosphatidylinositol 4,5-biphosphate (PIP2) counteracts desensitization to [Ca2+]i in inside-out patches and rundown of TRPM4 currents in whole-cell patch-clamp experiments. PIP2 shifted the voltage dependence of TRPM4 activation towards negative potentials and increased the channel's Ca2+ sensitivity 100-fold. Conversely, activation of the phospholipase C (PLC)-coupled M1 muscarinic receptor or pharmacological depletion of cellular PIP2 potently inhibited currents through TRPM4. Neutralization of basic residues in a C-terminal pleckstrin homology (PH) domain accelerated TRPM4 current desensitization and strongly attenuated the effect of PIP2, whereas mutations to the C-terminal TRP box and TRP domain had no effect on the PIP2 sensitivity. Our data demonstrate that PIP2 is a strong positive modulator of TRPM4, and implicate the C-terminal PH domain in PIP2 action. PLC-mediated PIP2 breakdown may constitute a physiologically important brake on TRPM4 activity.

International Union of Pharmacology. XLIX. Nomenclature and Structure-Function Relationships of Transient Receptor Potential Channels

include a 25-amino acid (aa) motif in some subfamilies (the TRP domain) containing a TRP box (EWKFAR) just C-terminal to S6. The TRP domain and box, as well as slight variations of these motifs, are present in all TRPC and TRPM channel genes, but not in other TRP channels. The N-terminal cytoplasmic domains of TRPC, TRPV, and TRPA channels contain ankyrin repeats, whereas those of the TRPC and TRPM channels contain proline-rich sequences in the region just C-terminal portion of the TRP domain, referred to as TRP box 2 (Montell, 2005). At present, no features, other than overall 6TM architecture/homology and cationic permeability, define the TRP family. Thus the definition of TRP channels will evolve as functions and structures are clarified. Genes for the TRP ion channel subunits were first defined in the Drosophila visual system. In the trp mutant, the light response (receptor potential) decays dur

Dissecting independent channel and scaffolding roles of the Drosophila transient receptor potential channel

Drosophila transient receptor potential (TRP) serves dual roles as a cation channel and as a molecular anchor for the PDZ protein, INAD (inactivation no afterpotential D). Null mutations in trp cause impairment of visual transduction, mislocalization of INAD, and retinal degeneration. However, the impact of specifically altering TRP channel function is not known because existing loss-of-function alleles greatly reduce protein expression. In the current study we describe the isolation of a set of new trp alleles, including trp 14 with an amino acid substitution juxtaposed to the TRP domain. The trp 14 flies stably express TRP and display normal molecular anchoring, but defective channel function. Elimination of the anchoring function alone in trp Δ 1272, had minor effects on retinal morphology whereas disruption of channel function caused profound light-induced cell death. This retinal degeneration was greatly suppressed by elimination of the Na+/Ca2+ exchanger, CalX, indicating that the cell death was due primarily to deficient Ca2+ entry rather than disruption of the TRP-anchoring function.

PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain

The subjective feeling of cold is mediated by the activation of TRPM8 channels in thermoreceptive neurons by cold or by cooling agents such as menthol. Here, we demonstrate a central role for phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in the activation of recombinant TRPM8 channels by both cold and menthol. Moreover, we show that Ca(2+) influx through these channels activates a Ca(2+)-sensitive phospholipase C and that the subsequent depletion of PI(4,5)P(2) limits channel activity, serving as a unique mechanism for desensitization of TRPM8 channels. Finally, we find that mutation of conserved positive residues in the highly conserved proximal C-terminal TRP domain of TRPM8 and two other family members, TRPM5 and TRPV5, reduces the sensitivity of the channels for PI(4,5)P(2) and increases inhibition by PI(4,5)P(2) depletion. These data suggest that the TRP domain of these channels may serve as a PI(4,5)P(2)-interacting site and that regulation by PI(4,5)P(2) is a common feature of members of the TRP channel family.

Identification of a tetramerization domain in the C terminus of the vanilloid receptor.

TRPV1 (transient receptor potential vanilloid receptor subtype 1) is a member of the TRP channel family gated by vanilloids, protons, and heat. Structurally, TRPV1 appears to be a tetramer formed by the assembly of four identical subunits around a central aqueous pore. The molecular determinants that govern its subunit oligomerization remain elusive. Here, we report the identification of a segment comprising 684Glu-721Arg (referred to as the TRP-like domain) in the C terminus of TRPV1 as an association domain (AD) of the protein. Purified recombinant C terminus of TRPV1 (TRPV1-C) formed discrete and stable multimers in vitro. Yeast two-hybrid and pull-down assays showed that self-association of the TRPV1-C is blocked when segment 684Glu-721Arg is deleted. Biochemical and immunological analysis indicate that removal of the AD from full-length TRPV1 monomers blocks the formation of stable heteromeric assemblies with wild-type TRPV1 subunits. Deletion of the AD in a poreless TRPV1 subunit suppressed its robust dominant-negative phenotype. Together, these findings are consistent with the tenet that the TRP-like domain in TRPV1 is a molecular determinant of the tetramerization of receptor subunits into functional channels. Our observations suggest that the homologous TRP domain in the TRP protein family may function as a general, evolutionary conserved AD involved in subunit multimerization.

TRP channel proteins and signal transduction.

TRP channel proteins constitute a large and diverse family of proteins that are expressed in many tissues and cell types. This family was designated TRP because of a spontaneously occurring Drosophila mutant lacking TRP that responded to a continuous light with a transient receptor potential (hence TRP). In addition to responses to light, TRPs mediate responses to nerve growth factor, pheromones, olfaction, mechanical, chemical, temperature, pH, osmolarity, vasorelaxation of blood vessels, and metabolic stress. Furthermore, mutations in several members of TRP-related channel proteins are responsible for several diseases, such as several tumors and neurodegenerative disorders. TRP-related channel proteins are found in a variety of organisms, tissues, and cell types, including nonexcitable, smooth muscle, and neuronal cells. The large functional diversity of TRPs is also reflected in their diverse permeability to ions, although, in general, they are classified as nonselective cationic channels. The molecular domains that are conserved in all members of the TRP family constitute parts of the transmembrane domains and in most members also the ankyrin-like repeats at the NH2 terminal of the protein and a "TRP domain" at the COOH terminal, which is a highly conserved 25-amino acid stretch with still unknown function. All of the above features suggest that members of the TRP family are "special assignment" channels, which are recruited to diverse signaling pathways. The channels' roles and characteristics such as gating mechanism, regulation, and permeability are determined by evolution according to the specific functional requirements.

Ectopic expression of the tetratricopeptide repeat domain of SPINDLY causes defects in gibberellin response.

The SPINDLY (SPY) protein of Arabidopsis is a negative regulator of gibberellin (GA) response. The SPY protein has 10 copies of the tetratricopeptide repeat (TPR) at the N terminus. TPR motifs function as protein-protein interaction domains. Several spy alleles are affected only in the TPR region suggesting that protein-protein interactions mediated by this domain are important for proper GA signaling. We have used a reverse genetics approach to further investigate the role of the TPR domain. The TPR domain of SPY was overexpressed in wild-type, gai, and spy plants. Expression of the TPR domain alone is not sufficient to rescue spy mutants. Expression of the TPR domain in a wild-type background produces phenotypes similar to those caused by loss-of-function spy mutants including resistance to GA biosynthesis inhibitors, short hypocotyl length, and early flowering. The dwarfing of the floral shoot internodes caused by the gai mutation was suppressed by expression of the TRP domain. Expression of the TPR domain had no effect on the abundance of endogenous SPY mRNA. The TPR domain was found to interact with SPY both in vitro and in yeast two-hybrid assays. These data indicate that the TPR domain of SPY can participate in protein-protein interactions and that these interactions are important for the proper functioning of SPY.