TRP Box Research Articles

Genome-Wide Identification and Phylogenetic Analysis of TRP Gene Family Members in Saurian.

The transient receptor potential plays a critical role in the sensory nervous systems of vertebrates in response to various mechanisms and stimuli, such as environmental temperature. We studied the physiological adaptive evolution of the TRP gene in the saurian family and performed a comprehensive analysis to identify the evolution of the thermo-TRPs channels. All 251 putative TRPs were divided into 6 subfamilies, except TRPN, from the 8 saurian genomes. Multiple characteristics of these genes were analyzed. The results showed that the most conserved proteins of TRP box 1 were located in motif 1, and those of TRP box 2 were located in motif 10. The TRPA and TRPV in saurian tend to be one cluster, as a sister cluster with TRPC, and the TRPM is the root of group I. The TRPM, TRPV, and TRPP were clustered into two clades, and TRPP were organized into TRP PKD1-like and PKD2-like. Segmental duplications mainly occurred in the TRPM subfamily, and tandem duplications only occurred in the TRPV subfamily. There were 15 sites to be under positive selection for TRPA1 and TRPV2 genes. In summary, gene structure, chromosomal location, gene duplication, synteny analysis, and selective pressure at the molecular level provided some new evidence for genetic adaptation to the environment. This result provides a basis for identifying and classifying TRP genes and contributes to further elucidating their potential function in thermal sensors.

Critical contributions of pre-S1 shoulder and distal TRP box in DAG-activated TRPC6 channel by PIP2 regulation

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) regulates the activities of numerous membrane proteins, including diacylglycerol(DAG)-activated TRPC3/6/7 channels. Although PIP2 binding is known to support DAG-activated TRP channel activity, its binding site remains unknown. We screened for PIP2 binding sites within TRPC6 channels through extensive mutagenesis. Using voltage-sensitive phosphatase (DrVSP), we found that Arg437 and Lys442, located in the channel’s pre-S1 domain/shoulder, are crucial for interaction with PIP2. To gain structural insights, we conducted computer protein–ligand docking simulations with the pre-S1 domain/shoulder of TRPC6 channels. Further, the functional significance of PIP2 binding to the pre-S1 shoulder was assessed for receptor-operated channel functions, cross-reactivity to DAG activation, and the kinetic model simulation. These results revealed that basic residues in the pre-S1 domain/shoulder play a central role in the regulation of PIP2-dependent gating. In addition, neutralizing mutation of K771 in the distal TRP box reversed the effect of PIP2 depletion from inhibiting to potentiating channel activity. A similar effect was seen in TRPV1 channels, which suggests that TRPC6 possesses a common but robust polarity switch mediating the PIP2-dependent effect. Overall, these mutagenesis studies reveal functional and structural insights for how basic residues and channel segments in TRP channels are controlled through phosphoinositides recognition.

12(S)-HETE mediates diabetes-induced endothelial dysfunction by activating intracellular endothelial cell TRPV1

Patients with diabetes develop endothelial dysfunction shortly after diabetes onset that progresses to vascular disease underlying the majority of diabetes-associated comorbidities. Increased lipid peroxidation, mitochondrial calcium overload, and mitochondrial dysfunction are characteristics of dysfunctional endothelial cells in diabetic patients. We here identified that targeting the lipid peroxidation product 12(S)-hydroxyeicosatetraenoic acid-induced [12(S)-HETE-induced] activation of the intracellularly located cation channel transient receptor potential vanilloid 1 (TRPV1) in endothelial cells is a means to causally control early-stage vascular disease in type I diabetic mice. Mice with an inducible, endothelium-specific 12/15-lipoxygenase (12/15Lo) knockout were protected similarly to TRPV1-knockout mice from type 1 diabetes-induced endothelial dysfunction and impaired vascular regeneration following arterial injury. Both 12(S)-HETE in concentrations found in diabetic patients and TRPV1 agonists triggered mitochondrial calcium influx and mitochondrial dysfunction in endothelial cells, and 12(S)-HETE effects were absent in endothelial cells from TRPV1-knockout mice. As a therapeutic consequence, we found that a peptide targeting 12(S)-HETE-induced TRPV1 interaction at the TRPV1 TRP box ameliorated diabetes-induced endothelial dysfunction and augmented vascular regeneration in diabetic mice. Our findings suggest that pharmacological targeting of increased endothelial lipid peroxidation can attenuate diabetes-induced comorbidities related to vascular disease.

Transfer of TRPV1 Sodium Binding Site into TRPV2

The transient receptor potential vanilloid 1 (TRPV1) channel is a nonselective cation channel activated by a wide variety of agonists, including acid, toxins, capsaicin, and noxious temperatures above 42°C. While recent cryo-EM structures have revealed the binding pockets for capsaicin, double-knot toxin (DkTx), and resiniferatoxin (RTx), no consensus has been reached on the structural basis of temperature activation. It was recently discovered that removal of sodium from the extracellular side of the membrane shifts a temperature-dependent opening transition such that TRPV1 is open at room temperature, indicating that sodium binding modulates activation by temperature.TRPV1's closest relative, TRPV2, is similar in sequence and structure, and can be rendered sensitive to the TRPV1-selective agonist RTx by only two point mutations, suggesting that the two channels have similar underlying gating mechanisms. In spite of these similarities, TRPV2 fails to respond to most TRPV1 agonists, including temperatures near 42°C, and TRPV2 is not activated by removal of extracellular sodium. To probe the link between temperature dependence and sodium sensitivity, and to localize the sodium ion binding site, we generated a series of chimeras in which transmembrane regions of TRPV1 were transferred into TRPV2. Transfer of the entire transmembrane domain and TRP box of TRPV1 into TRPV2 resulted in chimeras that became activated upon sodium removal, supporting the feasibility of our strategy. Although none of chimeras in which small regions of TRPV1 were transferred into TRPV2 were activated upon sodium removal, several exhibit potentiation when sodium is removed in the presence of subthreshold concentrations of agonist, indicating the successful transfer of the sodium binding site. Ongoing experiments are focused on determining the minimal TRPV1-like sodium binding site and characterizing the temperature-activation thresholds of these chimeric proteins to determine whether TRPV1-like temperature sensitivity is conferred by the sodium-binding domain.

Tryptamine-Based Derivatives as Transient Receptor Potential Melastatin Type 8 (TRPM8) Channel Modulators.

Pharmacological modulation of the transient receptor potential melastatin type 8 (TRPM8) is currently under investigation as a new approach for the treatment of pain and other diseases. In this study, a series of N-substituted tryptamines was prepared to explore the structural requirements determining TRPM8 modulation. Using a fluorescence-based screening assay, we identified two compounds acting as an activator (2-(1H-indol-3-yl)-N-(4-phenoxybenzyl)ethanamine, 21) or an inhibitor (N,N-dibenzyl-2-(1H-indol-3-yl)ethanamine, 12) of calcium influx in HEK293 cells. In patch-clamp recordings, compound 21 displayed a significantly higher potency (EC50 = 40 ± 4 μM) and a similar efficacy when compared to menthol; by contrast, compound 12 produced a concentration-dependent inhibition of menthol-induced TRPM8 currents (IC50 = 367 ± 24 nM). Molecular modeling studies using a homology model of a single rat TRPM8 subunit identified a putative binding site located between the VSD and the TRP box, disclosing differences in the binding modes for the agonist and the antagonist.

Activators of TRPM2: Getting it right.

Activators of TRPM2: Getting it right.

Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels.

Although PI(4,5)P2 is believed to play an essential role in regulating the activity of numerous ion channels and transporters, the mechanisms by which it does so are unknown. Here, we used the ability of the TRPV1 ion channel to discriminate between PI(4,5)P2 and PI(4)P to localize the region of TRPV1 sequence that interacts directly with the phosphoinositide. We identified a point mutation in the proximal C-terminal region after the TRP box, R721A, that inverted the selectivity of TRPV1. Although the R721A mutation produced only a 30% increase in the EC50 for activation by PI(4,5)P2, it decreased the EC50 for activation by PI(4)P by more than two orders of magnitude. We used chemically induced and voltage-activated phosphatases to determine that PI(4)P continued to support TRPV1 activity even after depletion of PI(4,5)P2 from the plasma membrane. Our data cannot be explained by a purely electrostatic mechanism for interaction between the phosphoinositide and the protein, similar to that of the MARCKS (myristoylated alanine-rich C kinase substrate) effector domain or the EGF receptor. Rather, conversion of a PI(4,5)P2-selective channel to a PI(4)P-selective channel indicates that a structured phosphoinositide-binding site mediates the regulation of TRPV1 activity and that the amino acid at position 721 likely interacts directly with the moiety at the 5' position of the phosphoinositide.

A Single-Residue Switch for High Temperature Dependence of Thermal TRPV3 Channels

Thermal TRP channels, a group of ion channels from the transient receptor potential family, play important functions in pain transduction and thermal sensation. The channels are directly activated by temperature, with strong temperature dependence. However, how temperature gates these channels remains poorly understood. One characteristic feature of some thermal TRP channels is hysteresis of gating. This is perhaps most prominent in vanilloid receptor TRPV3. The channel is initially only responsive to extreme heat with low activity, and only after repeated stimulation it becomes vigorously responsive to warm temperatures. Importantly, the hysteresis of activation is accompanied with a profound loss of the high temperature dependence of the channel, suggesting that the hysteresis involves structural changes in temperature sensing. Elucidation of such changes will therefore provide an opportunity to unravel the molecular mechanism underlying temperature sensing of thermal channels. Here we examine the molecular basis underlying hysteresis of heat activation of TRPV3. We show that the hysteresis is mediated by an N terminal domain proximal to membranes, the same region that we have previously identified for temperature sensing in heat sensitive vanilloid receptors. Upon exchange of the region, the heat activation of the channel becomes reversible and the temperature dependence becomes considerably reduced as the wild type channels after sensitization. Interesting much of the hysteresis effect can be attributed to a single residue near the TRP box. The position of the residue suggests a mechanism of temperature-dependent gating of thermal TRP channels involving an intracellular region assembled around the TRP domain.

Dissecting domain-specific evolutionary pressure profiles of transient receptor potential vanilloid subfamily members 1 to 4.

The transient receptor potential vanilloid family includes four ion channels–TRPV1, TRPV2, TRPV3 and TRPV4–that are represented within the vertebrate subphylum and involved in several sensory and physiological processes. These channels are related to adaptation to the environment, and probably under strong evolutionary pressure. Using multiple sequence alignments as source for evolutionary, bioinformatics and statistical analysis, we have analyzed the evolutionary profiles for TRPV1, TRPV2, TRPV3 and TRPV4. The evolutionary pressure exerted over vertebrate TRPV2 sequences compared to the other channels argues for a positive selection profile for TRPV2 compared to TRPV1, TRPV3 and TRPV4. We have analyzed the selective pressure on specific protein domains, observing a common selective pressure trend for the common TRPV scaffold, consisting of the ankyrin repeat domain, the membrane proximal domain, the transmembrane domain, and the TRP domain. Through a more detailed analysis we have identified evolutionary constraints involved in the subunit contact at the transmembrane domain level. Performing evolutionary comparison, we have translated specific channel structural information such as the transmembrane topology, and the interaction between the membrane proximal domain and the TRP box. We have also identified potential common regulatory domains among all TRPV1-4 members, such as protein-protein, lipid-protein and vesicle trafficking domains.

Mutation of I696 and W697 in the TRP box of vanilloid receptor subtype I modulates allosteric channel activation

The transient receptor potential vanilloid receptor subtype I (TRPV1) channel acts as a polymodal sensory receptor gated by chemical and physical stimuli. Like other TRP channels, TRPV1 contains in its C terminus a short, conserved domain called the TRP box, which is necessary for channel gating. Substitution of two TRP box residues-I696 and W697-with Ala markedly affects TRPV1's response to all activating stimuli, which indicates that these two residues play a crucial role in channel gating. We systematically replaced I696 and W697 with 18 native l-amino acids (excluding cysteine) and evaluated the effect on voltage- and capsaicin-dependent gating. Mutation of I696 decreased channel activation by either voltage or capsaicin; furthermore, gating was only observed with substitution of hydrophobic amino acids. Substitution of W697 with any of the 18 amino acids abolished gating in response to depolarization alone, shifting the threshold to unreachable voltages, but not capsaicin-mediated gating. Moreover, vanilloid-activated responses of W697X mutants showed voltage-dependent gating along with a strong voltage-independent component. Analysis of the data using an allosteric model of activation indicates that mutation of I696 and W697 primarily affects the allosteric coupling constants of the ligand and voltage sensors to the channel pore. Together, our findings substantiate the notion that inter- and/or intrasubunit interactions at the level of the TRP box are critical for efficient coupling of stimulus sensing and gate opening. Perturbation of these interactions markedly reduces the efficacy and potency of the activating stimuli. Furthermore, our results identify these interactions as potential sites for pharmacological intervention.

Mutations in the TRP Domain Differentially affect the Function of TRPM8

Sensing potential harmful stimuli is crucial for the survival of the organisms. The detection of these stimuli relies on specialized proteins that localize on the surface of certain sensory cells. The Transient Receptor Potential ion channels are involved in sensing some of those stimuli such as temperature, pH and different chemical compounds. Upon activation, they evoke ionic currents that trigger action potentials in primary sensory neurons.An important unsolved question regarding TRP channel's function, is how the energy of the activating stimuli is coupled to channel opening. Several lines of evidence have pointed to the cytosolic C-terminus of these channels, known as the TRP domain, as a pivotal coupling region.To further understand the role of this domain in channel function, we have designed a set of chimeras in the cold menthol receptor (TRPM8) that incorporate the corresponding region of the heat-activated TRPV1 channel. We focused in the small region between the last transmembrane segment (S6) and the TRP Box. Our data indicate that this small region is essential for the function of TRPM8 and TRPV1. within this region, we have found a mutation that turns TRPM8 into constitutively active (Y981E). Strikingly, this constitutive activity could become regulated upon the addition of another mutation within the same region (V986I). The resulting channel shows the same current amplitude as the wild type, but the response to voltage is faster. In addition mutations in the same region hamper TRPM8 function D991K. Based on the available TRPM8 structural model we interpret the effect of these mutations on the channel function and we propose a possible mechanism for the gating process of TRPM8.

Conservation of Tubulin-Binding Sequences in TRPV1 throughout Evolution

BackgroundTransient Receptor Potential Vanilloid sub type 1 (TRPV1), commonly known as capsaicin receptor can detect multiple stimuli ranging from noxious compounds, low pH, temperature as well as electromagnetic wave at different ranges. In addition, this receptor is involved in multiple physiological and sensory processes. Therefore, functions of TRPV1 have direct influences on adaptation and further evolution also. Availability of various eukaryotic genomic sequences in public domain facilitates us in studying the molecular evolution of TRPV1 protein and the respective conservation of certain domains, motifs and interacting regions that are functionally important.Methodology and Principal FindingsUsing statistical and bioinformatics tools, our analysis reveals that TRPV1 has evolved about ∼420 million years ago (MYA). Our analysis reveals that specific regions, domains and motifs of TRPV1 has gone through different selection pressure and thus have different levels of conservation. We found that among all, TRP box is the most conserved and thus have functional significance. Our results also indicate that the tubulin binding sequences (TBS) have evolutionary significance as these stretch sequences are more conserved than many other essential regions of TRPV1. The overall distribution of positively charged residues within the TBS motifs is conserved throughout evolution. In silico analysis reveals that the TBS-1 and TBS-2 of TRPV1 can form helical structures and may play important role in TRPV1 function.Conclusions and SignificanceOur analysis identifies the regions of TRPV1, which are important for structure – function relationship. This analysis indicates that tubulin binding sequence-1 (TBS-1) near the TRP-box forms a potential helix and the tubulin interactions with TRPV1 via TBS-1 have evolutionary significance. This interaction may be required for the proper channel function and regulation and may also have significance in the context of Taxol®-induced neuropathy.

Peptidyl-Prolyl Isomerase FKBP52 Controls Chemotropic Guidance of Neuronal Growth Cones via Regulation of TRPC1 Channel Opening

Immunophilins, including FK506-binding proteins (FKBPs), are protein chaperones with peptidyl-prolyl isomerase (PPIase) activity. Initially identified as pharmacological receptors for immunosuppressants to regulate immune responses via isomerase-independent mechanisms, FKBPs are most highly expressed in the nervous system, where their physiological function as isomerases remains unknown. We demonstrate that FKBP12 and FKBP52 catalyze cis/trans isomerization of regions of TRPC1 implicated in controlling channel opening. FKBP52 mediates stimulus-dependent TRPC1 gating through isomerization, which is required for chemotropic turning of neuronal growth cones to netrin-1 and myelin-associated glycoprotein and for netrin-1/DCC-dependent midline axon guidance of commissural interneurons in the developing spinal cord. By contrast, FKBP12 mediates spontaneous opening of TRPC1 through isomerization and is not required for growth cone responses to netrin-1. Our study demonstrates a novel physiological function of proline isomerases in chemotropic nerve guidance through TRPC1 gating and may have significant implication in clinical applications of immunophilin-related therapeutic drugs.

Two amino acid residues determine 2-APB sensitivity of the ion channels TRPV3 and TRPV4

Temperature-activated transient receptor potential ion channels (thermoTRPs) are polymodal detectors of various stimuli including temperature, voltage, and chemicals. To date, it is not known how TRP channels integrate the action of such disparate stimuli. Identifying specific residues required for channel-activation by distinct stimuli is necessary for understanding overall TRP channel function. TRPV3 is activated by warm temperatures and various chemicals, and is modulated by voltage. One potent activator of TRPV3 is 2-aminoethyl diphenylborinate (2-APB), a synthetic chemical that modulates many TRP channels. In a high-throughput mutagenesis screen of approximately 14,000 mutated mouse TRPV3 clones, we found 2 residues (H426 and R696) specifically required for sensitivity of TRPV3 to 2-APB, but not to camphor or voltage. The cytoplasmic N-terminal mutation H426N in human, dog, and frog TRPV3 also effectively abolished 2-APB activation without affecting camphor responses. Interestingly, chicken TRPV3 is weakly sensitive to 2-APB, and the equivalent residue at 426 is an asparagine (N). Mutating this residue to histidine induced 2-APB sensitivity of chicken TRPV3 to levels comparable for other TRPV3 orthologs. The cytoplasmic C-terminal mutation R696K in the TRP box displayed 2-APB specific deficits only in the presence of extracellular calcium, suggesting involvement in gating. TRPV4, a related thermoTRP, is 2-APB insensitive and has variant sequences at both residues identified here. Remarkably, mutating these 2 residues in TRPV4 to TRPV3 sequences (N426H and W737R) was sufficient to induce TRPV3-like 2-APB sensitivity. Therefore, 2-APB activation of TRPV3 is separable from other activation mechanisms, and depends on 2 cytoplasmic residues.

Identification of molecular determinants of channel gating in the transient receptor potential box of vanilloid receptor I

Transient receptor potential vanilloid receptor subtype I (TRPV1) is an ion channel gated by physical and chemical stimuli that belongs to the TRPV protein family. TRPV receptors contain a highly conserved, 6-mer segment near the channel gate, known as the TRP box, whose function remains unknown. Here, we performed an alanine scanning mutagenesis of the TRP box of TRPV1 (IWKLQR) and found that mutation of this motif affected channel gating by raising the free energy of channel activation. Functional characterization of TRPV1 mutants showed that substitution of I696, W697, and R701 by alanine severely affected voltage- and heat-dependent activation and notably reduced the capsaicin responsiveness and tachyphylaxia, while mutation of K698, L699, and Q700 had minor effects. In addition, mutation of I696 to alanine promoted a strong outward rectification at negative membrane potentials, and slowed the kinetics of channel activation. Taken together, our findings suggest that modification of I696, W697, and R701 to alanine altered channel function by affecting events downstream of the initial stimuli-sensing step and imply that intersubunit interactions within the TRP box play an important role in TRPV1 gating.

TrpY Regulation of trpB2 Transcription in Methanothermobacter thermautotrophicus

TrpY binds specifically to TRP box sequences upstream of trpB2, but the repression of trpB2 transcription requires additional TrpY assembly that is stimulated by but not dependent on the presence of tryptophan. Inhibitory complex formation is prevented by insertions within the regulatory region and by a G149R substitution in TrpY, even though TrpY(G149R) retains both TRP box DNA- and tryptophan-binding abilities.

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

Regulation of tryptophan operon expression in the archaeon Methanothermobacter thermautotrophicus.

Conserved trp genes encode enzymes that catalyze tryptophan biosynthesis in all three biological domains, and studies of their expression in Bacteria and eukaryotes have revealed a variety of different regulatory mechanisms. The results reported here provide the first detailed description of an archaeal trp gene regulatory system. We have established that the trpEGCFBAD operon in Methanothermobacter thermautotrophicus is transcribed divergently from a gene (designated trpY) that encodes a tryptophan-sensitive transcription regulator. TrpY binds to TRP box sequences (consensus, TGTACA) located in the overlapping promoter regions between trpY and trpE, inhibiting trpY transcription in the absence of tryptophan and both trpY and trpEGCFBAD transcription in the presence of tryptophan. TrpY apparently inhibits trpY transcription by blocking RNA polymerase access to the site of trpY transcription initiation and represses trpEGCFBAD transcription by preventing TATA box binding protein (TBP) binding to the TATA box sequence. Given that residue 2 (W2) is the only tryptophan in TrpY and in TrpY homologues in other Euryarchaea and that there is only one tryptophan codon in the entire trpEGCFBAD operon (trpB encodes W175), expression of the trp operon may also be regulated in vivo by the supply of charged tRNA(Trp) available to translate the second codon of the trpY mRNA.

Half-site arrangement of hybrid glucocorticoid and thyroid hormone response elements specifies thyroid hormone receptor complex binding to DNA and transcriptional activity.

Thyroid hormone receptors bind to thyroid hormone response elements (TREs) as heterodimers with 3,5,3'-L-triiodothyronine (T3) receptor auxiliary protein (TRAP) and retinoid X receptors (RXRs). Currently, it is not known whether TR/TRAP or TR/RXR heterodimers need to bind to both TRE half-sites and whether there is a preferred orientation for TR/RXR heterodimer binding to TREs or transcriptional activation. Accordingly, we created a mutant TR alpha (TR-P box) by changing 3 amino acids in the P box region of the first zinc finger of the DNA-binding domain to that of the glucocorticoid receptor (GR), and we examined wild-type TR alpha and TR-P box complex binding to hybrid response elements containing TRE and glucocorticoid receptor element (GRE) half-sites arranged as a direct repeat with a four-nucleotide gap. TR-P box/RXR heterodimers selectively bound to the hybrid response elements in which GRE half-site was the downstream half-site, whereas TR alpha/RXR bound to hybrid response elements in which GREs were in either position. Additionally, TR/TRAP or TR/RXR heterodimer required two half-sites for binding to DNA, with strong binding to at least one of the half-sites. Last, co-transfection assays and methylation interference studies using the hybrid response elements suggest that the sequential arrangement of strong and weak half-sites in the TRE may be a critical determinant of TR/RXR heterodimer binding and transcriptional activation.