TRP (transient receptor potential) ion channel family: structures, biological functions and therapeutic interventions for diseases

TRPC

TRPC channels were the first identified members of the TRP family.²¹ TRPC channels are nonselective cation channels.²² They range from a few tens to a few hundred kilodaltons.²³ In 1995, the full sequence of the first human homolog (TRPC1) was reported.²⁴ Since then, seven mammalian TRPC proteins (TRPC1–7) have been described.¹⁴ Based on aa similarities, mammalian TRPCs fall into four subsets: TRPC1, TRPC2, TRPC3/6/7, and TRPC4/5.²⁵ TRPC channel proteins are expressed in excitable and non-excitable cells.²⁶ TRPCs have a broad tissue-specific distribution and are therefore involved in various pathophysiological functions.²⁷

TRPV

TRPV channels are a part of the TRP channel superfamily and named for their sensitivity to vanilloid and capsaicin.^28,29 TRPVs are divided into two types.³⁰ TRPV1–4 can be thermally activated and are therefore called thermal TRP channels.³¹ One of the first clues toward understanding the functional diversity of TRPV channels came from the structural studies of the N-terminal ankyrin repeat domain.³² In most tissues, these domains serve as sensors for different pain stimuli (heat, pressure, and pH) and contribute to the homeostasis of electrolytes, the maintenance of barrier functions, and the development of macrophages.^33,34 Given fundamental role in a multitude of physiological and pathophysiological processes, TRPV channels are promising targets for drug development.

TRPA

In 1999, the first human TRPA protein, TRPA1, was discovered during a screening of downregulated genes following the oncogenic transformation of fibroblasts.³⁵ TRPA1 is the only TRPA protein present in humans.^36,37 TRPA1 was previously called ANKTM1 because the protein consists of numerous N-terminal ankyrin repeats.³⁸ TRPA1 is a sensor for diverse noxious external stimuli such as intense cold, irritating compounds, mechanical stimuli, reactive chemicals, and endogenous signals associated with cellular damage.³⁹ This diversity of functions and expression in nociceptive nerve fibers, epithelial cells, and various other cells make this channel relevant to a wide range of diseases and an attractive therapeutic target.⁴⁰

TRPM

Since the first cloning of TRPM1 in 1998, tremendous progress has been made in the identification of novel members of the TRPM subfamily and their functions.⁴¹ The TRPM subfamily consists of eight members; TRPM1–TRPM8.⁴² TRPMs have been involved in several physiological and pathological processes, including cellular proliferation,⁴³ temperature sensing,⁴⁴ vascular development,⁴⁵ cancer progression,⁴⁶ neurological diseases,⁴⁷ endothelial dysfunction,⁴⁸ inflammation,⁴⁹ type II diabetes,⁵⁰ and many other processes. TRPM channels possess a large cytosolic domain consisting of 732 and 1611 aa for each subunit, making them the largest members of the TRP superfamily.⁵¹

TRPN

The TRPN subfamily was named based on the founding member, Drosophila NOMPC.⁵² Mammals do not encode any TRPN members.⁵³ TRPNs are found in worms, flies, and zebrafish.⁵⁴ Genetic screening, calcium imaging, and electrophysiological analysis have identified TRPN as a mechanically gated channel in eukaryotes.⁵⁵ A TRPN channel protein is essential for sensory transduction in insect mechanosensory neurons and in vertebrate hair cells.⁵⁶ However, without a molecular marker for the protein, its exact location and role in transduction are uncertain.

TRPP and TRPML

TRPP and TRPML belong to group 2 of TRP channels and have limited homology similarity to group 1. Furthermore, the large loop between transmembrane domains (TMD) 1 and 2, which discriminates them from group 1.²⁵ Invertebrate TRPPs have been described in C. elegans,⁵⁷ Drosophila (AMO),⁵⁸ and the sea urchin (suPC2).⁵⁹ The three human genes encoding for the TRPP protein family are polycystic kidney disease 2 (PKD2 and TRPP2), PKD2-like 1 (PKD2L1 and TRPP3), and PKD2-like 2 (PKD2L2 and TRPP5).¹⁷ TRPPs may comprise the most primitive TRP subfamily given that as microbial homologs of TRPP2 are found in yeast.⁶⁰ In addition to functioning as a cation influx channel, TRPP2 is also localized in the endoplasmic reticulum (ER) membrane where it is proposed to serve as a new type of Ca²⁺-release channel.⁶¹

The mucolipin family of the ion channel TRP superfamily (TRPML) includes three members: TRPML1, TRPML2, and TRPML3.⁶² The molecular weights of TRPMLs are around 65 kDa.⁶³ TRPML1, was cloned during the search for the genetic determinants of the lysosomal storage disease MCOLN.⁶⁴ Mutations of this protein are characterized by severe neurodegeneration. Defects in TRPML function are predicted to have important effects on organelle acidification, vesicle fusion, endosome maturation, and signaling; thus, suggesting that this protein family plays a key role in normal and pathological conditions.^65,66

TRPVs

The earliest structural information obtained for the TRPV subfamily was the X-ray crystal structures of the isolated cytoplasmic ankyrin repeat domain from TRPV2 in 2006, immediately followed by similar structures for other TRPV channels.^70,71 As is true for all TRP channels, TRPV channels are tetrameric and each monomer features the classic six transmembrane helix architecture of voltage-gated ion channels in its TMD.⁷² The overall sequence similarity of TRPVs is more than 40%. TRPV protein has an ~400–450-residue N-terminal domain with 3–6 intracellular ankyrin repeats, which are essential for channel function.^70,73 An ~150-residue C-terminus that acts as a platform for interactions with other proteins and ligands.⁷⁴

Liao et al. used advances in EM to determine the structure of the mammalian TRPV1 channel at a resolution of 3.4 Å.⁷⁵ S1–S4 form a bundle similar to the voltage-sensing domain in voltage-sensitive ion channels.⁷² S5 and S6 extend from the S1–S4 bundle, and the conserved “TRP structural domain” interacts with the S4–S5 linker.^72,76 TRPV1 has a wide extracellular “mouth” with short selective filtering.⁷⁵ This domain-swapped pore domain consists of S5 and S6, which form the central pore and lower gate, whereas a short loop and helix between S5 and S6, termed the pore helix (PH), forms the upper gate or selectivity filter.⁷² The dynamic communication between the upper and lower gates may be the basis for the integration of various physiological signals.⁷⁶ Notably, the motion of S6 in the TRPV1 structure can be roughly described as small, medium, or large. When the motion is small, the π-helix is retained, and the channel gate is formed by I679.⁷⁷ In the intermediate category, the π-helix transitions to the α-helix, and the gate is formed by M682.⁷⁷ When S5/S6 motion is large, the π-helix conformation is observed, and the open gate (9.8 Å) is formed by I679.⁷⁷ However, transitions between π and α have also been observed in TRPV2 and V3 structures, but the functional consequences of such transitions remain to be fully understood.^78,79

TRPCs

Given the great advancements in single-particle cryo-EM technology, high-resolution structures of TRPCs became available in 2018.⁸⁰ TRPC2 is thought to be a pseudogene in humans.⁸¹ The overall architecture of the homologous TRPC3, TRPC4, TRPC5, and TRPC6 is consistent with that of TRP.^82–84 The structure of TRPCs is similar to the voltage sensor structural domains of voltage-gated K⁺, Na⁺ and Ca²⁺ channels, with S5–S6 forming a structurally conserved ion conductance or pore domain common to all TRP channels, voltage-gated channels, inwardly-rectifying K channels, and bacterial K and Na channels.^85,86 All TRPC structures contain a three-helix region designated as the pre-S1 elbow and pre-S1 helix before the transmembrane S1 helix.^87,88 This region is halfway embedded in the membrane, with its N-terminal exposed to the cytoplasmic side to connect with a long stretch of tightly folded linker helices located at the proximal N-terminus of each protomer.⁸⁹ Immediately before the linker helices are the four ankyrin-like repeats that form the outskirt of the cytoplasmic architecture, which completely surrounds the four helical bundles composed of the second C-terminal helix.⁹⁰

Currently, the resolved region of the cytoplasmic structural domain of TRPC4 reaches approximately 80 Å, which is relatively short compared with that of other TRP channels.⁹¹ Helices S5 and S6 are exchanged with the respective helix of the adjacent protomer and form a pore at the center of the quaternary channel with a negatively charged extracellular opening; residue E555 at the top end of the pore tower is conserved in TRPC1, TRPC4 and TRPC5 and forms a salt bridge with R556 of the adjacent protomer.⁹¹ Fan et al.⁹² proposed that TRPC3 is in a lipid-occupied closed state with a structure of 3.3 Å. TRPC3 has four elbow-like membrane reflux helices before the first transmembrane helix.⁹² The third transmembrane helix, S3, is very long, forms a unique transmembrane structural domain and constitutes an extracellular structural domain that may act as a sensor for external stimuli.⁹²

TRPA

The TRPA1 gene encodes a large protein consisting of about 1100 aa (1119 aa in humans, 1125 aa in rats, 1115 aa in mice, 1120 aa in zebrafish, 1197 aa in Drosophila, and 1193 aa in C. elegans), with an estimated molecular mass between 120 and 130 kDa.⁹³ TRPA1 has distinctively large intracellular NH2 and COOH termini, which together account for about 80% of its molecular mass.⁹⁴ Each subunit of TRPA1 consists of six transmembrane alpha sheets (S1–S6) plus a re-entrant pore loop between S5 and S6; homotetramers are formed by “domain exchange” interactions and establish a conserved transmembrane core.^95,96 The distinguishing feature of TRPA1 is its long N-terminus with 14–18 predicted ankyrin repeats, each consisting of 33 aa.^97,98 A structural domain containing a sequence of five ankyrin repeats surrounds the coiled coil and connects to another extended feature, which forms a crescent-shaped element.⁹⁵ TRPA1 also possesses a distinct tetrameric coiled coil at the center of the channel, beneath the permeation pore. This stalk-like structure is stabilized by the interaction of positively charged residues on the exterior surface of the coiled coil with inositol polyphosphates.⁹⁴ These interactions are essential for TRPA1 channel activity.^94,99,100

TRPMs

In late 2017, several TRPM structures were solved using single-particle cryo-EM.¹⁰¹ TRPMs have four shared homologous regions: a TMD consisting of six transmembrane helices, a TRP helix, and C-terminal domains containing the conserved TRP domain followed by a coiled-coil region that differs among members.^16,102 Despite their common structural features, the members of the TRPM subfamily are less conserved than those of other subfamilies. Several TRPM channels, such as TRPM2, uniquely contain a functional nucleoside diphosphate linked to another moiety/ADP ribose domain and a kinase domain that resembles to protein kinase A to a certain extent.^103,104 TRPM2 is structurally characterized by its C-terminal NUDT9-H structural domain, a homologue of the human ADP-ribose pyrophosphatase NUDT912.¹⁰⁵ In other words, these TRPMs combine features of ion channels and enzymes and are thus referred to by some as “chanzymes”.^106–108 The ion conductance pore of TRPM2 is restricted at two locations, a selective filter located close to the extracellular entrance, which is formed by a hinge connecting the PH and the P ring, and a gate lined by S6 near the inner end of the cell.¹⁰⁵

TRPM4 consists of three layers, namely, the N-terminal nucleotide-binding domain, ankyrin repeat domain, and the C-terminal coiled-coil helix, which form the base layer of TRPM4.¹⁰⁹ The middle layer consists of a linking helix structure containing 12 helices and forming a scaffold for multiple interactions between structures within the subunit.¹⁰⁹ The S1–S6 and TRP structural domains embedded in lipid form the top layer of the channel.¹⁰⁹ The C-end coiled spiral structure forms a parallel disc-shaped coil.¹¹⁰ The discoid coil is surrounded by a large, intertwined cytoplasmic structural domain consisting of four N-terminal TRPM homology regions (MHR1–4), which are highly conserved in the TRPM subfamily.^95,102,111 Of these, MHR1–2 contain an eight-stranded β-sheet surrounded by eight α-sheets.¹¹⁰ By contrast, MHR3–4 are composed of stacked α-sheets and connected to the transmembrane structural domain via MHR4, which clasps the TRP structural domain; as a result, an interaction occurs between the cytoplasmic structural domain and the transmembrane core.^109,112 In 2018, Autzen et al.¹¹² compared the structures of Ca²⁺-free and Ca²⁺-bound TRPM4 and observed an additional density in the hydrophilic pocket on the cytoplasmic side of the S1–S4 structural domain (this density represents a true bound Ca²⁺).

The mouse TRPM8 channel is a three-layer homologous structure. The top transmembrane channel region consists of the pre-S1 structural domain, TMD, and TRP structural domain.¹¹³ The TMD consists of a voltage-sensor-like structure (VSLD) consisting of transmembrane helices S1 to S4, and a pore domain formed by S5 and S6 helices,^113,114 similar to the previously identified TRPV structure.^75,79 The TMD of TRPM8 also has several features that distinguish it from the structure of other TRP ion channels. First, relative to the structure of apo TRPV1, the PH of TRPM8_FA is positioned 12 Å away from the central axis, tilted by 8°, and shifted by 5 Å toward the outside of the cell.¹⁰¹ Second, no non-α helical elements (e.g., 310 or π helices) can be found in the TMs of TRPM8, which in other TRP channels are proposed to provide helical bending points for channel gating.¹¹⁵ Finally, although TRPV channels have a pre-cellular S1 helix, TRPM8_FA contains three additional helices between S1 and the presumed pre-cellular S1 helix in the membrane region.¹⁰¹ Diver et al.¹¹⁶ observed a substantial rearrangement in the S4–S5 linker in the TRPM8 structure, and it repositioned the S1–S4 and pore domains relative to the TRP helix.

TRPPs

The TRPP subfamily has three members (TRPP2, TRPP3, and TRPP5).¹¹⁷ The PKD1 and PKD2 genes encoding polycystin-1 and polycystin-2 proteins, respectively, were originally named TRPP1 and TRPP2.^118–120 However, members of the PKD1 family do not belong to the TRP family. PKD proteins are divided into two groups based on sequence homology: typical isoforms with 6 TMs (PKD2 subfamily), such as TRPP2, TRPP3, and TRPP5; and another group with 11 TMs and a short intracellular C-terminal tail (PKD1 subfamily), represented by PKD1, PKD1L1, PKD1L2, PKD1L3 and PKD-REJ7.¹²¹ The TRPP protein can form homotetramers and heterotetramers,^122,123 such as the PKD1/TRPP2 and PKD1L3/TRPP3 complexes, with members of the PKD1 family.^124–126

PKD1 is a 4,303-aa, 465-kDa integral membrane protein containing a large N-terminal extracellular structural domain and an intracellular C-terminal structural domain.¹²⁷ Unlike PKD1, TRPP2 is a 968-aa, 110-kDa protein with 6 TMs and a pore-forming loop,¹²⁸ and has intracellular amino and carboxyl termini.¹²⁹ Shen et al.¹³⁰ showed that TRPP2 is a Na/K conducting channel with low permeability and small single-channel conductance to Ca²⁺. The complexes of PKD1 and TRPP2 exhibit a 1:3 structural ratio.¹³¹ TRPP2 and PKD1 bind directly through their C-termini to form a complex containing three TRPP2 and one PKD1.¹³² This association involves a coiled-coil structure at the C-terminus of both proteins.¹²⁵ R807X, E837X, and R872X of TRPP2 and R4227X and Y4236X of PKD1 can lead to deletion of the coiled-coil structural domain, which results in the mutation of ADPKD.^133,134

TRPP3 (polycystin-2 like 1 (PKD2L1)) and TRPP2 share high sequence similarity (79% homology and 62% identity).¹³⁵ Unlike TRPP2, the PH and S6 of TRPP3 are involved in the opening of the upper and lower gates to adopt an open conformation.¹³⁶ Su et al.¹³⁶ suggested that the simultaneous occurrence of gate opening and voltage-sensing domain (VSD) conformational changes in TRPP3 is caused by coupling between the VSD and pore domains. PKD1L3/TRPP3 complexes form calcium-permeable, non-selective cation channels.^137,138 The structure of PKD1L3/TRPP3 in the apo and Ca²⁺-loaded states shows two Ca²⁺ binding sites at selectivity filter and VSD_III respectively.¹³⁹ It also has a 1:3 stoichiometry TRP structure protected by Lys2069 of PKD1L3 and Asp523 from the three subunits of TRPP3.¹³⁹

TRPN and TRPMLs

Drosophila NOMPC protein is the first member of the TRPN subfamily.¹⁴⁰ The Drosophila NOMPC protein shares significant homology with the TRP superfamily.⁵² The N-terminus of NOMPC contains 29 ankyrin repeats, the largest number among TRP channels.¹⁴¹ Unlike other TRP family proteins, the ankyrin repeat structure of NOMPC is a quadruple structure, with each structural domain acting like a helical spring.¹⁴² The TMD of each NOMPC subunit consists of six transmembrane α-helices (S1–S6) and a re-entrant pore loop located between S5 and S6.¹⁴² The TRP structural domain is sandwiched between the linker helix and the S4–S5 linker. The short helix following the TRP structural domain is wrapped around the elbow in front of S2 and S1.¹⁴² However, the C-terminus is mostly unstructured, with a short helix fragment interacting with the linker domain.¹⁴² No TRPN has been found in mammals.

The TRPML subfamily is defined by a human protein, mucolipin 1.¹⁴³ Mucolipin 1 (TRPML1) is a 580 aa-long protein probably restricted to intracellular vesicles.¹⁴⁴ TRPML1 contains two proline-rich regions, namely, a lipase serine active site in the N-terminus and a dileucine motif suggestive of lysosomal targeting in the C-terminus.⁶⁴ TRPML1 is characterized by its longer S2 helix than that of PKD2 and protrudes from the membrane bilayer-embedded portion of the cell membrane, with an extension of the N-terminal S1 helix (pre-S1) that includes two small alpha helices (α1 and α2) rich in many basic aa.¹⁴⁵ Several aromatic and hydrophobic residues in the PH 1, S5, and S6 helices of TRPML1 and the S6 helix of the adjacent subunit form a hydrophobic cavity to accommodate the agonist.¹⁴⁵ Phosphatidylinositol-3,5-bisphosphate binds to the N-terminal end of TRPML1, that is away from the pore, and a helix-turn-helix extension between the S2 and S3 segments may link ligand binding to pore opening.¹⁴⁶ Two other members of the TRPML subfamily, namely, TRPML2 and TRPML3, are encoded by MCOLN2 and MCOLN3 genes, respectively.¹⁴⁷ Similar to TRPML1, they are active in late endosomes and lysosomes.¹⁴⁸ TRPML3 is divided into an extracellular structural domain (ECD), a TMD, and a cytosolic structural domain.¹¹⁵ The ECD consists of two β-sheets and two extracellular helices that form a ring that caps the extracellular side of the channel and is structurally similar to the ECD of TRPML1 and PKD2.^{115,130,149,150} Unlike PKD2, in which the ECD interacts extensively with the TMD, in TRPML3, minimal interaction exists between the ECD and the TMD.^115,151

Activation of TRPA1 induces pain

TRPA1 plays a role in various forms of pain, including neuropathic cold pain, inflammatory pain, and hereditary episodic pain syndromes.^215,216 As shown by dermal experiments, the activation of TRPA1 by certain compounds triggers pain and heat sensations.^215–217 Sustained activation of TRPA1 by endogenous substances induces chronic pain involved in various disorders,²¹⁸ including fibromyalgia, osteoarthritis, inflammation, diabetes, obesity, migraine, neuropathy, bronchitis, and emphysema.²¹⁹

Chembridge-5861528 blocks TRPA1 and relieves mechanical nociception after nerve injury.²²⁰ Koh et al.²²¹ observed that inhibition of TRPA1 expression reduced the development of mechanical, thermal, and cold heterosensitivity. The experimental results of Sagalajev et al.²²² showed that injury-induced oxidative stress in the amygdala stimulated TRPA1 to promote neuropathic pain behavior. This pain perception involved the inhibition of medullary serotonergic feedback inhibition acting on spinal 5-HT1A receptors, which was reversed by TRPA1 antagonists.^223,224 In Schwann cells, activation of TRPA1 induces NADPH oxidase 1-dependent H₂O₂ release, maintains macrophage infiltration of injured nerves, and sends paracrine signals to activate TRPA1 in phagocytosed nociceptors to maintain mechanical nociception (Fig. 4).²²⁵

Activation of TRPA1 channels leads to an influx of sodium and calcium ions, which induces the depolarization of nociceptive nerve ending that is needed to generate the centrally propagating nociceptive signal.²²⁶ TRPA1 is expressed not only on peripheral but also on central terminals of nociceptive primary afferent nerve fibers.²²⁷ On central terminals located within the spinal dorsal horn, TRPA1 amplifies glutamate release and thereby transmission of nociceptive signals to spinal interneurons and projection neurons.^228,229

Topical administration of selective TRPA1 agonists in vivo induced nociceptive behavior in experimental animals and pain sensation in human beings.^216,230 Bautista et al.²³¹ used TRPA1-deficient mice to better demonstrate that environmental stimulants and endogenous analgesics depolarize nociceptive receptors via TRPA1 to elicit inflammatory pain. TRPA1 antagonists showed no effect on all pain. Intrathecal application of a low dose of TRPA1 antagonist attenuated formalin-induced tactile allodynia but had no effect on acute pain behavior.²³² In addition, systemic administration of HC-030031 (TRPA1 antagonist) showed to have anti-isokinetic and inflammatory pain efficacy in a CFA model.¹⁵⁵ Interestingly, low doses of intrathecal HC-030031 can also able to attenuate pain.²³³ By contrast, a similar dose of a TRPA1 antagonist had no effect on healthy rats.^232,234

TRPVs in pain

TRPV1 is a heat-activated cation channel modulated by inflammatory mediators and contributes to acute and chronic pain.²³⁵ About 30% of the TRPA1-expressing neurons co-express TRPV1.⁹³ TRPV1, which is widely expressed in bipolar neurons, transmits sensory information from the periphery to the somatosensory cortex via the spinal cord.^236,237 Stimulation of TRPV1 evokes a burning sensation, which reflects the central role of the channel in pain.²³⁸

Trpv1 knockout mice in an inflammatory model exhibited attenuated thermal nociception, which sparked great interest among researchers to develop TRPV1 antagonists with analgesic properties.^239,240 TRPV1 is involved in toxic chemicals and heat-induced pain and promotes peripheral sensitization.²⁴¹ Analgesics may enhance nociceptive receptor activity by increasing TRPV1 expression.²⁴²

In the case of cutaneous and visceral pain, the downregulation of TRPV1 activity produces antinociceptive effects.^243,244 TRPV1 in skin non-neuronal cells may release pro-inflammatory agents acting on nociceptive endings, which sensitizes neuron-expressed TRPV1 channels and triggers pain signals.^245,246

TRPV4 primarily mediates inflammatory pain and neuropathic pain. Colonic mechanical pain and nociception were significantly relieved in Trpv4 knockout mice.²⁴⁷ In mice, topical treatment with a TRPV4-selective inhibitor decreased UVB-evoked pain behavior, epidermal tissue damage, and endothelin-1 expression.²⁴⁸ In sunburned humans, elevated expressions of epidermal TRPV4 and endothelin-1 were observed.²⁴⁹ Activation of TRPV4 induced the transcriptional regulation and release of cytokines and tachykinins (substance P (SP) and calcitonin gene-related peptides (CGRP)),²⁴⁷ which ultimately led to chronic pain.²⁵⁰ Elevated prostaglandin E₂ (PGE2) is also thought to contribute to TRPV4-mediated nociceptive behavior,²⁵¹ which is important in pathological pain states associated with multiple diseases.²⁵² In addition, TRPV4 plays a role in chronic DRG compression-induced neuropathic mechanical pain via the p38 MAPK pathway.²⁵³

Although studies on TRPVs related to pain have mainly focused on TRPV1 and TRPV4. TRPV2 and TRPV3 are also promising targets. TRPV2 is widely expressed in neuronal and non-neuronal cells.^254,255 Increased expression of TRPV2 is associated with thermal hyperalgesia and mechanical thermal after oral mucosal incision.²⁵⁶ However, the role of TRPV2 in sensory neurons is currently unknown, and a limited number of studies have been conducted in relation to pain.

By contrast, TRPV3 does not act as extensively as other channels, and its studies have focused on skin diseases.²⁵⁷ TRPV3 has been suggested as a candidate transducer contributing to pain hypersensitivity associated with inflammatory states.^258,259 Analgesic effects were observed in a model of pruritus treated with selective and potent inhibitors of the TRPV3 channel by Han et al.²⁶⁰. TRPVs are a promising therapeutic target in pain research.

TRPMs in pain

Numerous studies have linked the TRPM8 channel to pain. The channel is mainly expressed on peripheral sensory neurons and is known as a cold temperature sensor. However, it is also expressed in deep viscera. The administration of TRPM8 antagonists, knockdown of the TRPM8 gene, or ablation of primary afferent neurons expressing TRPM8 can be effectively relieve the cold pain associated with tissue and nerve damage.^261,262 The experimental results of Zuo et al.²⁶³ demonstrated that TRPM8 played a role in cold soreness and hyperalgesia following chronic trigeminal nerve injury. Activation of TRPM8 effectively reduces the painful behavior caused by chemical irritation, toxic heat, and inflammation.^264,265 TRPM8 can also amplify the analgesic effects of their agonists by forming complexes with 5-HT-1B receptors.²⁶⁶ In addition, when TRPM8 od TRPA1 and TRPV1 are co-activated, an analgesic effect is produced.²⁶⁷

In addition to TRPM8, significant advances have been made in the role of TRPM2 in pain. Exposure of TRPM2 to H₂O₂ significantly increased mRNA and protein levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and a chemokine (CXCL2).^268,269 Remarkably, the inhibition of TRPM2 expression can affect the elevated levels of cytokines and chemokines.²⁶⁹ TRPM2 may play a role in inflammatory pain. In animal experiments, TRPM2-deficient mice were unresponsive to mechanical and thermal nociception in a model of keratin-induced inflammation and exhibited reduced edema.²⁷⁰ Therefore, TRPM2 contributes to inflammatory pain by promoting peripheral immune functions.

TRPM2 and TRPM8 are the most relevant members of the family of TRPMs regarding pain, but the role of other members of the family in pain should not be underestimated. Pharmacological and genetic studies have shown that TRPM3 is associated with various pain patterns. Su et al.²⁷¹ concluded that TRPM3 plays a role in thermal hyperalgesia and spontaneous pain but not in cold and mechanical hyperalgesia. The heat-activated TRPM3 ion channel is a potential target for new analgesics, but its mechanism in relation to pain is unknown and needs to be explored.

TRPs are arguably the most widely studied analgesic targets today. TRPV1 has been validated as a target for analgesic drugs in preclinical studies,²⁷² and its side effects should not be underestimated. Whether other pain-related TRP channels act as the sole mediators of nociception, and the mechanisms of analgesia remains to be determined.

Alleviating IBD symptoms: emerging TRP targets

Traditional strategies for the treatment of IBD have relied on the frequent administration of high doses of drugs, including antibiotics, anti-inflammatory agents (such as 5-aminosalicylic acid and corticosteroids), and biological and immunosuppressive agents (azathioprine, 6-mercaptopurine, methionine, cyclosporin-A, and tacrolimus),²⁸⁴ to reduce inflammation.²⁸⁵ The emergence of monoclonal antibodies as biological therapies has significantly increased the treatment options for IBD.²⁸⁶ Numeros TNF-α antibodies, such as adalimumab or certolizumab, are used in the clinical treatment of IBD.²⁸⁷ Antibodies targeting IL-12 and IL-23, such as ustekinumab and natalizumab, have also been proposed for clinical approval.²⁸⁸ These drugs can effectively relieve early inflammatory symptoms, but their long-term use can cause toxicity and damage organs. These problems may be effectively avoided when certain TRP modulators are used.

TRPVs in IBD

Activation of TRPV4 receptors in the GI tract can trigger inflammation.²⁸⁹ TRPV4 is expressed and functional in human colon samples, human intestinal epithelial cell lines (Caco-2 and T84), and inflamed colons of mice; 4α-phorbol-12,13-didecanoate (4αPDD) activated TRPV4 in the GI tract and increased the intracellular calcium ion concentration, which ultimately leads to chemokine release and colitis.²⁹⁰ Cenac et al.²⁹¹ demonstrated that 4αPDD selectively activated TRPV4 in colonic sensory neurons, which affected visceral nociception and hypersensitivity. Fichna et al.²⁹² first observed the significantly higher expression of TRPV4 mRNA in the tissues of IBD patients compared with those of non-IBD patients. Activation of TRPV4 receptors in the GI tract has a pro-inflammatory effect, and selective blockade of TRPV4 in an animal model of IBD alleviated colitis and the pain associated with intestinal inflammation. De Petrocellis et al.²⁹³ showed that activation and desensitization of TRPV channels mediated intracellular Ca²⁺ concentrations. Interference with TRPV1–4 channel activity and expression may have multiple potential therapeutic applications for IBD.

Increased TRPV1 nerve fibers in patients with IBD correlated with abdominal pain severity.²⁹⁴ Upregulation of TRPV1 protein expression in IBD patients affected nerve endings and immune cells, which suggests the importance of TRPV1 in colonic immune regulation of the inflammatory response and in the pathogenesis of IBD.²⁹⁵ Over 20% of patients with IBD have significantly elevated TRPV1 expression in the rectum, which is a key point in mediating inflammatory pain.²⁹⁶ The process of producing neuropathic pain in patients with IBD is associated with the activation of TRPV1 because it promotes the release of neuropeptides, such as SP and CGRPs from peripheral terminals (Fig. 5).²⁹⁷ Moreover, increased levels of TRPV1 were observed in patients with acute exacerbations of IBD and in experimental models of acute colitis.²⁹⁸ Pretreatment of the IBD model with the TRPV1 antagonist JYL1421 reduced visceral hypersensitivity, inhibited colorectal dilation, and improved microscopic colonic inflammation.²⁹⁹ All these findings support the idea that TRPVs at least partially participate in the regulation of IBD.

Activation of TRPM8 for the treatment of IBD

TRPM8 mRNA is highly expressed in colonic DRG neurons, and TRPM8 protein is distributed throughout the nerve fibers of the colon wall. Harrington et al.³⁰⁰ suggested that TRPM8, which is present in colonic sensory neurons, couples to TRPV1 and TRPA1 and inhibits their downstream chemosensory and mechanosensory effects. In animal models of post-inflammatory colonic allergy, increased TRPM8 expression significantly attenuated hypersensitivity to mechanical stimuli and reduced the symptoms of colonic allergy.³⁰¹

Inflammation in mouse colitis models can be alleviated by the activation of TRPM8. Selective activation of TRPM8 reduces the release of inflammatory neuropeptides, inhibits the release of pro-inflammatory cytokines and chemokines, and prevents the accumulation of leukocytes in the colon.³⁰² Inhibitors of phospholipase A2 (PLA2) reduce the response of TRPM8 to cold and agonists, and lysophospholipids as products of PLA2 have the opposite effect on TRPM8.³⁰³ Notably, PLA2 levels are elevated in patients with CD and UC,³⁰⁴ whereas levels of lysophospholipids are clearly decreased.³⁰⁵ These changes may also have implications for the function of TRPM8 during colitis.

TRPA1 agonists

Ample evidence indicates that TRPA1 plays a major role in the development of IBD.⁴ 2,4,6-Trinitrobenzene sulfonic acid (TNBS) induces colitis with colonic dilation and increases sensitivity to chemical stimuli; TRPA1 expression is upregulated in the colonic afferent DRG neurons.³⁰⁶ Intrathecal administration of the TRPA1 antisense oligodeoxynucleotide inhibitor resulted in the downregulation of TRPA1 expression in the DRG and inhibited IBD-induced nociceptive hypersensitivity, colonic dilation, and allyl intracolonic isothiocyanate.³⁰⁷ Dinitrobenzene sulfonic acid (DNBS) binds to the intracytoplasmic N-terminal cysteine residue of the TRPA1 protein; thus, effective treatment of DNBS-induced colitis is achieved by antagonizing TRPA1 or genetic deletion of TRPA1, and therefore, TRPA1 is considered a direct target of DNBS-induced IBD.³⁰⁸

Application of TRPA1 agonists alleviated DNBS-induced colitis inflammation in mice. Activation of TRPA1 restored the integrity of the colonic epithelial mucosa, inhibited the production of nitric oxide, IL-10, and IFN-γ, and significantly reduced nitrite levels.³⁰⁹ These data suggest the significant role of TRPA1 in restoring intestinal mucosal integrity and reducing inflammatory factors after IBD. It may also be used to provide a new therapeutic strategy for the relief of IBD.

Complications of IBD: NAFLD

NAFLD is one of the most prevalent complications of IBD,³¹⁰ and it may occur at any stage of the natural course of the disease.³¹¹ The prevalence of NAFLD ranges from 1.5% to 55% in UC and from 1.5% to 39.5% in CD (with an overall mean prevalence of 23%).³¹² Patients with IBD with NAFLD tend to have stable liver disease that is stable for 4–6 years.³¹³ NAFLD is also the most common liver disease, with a prevalence of up to 25% worldwide³¹⁴; thus, it imposes a significant economic burden on patients and leads to a health-related decline in quality of life.³¹⁵ It is characterized by fibrosis, inflammation, fatty deformation, and lipid deposition in hepatic parenchymal cells.

TRPV as a therapeutic target in NAFLD

Activation of TRPV1 increases energy metabolism and prevents the accumulation of visceral fat.³¹⁶ Monoacylglycerols (MGs) with unsaturated long-chain fatty acids are new TRPV1 agonists that along with capsaicin, are found in food and capsaicin to prevent the accumulation of visceral fat. Iwasaki et al.³¹⁷ tested a diet of MGs that activated TRPV1 and found a reduction in the weight of white adipose tissue and lower levels of serum glucose, total cholesterol, and free fatty acids. Activation of TRPV1 also enhanced energy expenditure and increased thermogenesis, which further reduced visceral fat accumulation in mice and prevented NAFLD.³¹⁸ Therefore, activation of TRPV1 is considered an important candidate for reducing the risk of diseases, such as NAFLD (Fig. 6).

Long-term activation of TRPV1 in tissues causes upregulation of hepatic uncoupling protein 2 (UCP2), which effectively prevented high-fat diet-induced NAFLD in mice.³¹⁹ Previous studies have shown that TRPV1 activation by dietary capsaicin resulted in the upregulation of UCP2 expression in a wild-type (WT) mouse liver model but not in Trpv1 knockout mice. These results suggest that the mechanism by which TRPV1 is activated to prevent exacerbation of fatty liver may be associated with increased UCP2-mediated hepatic β-oxidation.³²⁰ Thus, dietary capsaicin may serve as a promising intervention for people at high risk of fatty liver disease. However, whether TRPV1 is involved in the regulation of lipid accumulation and the underlying mechanisms involved in this process are currently unclear.

Song et al.³²¹ observed that ruthenium red or a synthetic siRNA against TRPV4 inhibited the proliferation of HSC-T6 cells, which caused downregulation of myofibroblast markers, including α-SMA and Col1α1. Seth et al.³²² reported that TRPV4 responded to the cytotoxic environment of the liver and negatively regulated cytochrome p450 enzymes (CYP2E1). Activation of TRPV4, which releases NO to act on neighboring hepatocytes, inactivates CYP2E1-induced redox toxicity (Fig. 6). TRPV4 acts as a cellular stress sensor in diseased tissues of patients with NAFLD, and thus, it constitutes an endogenous defense molecule that may act as a novel regulator in a protective manner for hepatocytes.

TRPM and TRPC channel involvement in NAFLD

Assessment of acetaminophen-induced liver injury based on blood liver enzyme concentrations and liver histology revealed significantly less severe liver injury in Trpm2 knockout mice than in WT mice.³²³ TRPM2 channels are integral in the mechanism of acetaminophen-induced hepatocellular death. Thus, TRPM2-mediated cell death is an important mechanism in the pathogenesis of NAFLD-induced liver injury (Fig. 6).³²⁴ However, blockade of TRPM7 inhibits the activation and proliferation of primary hematopoietic stem cells and induces apoptosis in activated cells; ER stress was identified as a possible underlying molecular basis.³²⁵ TRPM2 and TRPM7 are valuable future therapeutic targets for NAFLD and serve as basis for discoveries related to in this disease.

Arterial flow-regulated diastolic function, which leads to atherosclerosis, is lower in patients with NAFLD than in the healthy population. TRPC3-related endothelial mechanisms that promote NAFLD drive the progression of atherosclerosis. In vitro, TRPC3 is inhibited in hepatic sinusoidal endothelial cells, which makes them less sensitive to ER stress-induced apoptosis; therefore, the pro-apoptotic influence of TRPC3 may increase other fibrotic factors in vivo.³²⁶ A positive correlation was found among enhanced expression of endothelial TRPC channels, development of early steatohepatitis, and atherosclerotic load in a mouse model of nonalcoholic fatty liver hyperlipidemia fed with a traditional Western-style diet.

Nishiyama et al.³²⁷ speculated that TRPC3 or TRPC6 may be involved in the fibrotic process of NAFLD. However, systemic deletion of either TRPC3 or TRPC6 does not attenuate liver fibrosis, inflammation, nor steatosis. These findings suggest that TRPC3 and TRPC6 are unlikely to be implicated in liver dysfunction and fibrosis in NAFLD mouse models.³²⁸ No effective therapy is available for NAFLD, and its exact pathogenesis remains to be elucidated. All these data indicate that TRP channels may be an interesting target for drug development for the treatment of NAFLD.

Extraintestinal complications of IBD: psoriasis

A distinct bidirectional association exists between psoriasis and IBD.³²⁹ At the epidemiological level, patients with psoriasis are at increased risk of developing IBD, and vice versa.³³⁰ On the skin, TRPs are interesting pharmacological targets because they allow for topical treatment and may reduce the risk of severe harmful effects.³³¹ TRP channels are widely expressed in various skin cell types, including keratinocytes,³³² melanocytes,³³³ nerve endings, and immune cells.²⁴⁵ Their functions involve hair growth,³³⁴ wound healing,³³⁵ skin cell proliferation and differentiation, immunity,³³⁶ skin barrier function,³³⁷ and skin inflammation and pruritus.^338,339 IBD and psoriasis are relatively common diseases that require long-term treatment. Immunosuppressive drugs are currently the most commonly used treatment in clinical practice and a significant overlap has been observed between effective drugs for the treatment of IBD and psoriasis. The development of potent targeted therapies for TRPs is changing the outlook for chronic inflammatory diseases such as IBD and psoriasis.

Remitting effect of inhibition of TRPVs on psoriasis

TRP channels contribute differently to various psoriasis symptoms. Activation of TRPV1 and TRPC4 was found to be detrimental to psoriasis, whereas TRPA1 was found beneficial in preclinical studies.³⁴⁰ Lee et al.³⁴¹ reported that the inhibition of TRPV1 activity, which activates ALX/FPR2 receptors, coupled with the control over CGRP release, significantly alleviated psoriasis-like pruritus and inflammation in an imiquimod (IMQ) animal model of psoriasis.

The TRPV1 and NaV1.8 neuroreceptors control the cutaneous immune response by interacting with dermal dendritic cells to inhibit the production of several key effector cytokines in psoriatic dermatitis and regulate the IL-23/IL-17 pathway.³⁴² Neurons expressing TRPV1 can mediate itch signaling.³⁴³ However, intrathecal injection of capsaicin (10 μg) disrupts the central terminals of TRPV1-expressing neurons.³⁴⁴ Therefore, we hypothesize that TRPVs are considered novel targets for the treatment of psoriasis by suppressing itching to relieve psoriasis symptoms.

Modulation of TRPM and TRPC channels

Cold activation of TRPM8 reduces pruritus caused by psoriasis.³⁴⁵ This mechanism possibly occurs through blocking of histaminergic and non-histaminergic pruritic pathways by pharmacological hypothermic cooling.³⁴⁶ Activation of TRPM8 also reduces inflammation-induced psoriasis due to its capability to strongly improve edema, inhibit mechanical nociception, and decrease levels of inflammatory cytokines (IL-1β, TNF-α, and IL-6); as a result, the inflammatory response is reduced.³⁴⁷ In conclusion, these studies provide strong evidence that TRPM8 is a major cold sensor for amelioration of pruritus and inflammation in various dermatological conditions; it enables the use of TRP channel modulators in the treatment of psoriasis.³⁴⁸

In the IMQ-induced psoriasis mouse model, activation of TRPM8 channels stimulated Ca²⁺ responses in cervical DRG neurons, reduced scratching behavior in IMQ mice,³⁴⁹ attenuated IMQ-induced morphological changes in lesions, and controlled the infiltration of dermal neutrophils, dendritic cells, and Th17 cells.³⁵⁰ Activation of TRPM8 channels also significantly inhibited the generation of iNOS, cyclooxygenase (COX)-2, TNF-α, and IL-6, and prevented the phosphorylation of IκBα, NF-κB p65, ERK, JNK, and p38 MAPKs.³⁵¹ Thus, activation of TRPM8 channels is thought to be effective in ameliorating the symptoms of IMQ-induced pruritus and psoriasis-like inflammation, which make it a promising strategy for the treatment of psoriasis.

The level of 5-HT is higher in the skin of patients with psoriasis than that in healthy individuals.³⁵² Specific inhibition of TRPC4 immediately controlled the neuronal activity induced by α-methyl-5HT in dermal nerve preparations.³⁵³ Interestingly, visual signs of skin inflammation, such as erythema and desquamation, were significantly improved after TRPC4 inhibition and were accompanied by a reduction in epidermal thickness and inflammatory cell infiltration. This effect was achieved by silencing TRPC4 in the DRG and showed an effective reduction in psoriatic pruritus even after repeated applications of IMQ.³⁵⁴ Therefore, TRPC4 antagonists may soon be used clinically for the treatment of acute pruritus and psoriasis.

2019 coronavirus disease (COVID 19)

The COVID-19 pandemic, which is caused by severe acute respiratory disease coronavirus 2 (SARS-CoV-2), has resulted in hundreds of millions of confirmed cases worldwide.³⁵⁶ Calcium channel blockers are protective against death from SARS-CoV-2 infection and inhibit the infectivity of SARS-CoV-2 in epithelial lung cells.³⁵⁷ Given that coronaviruses require Ca²⁺ ions to enter host cells, Ca²⁺ are considered essential for viral entry into host cells.³⁵⁷ Interestingly, most TRP channels are highly permeable to Ca²⁺.^358,359 Therefore, the researchers hypothesized that TRP channels are involved in certain processes in COVID-19.

The report by Leidinger et al. points to the idea that TRPC6 channels are involved in physiology of normal human lungs and in COVID-19 pneumonia.³⁶⁰ They suggested that TRPC6 may exacerbate SARS-2-induced inflammation, but the molecular mechanism remains to be elucidated. Several patients with COVID-19 will develop acute respiratory distress syndrome (ARDS).^361,362 Khodadadi et al.³⁶³ found increased expression of TRP cation channels after the treatment of ARDS. Drugs may exert their protective effect against ARDS through TRPs.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) has also been defected in some COVID-19 patients.^364–366 Clinical studies have shown that TRPM3 channel activity is impaired in patients with post-COVID-19 conditions, which suggests impaired ion mobilization; such condition may impede cellular function and lead to chronic post-infection symptoms such as ME/CFS.³⁶⁷

Symptoms of COVID-19 associated with TRPA1/TRPV1 include the following: cough,³⁶⁸ smell, and taste disturbances,^369–372 loss of appetite,³⁷³ nausea,³⁷⁴ vomiting,³⁷⁵ inflammatory responses, and pain.⁴⁰ However, several clinical studies targeted TRP channels for the treatment of COVID-19. TRPA1 and TRPV1 may provide a breakthrough in the treatment of symptoms of COVID-19.

Given that a common denominator of COVID-19-related symptoms is impaired redox homeostasis, which is responsible for reactive oxygen species (ROS) accumulation, several TRP channels are also involved in oxidative stress. Bousquet et al.³⁷⁶ hypothesized the involvement of TRPA1 and TRPV1 in COVID-19. In another direction of thought, harmful stimuli activate TRPV1 and increase the release of SP and pro-inflammatory cytokines,³⁷⁷ which exacerbate the symptoms of patients infected with COVID-19.

TRPV4 channels are involved in the recruitment of neutrophils and macrophages during lung injury.³⁷⁸ In addition, TRPV4 has been associated with pulmonary edema and pulmonary fibrosis.³⁷⁹ Therefore, TRPV4 is a novel target for COVID-19 treatment.³⁸⁰ TRPs are involved in pathophysiological processes that largely overlap with the symptoms of COVID-19. Published papers on COVID-19 have proliferated, but the mechanisms underlying the disease and TRPs remain inconclusive. TRP channels represent promising targets for the relief of multiple symptoms of COVID-19.

Cough

Cough is a reflex action of the respiratory tract for clearing the upper airways.³⁸¹ It is globally prevalent across all age groups. Stimulation of bronchopulmonary vagal C-fibers by inflammatory mediators can also cause cough.^382,383 TRPV1 is expressed in the C-fiber sensory nerves of the vagus in the lungs and is involved in the afferent sensory loop of the cough reflex.³⁸⁴ Trevisani et al.³⁸⁵ noted the pathogenic role of TRPV1 in cough in animal experiments. Iodo-resiniferatoxin (a potent TRPV1 inhibitor) significantly reduced cough induced by inhalation of capsaicin or citric acid in conscious guinea pigs.³⁸⁶ Therefore, inhibition of TRPV1 expression is a potential target for cough treatment.

TRPV4 plays a role in increasing sensitivity in the afferent arm the cough reflex.³⁸⁷ Selective activation of TRPV4 caused depolarization of airway sensory nerves and cough symptoms in experimental animals. Conversely, TRPV4 antagonists can reverse this phenomenon.^388,389 In addition, the selective P2X3 antagonist AF-353 inhibits TRPV4-induced neural depolarization and coughing in animals and humans, which suggests that TRPV4 can cause ATP release and relieve cough.³⁸⁸ Thus, activation of TRPV4, through the release of ATP is likely to be a pathological cause of the cough.

The TRPA1 receptor (not activated by capsaicin) has become a topic of interest in the field of cough treatment because it is known to be activated by ligands such as acrid smoke.³⁹⁰ Birrell et al.³⁹¹ found in normal humans and guinea pigs that inhalation of cinnamaldehyde (a TRPA1 agonist) produced a dose-related increase in cough. Glenmark’s compound GRC 17536, a potent and specific TRPA1 antagonist, effectively suppressed the cough induced by a citric acid challenge in guinea pigs.³⁹² A clinical study was subsequently conducted but was terminated due to the lack of clinical efficacy.

TRPM2 may participate in oxidative stress at the cough center by regulating cough.³⁹³ In the case of TRPM8, menthol prevents coughing by activating TRPM8.³⁹⁴ Neurophysiological, immunohistochemical and molecular analyses suggest that the cough suppressant effect of peppermint occurs via a reflex initiated from nasal TRPM8.^395,396 Based on the properties of TRPs, TRPM8 is undoubtedly one of the targets of cough. Most clinical studies targeting TRPs for the treatment of cough have ended with no drug efficacy. Future research should focus on new strategies to targeting this channel.

Chronic obstructive pulmonary disease (COPD) and asthma

Asthma and COPD both cause airway obstruction and are associated with chronic inflammation of the airways;³⁹⁷ they are also very common respiratory diseases.^398,399 A key risk factor for developing respiratory disease is smoking.^400,401 The use of TRPV1 and TRPV4 antagonists inhibits cigarette smoke-induced ATP release from human bronchial epithelial cells and may be associated with COPD-influenced increases in pulmonary ATP.⁴⁰² Activation of TRPV4 and opening of pannexin 1 channels leads to ATP release, which contributes to the pathogenesis of COPD.⁴⁰³ Baxter et al. also noted that TRPV4 gene expression was increased in the lung tissue of COPD patients.⁴⁰² At the genetic level, 7 of the 20 single-nucleotide polymorphisms of TRPV4 tested have been associated with COPD, which suggests a potential relationship between TRPV4 protein structure and disease pathogenesis.⁴⁰⁴

Contraction of airway smooth muscle in human and guinea pig tissues was observed in vitro with TRPV4 agonists, and this condition may exacerbate asthma.⁴⁰⁵ McAlexander et al. suggested that activation of TRPV4 leads to airway constriction in humans and is entirely dependent on the production of cysteine leukotrienes.⁴⁰⁶ Therefore, activation of TRPV4 may exacerbate symptoms in patients with asthma.

Several endogenous TRPV1 agonists are present in diseased lungs, such as those affected by COPD and asthma. The products of lipoxygenase metabolism of arachidonic acid, such as 12-(S)- and 15-(S)-hydroperoxyeicosatetraenoic acid, are released during asthma attacks and have a stimulatory effect on TRPV1.⁴⁰⁷ Another endogenous agonist of TRPV1, anandamide, is also synthesized in the lungs.⁴⁰⁸ The increased sensitivity of patients with COPD or asthma to inhaled capsaicin may explain the involvement of TRPV1 in the exacerbation of symptoms of these diseases.⁴⁰⁹

In contrast to TRPV1 and TRPV4, little is known about TRPV2.⁴¹⁰ TRPV2 acts in the early stages of receptor-mediated phagocytosis by macrophages.⁴¹¹ Its relevance to alterations in macrophage responsiveness and host defense in COPD still needs to be determined.

Andre et al.⁴¹² demonstrated that bronchial ring constriction produced by cigarette smoke extracts and acrolein or crotonaldehyde was inhibited by TRPA1 inhibitors. Furthermore, TRPA1 activation and increased Ca²⁺ influx promoted neuropeptide release from the isolated guinea pig airway tissue. These data suggest that airway neurogenic inflammation is mediated via TRPA1 stimulation, and suggest that TRPA1 may play a role in the pathogenesis of COPD.

Notably, TRPC6 mRNA expression significantly increases in alveolar macrophages from COPD patients, whereas there was no difference has been observed in the expressions of other TRPC subfamily members.^413,414 TRPC6 is a potential candidate target for COPD, but its role in the regulation of macrophage function under physiological and pathophysiological conditions remains to be determined.

A novel truncated functional TRPM8 variant activated by cold and menthol has been identified in human bronchial epithelial cells.⁴¹⁵ Whether TRPM8 expression is altered in the airway nerves or airway epithelium of individuals with cold-induced asthma and disease exacerbation remains to be elucidated.⁴¹⁶

Other respiratory diseases

Idiopathic pulmonary fibrosis (IPF) is a prototype of chronic, progressive, and fibrotic lung disease.⁴¹⁷ Rahaman et al.⁴¹⁸ determined that TRPV4 activity was upregulated in lung fibroblasts from IPF patients, and mediated the onset of lung fibrosis. TRPV4 also plays an important role in the formation of pulmonary edema and acute lung injury.^378,419 TRPV4 agonists caused pulmonary endothelial permeability, which was inhibited by the non-selective TRP blocker ruthenium red and was absent in Trpv4 knockout mice.⁴²⁰ Evidence showing that activation of TRPV4 may be involved in disrupting the integrity of the epithelial and endothelial barriers suggests the possible role of TRPV4 in edema formation. Studies also reported that TRPC6 is involved in pulmonary edema formation.⁴²¹ Selective TRPC6 blockers were found to inhibit pulmonary ischemia-reperfusion-induced edema.⁴²²

A number of TRP channel modulators have entered clinical trials and significant progress has been made in describing the physiopathological function of TRP channels in the respiratory system.

TRPC

TRPC is involved in chemokine ligand 2-mediated neuroprotection. Specific blockade of TRPC1 and TRPC5 inhibited ERK/cAMP response element binding protein activation and provided neuroprotection.⁴²⁶ TRPCs have also been linked to Parkinson’s disease (PD) and Alzheimer’s disease (AD). TRPC1 overexpression may exert PD protection by mediating an inward calcium flow, inhibiting cytochrome c release in mitochondria,⁴²⁷ and reducing MPP-induced neurotoxicity and apoptosis.⁴²⁸ In AD, each member of the TRPC channel performs a different role. TRPC1, TRPC3, and TRPC4 are found to be involved in neuroprotection,^429,430 TRPC3 may be involved in the dysregulation of tau proteins in AD, and TRPC5 possibly participates is involved in neurodegeneration.⁴³¹

In addition to these disorders, the TRPC channels are involved in neurological disorders, such as autism, bipolar disorder (BD), and mental retardation.⁴³² Griesi-Oliveira et al.⁴³³ found that the sequence of the TRPC6 gene on chromosome 11 was disrupted in autistic patients, resulting in haploinsufficient function. Later, they studied the effects of TRPC6 disruption on pluripotent stem cell-derived cortical neuronal cells. They observed that alterations in neurons of autistic patients were associated with disrupted expression of TRPC6.⁴³² A behavioral study of Trpc6 knockout mice by Griesi et al. found no alterations in social or repetitive behavior, but a reduction in exploratory behavior may be associated with clinical signs of autism.⁴³³

Evidence from functional studies points to the involvement of TRPC3 and TRPC7 in BD.⁴³⁴ Clinical results demonstrated reduced TRPC7 expression in a subgroup of BD patients.⁴³⁵ Interestingly, this reduction was inversely correlated with the basal level of Ca²⁺ in B lymphocyte lines (BLCLs) from BD patients. Andreopoulos et al.⁴³⁶ treated BD rats with chronic lithium and TRPC3 protein levels were significantly reduced, whereas mRNA levels were not.⁴³⁷ In a study by Roedding et al. investigated whether TRPC3 expression and function were affected by oxidative stress in BD and control BLCLs; chronic mitochondrial-generated oxidative stress reduced TRPC3 protein levels and Ca²⁺ influx through these channels, but no significant difference was observed between the two groups.⁴³⁸ Thus, TRPC3 is probably not an ideal target for BD.

Depression is a common psychological disorder in the society and is also a neurological disorder.⁴³⁹ Hypericin acts as a non-selective neurotransmitter reuptake inhibitor and has antidepressant effects.⁴⁴⁰ It produces a partial antidepressant effect by activating TRPC6 channels, increasing sodium permeability in the presynaptic membrane, reducing the sodium gradient driving neurotransmitter reuptake through the transporter, and increasing the levels of monoamine neurotransmitters in the synaptic cleft.⁴⁴¹ To a certain extent, depression is also associated with brain-derived neurotrophic factors (BDNF).⁴⁴² TRPC channel-induced calcium transients regulate the density and morphology of dendritic spines in BDNF, to produce antidepressant effects.^441,443

TRPC channels play an important role in vasospasm in hemorrhagic stroke, neuronal death and survival in ischemic stroke, and thrombin-induced pathological changes in astrocytes, and triggering of stroke by affecting blood pressure and atherosclerosis.⁴⁴⁴ In Ca²⁺ imaging experiments, Ca²⁺ influx through TRPC6 activated MAPK signaling and promoted the neuroprotective chemokine CXCL1,⁴⁴⁵ which protects nerves following stroke. The unique channel characteristics and downstream pathways of TRPCs render them potential new targets for the treatment of neurological diseases.

TRPVs

TRPA1

The toxic effects of amyloid-β on astrocytes triggered by TRPA1 channel activation are key to the progression of AD.⁴⁶⁰ Furthermore, mitochondria have an important role in the pathogenesis of AD.⁴⁶¹ TRPA1 channels reduces the intracellular Ca²⁺ concentration, apoptosis and intracellular ROS, and this may have a role in the treatment of AD.⁴⁶² TRPA1 activity, if inhibited, will exacerbate astrocytic lesions, inhibit the production of pro-inflammatory cytokines as well as the activity of astrocytes and the transcription factors NF-κB and nuclear factor of calcineurin-activated T cells (NFAT), and play a key role in the pathogenesis of AD.¹⁹⁴

Prolonged activation of TRPA1 by trans-cinnamaldehyde leads to increased epileptic activity.⁴⁶³ In contrast, HC030031 (a TRPA1 antagonist) promotes anticonvulsant effects, and preventes TRPA1 upregulation during seizures.⁴⁶⁴

Increased NADPH oxidase and superoxide dismutase activities and TRPA1 endogenous agonist levels (hydrogen peroxide and 4-hydroxynonenal) were reported in a mouse model of progressive multiple sclerosis-experimental autoimmune encephalomyelitis.⁴⁶⁵ Thus, TRPA1 may be involved in depression and anxiety-like behaviors. TRPA1 also exerts tonic control and promotes anxiety and depression.⁴⁶⁶ Antagonists of TRPA1 are a potential drug category for the treatment of anxiety and mood disorders.

In hypoxia, endothelial TRPA1 channels cause vasodilation, which reduces ischemic damage, a set of phenomena thought to be associated with ischemic stroke.⁴⁶⁷ In combination with these findings, TRPA1 can be included as a candidate for therapeutic targets in neurological diseases.

TRPM

TRPM2-mediated cellular and molecular crosstalk with amyloid β peptide or oxidative damage results in the formation of AD lesions.⁴⁶⁸ TRPM2 plays an important role in mediating cell death induced by various oxidative stress-induced pathological factors, including ischemia and reperfusion, the neurotoxic amyloid β-peptide, and MPTP/MPP⁺, which lead to neuronal death in the brain.^324,469 Activation of TRPM2 alters intracellular ion homeostasis, which leads to abnormal initiation of various cell death pathways, making it an important mechanism in the pathogenesis of ischemic stroke and neurodegenerative diseases.⁴⁷⁰

Hazalin et al.⁴⁷¹ found that inhibition of TRPM4 expression at the mRNA and protein levels improved cognitive deficits in chronically underperfused rats, but did not affect their motor function. It has also been suggested that TRPM4 plays a key role in the treatment of ischemic stroke.⁴⁷² Chen et al.⁴⁷³ examined the link between TRPM4 and ischemic stroke through in vivo assessment and observed that inhibition of TRPM4 improved BBB integrity after reperfusion in ischemic stroke.

The TRP channel may represent a promising therapeutic strategy for neurological disorders. Future research and development should focus on the TRPC channel because it is more widely distributed in the nervous system and existing studies are not sufficiently advanced.

Cardiac hypertrophy and heart failure

Cardiac hypertrophy and heart failure are closely related. Ang II-induced cardiac hypertrophy is the key to the pathological process of heart failure.^475,476 Using siRNA, Onohara et al.⁴⁷⁷ demonstrated that TRPC3 and TRPC6 were required for Ang II-induced NFAT translocation in cultured rat myocytes, which is a key step in cardiac hypertrophy.⁴⁷⁸ Furthermore, Ang II stimulation resulted in a significant increase in apoptosis in cultured neonatal rat cardiomyocytes transfected with TRPC7, accompanied by a decrease in atrial natriuretic factor expression and destruction of actin fibers.⁴⁷⁹ Ang II/phenylephrine-infused TRPC3 transgenic mice showed increased activation of NFAT in vivo, cardiomyopathy, and increased hypertrophy following stimulation with neuroendocrine agonists or pressure overload.⁴⁸⁰ Mice lacking TRPC1 channels maintained cardiac function, which prevented cardiac hypertrophy, when subjected to hemodynamic stress and neurohormonal overload.⁴⁸¹ TRPV3 expression also increased in Ang II-induced cardiomyocyte hypertrophy in vitro, accompanied by an increased intracellular calcium ion concentration, which promoted the expression of calcineurin and phosphorylated CaMKII protein and enhanced nuclear translocation of NFATc3.⁴⁸²

Feng et al.⁴⁸³ observed clinically that TRPM4 currents were more than twofold larger in fibroblasts from heart failure patients than from controls, and this finding implies the possible role of TRPM4 in cardiac fibrosis in heart failure. Moreover, dietary activation of TRPV1 by capsaicin may prevent the cardiac hypertrophy caused by a high-salt diet by improving cardiac mitochondrial dysfunction.⁴⁸⁴

High blood pressure

High blood pressure is associated with vasoconstriction.^485,486 Evidence showing the involvement of TRP channels in the regulation of vasoconstriction is lacking. Furthermore, impaired TRPV1 signaling may contribute to the pathogenesis of hypertension.⁴⁸⁷ Anandamide activates TRPV1 on perivascular sensory nerves, which leads to the release of CGRP to reduce vasoconstriction.⁴⁸⁸ Anandamide also inhibits vasoconstriction by activating the TRPV1 receptor in the endothelium, which causes the acute release of NO.⁴⁸⁹

TRPV4 and TRPM4 also have a regulatory effects on systemic blood pressure. Plasma adrenaline concentrations and urinary excretion of catecholamine metabolites, which may reduce hypertension, greatly increased in Trpm4 knockout mice.⁴⁹⁰ Furthermore, in Trpv4 knockout mice, the odds of hypertension induced by inhibition of NOS were greater, which was mainly related to the involvement of TRPV4 in vasodilation,⁴⁹¹ this finding suggests the vasoprotective effect of endothelial TRPV4.

Atherosclerosis

Atherosclerosis is the process of plaque formation that leads to the development of a number of CVDs.^492,493 In addition, atherosclerosis may be associated with the apoptosis of macrophages.⁴⁹⁴ Tano et al. discovered that Trpc3 knockout mice exhibited reduced macrophage apoptosis and necrosis and increased collagen content.⁴⁹⁵ Specific inhibition of TRPA1 exacerbated oxidized low-density lipoprotein-induced lipid accumulation in macrophages, and atherosclerotic lesions and hyperlipidemia aggravated.⁴⁹⁶ Upregulation of TRPM2 enhanced 5-HT vascular reactivity during the development of atherosclerosis.⁴⁹⁷

Vascular complications

Autosomal dominant PKD is caused by mutations in PKD1 and PKD2 and often associated with vascular abnormalities.^498,499 PKD1 and PKD2 encode polycystins TRPP1 and TRPP2, respectively.¹¹⁹ Abnormal intracellular Ca²⁺ regulation associated with PKD2 haploinsufficiency reduces vascular smooth muscle cell contractions.⁵⁰⁰ This condition is essential for maintaining the structural and functional integrity of blood vessels.⁵⁰¹ Overall, these observations suggest that TRP channels may be an avenue for mitigating CVD.

Cancer

TRP channels play a key role in cancer progression.⁵⁰⁴ Changes in the expression of TRP channels have been found in a number of cancers, including prostate,⁵⁰⁵ breast,⁵⁰⁶ colorectal,⁵⁰⁷ pancreatic,⁵⁰⁸ liver,⁵⁰⁹ and gastric cancers.⁵¹⁰ Such finding was mainly due to the inhibition of cell migration and invasion,⁵¹¹ and tumor cell growth and proliferation caused by TRP channels.⁵¹² Researchers need to further elucidate the functional role of TRP channels in cancer and their changes.

Obesity

Obesity is characterized by excessive fat accumulation in the body.¹⁸¹ Notably, TRP channels are expressed in adipose tissue.⁵¹³ In response to exogenous stimulation, TRP channel receptors activate the sympathetic nervous system norepinephrine brown adipose tissue axis to enhance thermogenesis and thereby suppress obesity.⁵¹⁴ Oral capsaicin reduces abdominal fat⁵¹⁵ and enhances brown fat thermogenesis, white fat browning,⁵¹⁶ fatty acid β-oxidation,⁵¹⁷ and energy expenditure in obese patients.⁵¹⁸ The function of these TRP channels identifies them as potential targets for obesity.

Kidney disease

TRPC6 is expressed in glomerular podocytes. Increased expression levels of TRPC6 were observed in patients with focal segmental glomerulosclerosis (FSGS),⁵¹⁹ membranous nephropathies,⁵²⁰ minimal change nephrosis,⁵²¹ and diabetic nephropathy.⁵²² TRPC6 mutations are involved in FSGS at the genetic level.⁵²³ TRPC 6 plays a cytoprotective role in membranous nephropathy.⁵²⁴ Aberrant expression of TRPC6 channels may be an important causative factor in kidney disease, but its clinical application needs further study.

Metabolic syndrome

Metabolic syndrome is a common metabolic disorder that results from the increased prevalence of obesity.⁵²⁵ As mentioned earlier, TRP channels are involved in CVD and obesity. The dysfunction of TRP channels is likely to contribute to increased cardiovascular risk in patients with metabolic syndrome.⁵²⁶ Although no experimental evidence indicates that TRP channels directly affect metabolic syndrome, TRP channels can be investigated as a medicinal target because of their role in related disorders.

Diabetes

TRP channels are related to several aspects of the pathology of diabetes, including insulin release from pancreatic beta cells and secondary diseases such as diabetic neuropathy, nephropathy, and vasculopathy.⁵²⁷ Chung et al.⁵²⁸ observed that the gene expression of TRPC4 was upregulated by 22%, and the protein expressions of TRPC1 and TRPC6 were downregulated by 50% in blood vessels of diabetic patients compared with non-diabetic controls. They suggested that diabetes affected capacitive calcium entry by regulating TRPCs.⁵²⁸ In addition, activation of TRPV4 affects insulin secretion and influences the level of insulin mRNA in cells.⁵²⁹ TRP channels have distinct clinical potential for the treatment or prevention of diabetes and its complications. However, given that TRP channels have a number of conflicting indications, clinical drug development will face a number of challenges.

TRPV1 agonists and antagonists

Various TRPV1 antagonists have been evaluated at various stages of clinical trials for different indications. TRPV1 antagonists show promise for pain treatment. ABT-102, a structurally novel TRPV1 antagonist, has been tested in phase I clinical trials for pain associated with inflammation, tissue damage, and ischemia.⁵⁵⁸ ABT-102 was clinically well tolerated by repeated dosage but caused a short-term increase in the mean core body temperature in healthy volunteers.⁵⁵⁹ Phase II clinical trials have been completed for the use of AZD1386, also an antagonist of TRPV1, for neuropathic pain, dental pain, nonerosive reflux disease, and osteoarthritis.⁵⁶⁰ However, in patients with osteoarthritis and nonerosive reflux disease, the clinical trial was terminated because there was no significant reduction in pain was observed.^561,562

Capsaicin in chili peppers bestows the sensation of spiciness and is a common component found in the fruits of Capsicum.^157,563 It is a classical TRPV1 agonist. Studies have reported the beneficial effects of capsaicin acting on TRPV1 in obesity, cardiovascular and GI, various cancers, neurogenic bladder, and skin diseases.⁵⁶⁴

A randomized trial assessing the effects of TRPV1 antagonist SB705498 on pruritus induced by histamine and cowhage in healthy volunteers has progressed to phase I.⁵⁶⁵ However, the results of SB705498 on atopic dermatitis showed that it is not clinically relevant for pruritus due to dermatitis. SB705498 has also been investigated for the treatment of migraine (ClinicalTrials.gov: NCT00269022), toothache (ClinicalTrials.gov: NCT00281684) and rectal pain (ClinicalTrials.gov: NCT00461682), all of which have now completed Phase II clinical trials.⁵⁶⁶ The results of these clinical studies have not been published to date.

Phase II clinical trials of XEND0501 for the treatment of chronic cough and COPD (ClinicalTrials.gov: NCT022233699 and NCT022233686) have been completed. Although XEND0501 was well tolerated, it was not clinically meaningful for patients with refractory cough.⁵⁶⁷

PAC-14028 completed a Phase II clinical trial for the topical treatment of pruritus, rosacea, and atopic dermatitis (ClinicalTrials.gov: NCT02052531, NCT02052999 and NCT02583022, respectively).⁵⁶⁸ In 2019, the results of the clinical Phase IIb trial of PAC-14028 cream met safety and efficacy metrics.⁵⁶⁹ In addition to the TRPV1 antagonists mentioned above, numerous chemical, biological and natural agents, including NEO-6860, JNJ-39439335 and others, have been introduced in clinical trials.

TRPA1 antagonists

Given its involvement in pain, itching, and respiratory syndromes, TRPA1 has been pursued as a promising drug target.⁵⁷⁰ GDC-0334 is a chemical-derived drug that reduced TRPA1 agonist-induced skin blood flow, pain and pruritus in a phase I clinical trial (ClinicalTrials.gov: NCT03381144).⁵⁷¹ GDC-0334 provides a therapeutic basis for assessing TRPA1 inhibition as a clinical treatment for asthma.

In 2012, Glenmark selected GRC 17536 for clinical development as a TRPA1 antagonist. In September 2014, Glenmark announced positive results obtained in the Phase II clinical trial.³⁹² GRC 17536 had statistically significant and clinically relevant response in patients with moderate to severe painful diabetic neuropathy.⁵⁷² GRC 17536 remains the only TRPA1 antagonist to have completed a Phase IIa study to date.⁵⁷³

In 2015, Orion announced that the TRPA1 antagonist ODM108 (acetylene-containing compound) entered phase I clinical trials for neuropathic pain.⁵⁷⁰ However, complex non-linear pharmacokinetics emerged and the clinical trial was discontinued in 2016. A key point of progress in TRPA1 research is the efficient oral utilization of the substance. A problem with all TRPA1 antagonists developed to date is that they are highly lipophilic and relatively insoluble. Addressing this issue is necessary for the drug development involving TRPA1.

Regulation of TRPM8

The TRPM8 channel is responsible for the sensation of cold environmental temperatures, it is also clinically associated with pain, itching, and obesity. PF-05105679 is a moderately potent TRPM8 blocker that has been evaluated for the treatment of cold pain sensitivity.⁵⁷⁴ Winchester et al.⁵⁷⁵ observed in clinical trials of PF-05105679 that the compound showed significant pain inhibition in the cold press test and no effect on core body temperature was observed. These results suggest that TRPM8 plays a role in acute cold pain signaling, and its dose does not result in a lower than normal body temperature.

Menthol is a natural source of TRPM8 agonists. Topical menthol activates nociceptors on the skin and then desensitizes them.⁵⁷⁶ When topically applied, menthol can also act as a vasorelaxant, but the mechanism is unclear.⁵⁷⁷ Menthol is also used clinically for migraines, neuropathy, and muscle pain. It provides a cooling effect, leaving human subjects with a cool sensation on the skin and significantly less discomfort than ice.⁵⁷⁸

Another TRPM8-related compound, isopulegol (Phase II, ClinicalTrials.gov: NCT02193438), had no positive biomechanical or neurophysiological effects on patients with oral dysphagia due to stroke or neurodegenerative disease no significant adverse events were observed.⁵⁷⁹

Peppermint oil is another TRPM8 agonist of a natural origin. Winchester et al.⁵⁷⁵ investigated the role of peppermint oil in a mouse model of colitis. The results showed that peppermint oil, activated TRPM8 and the treated mice showed improvements in macroscopic and microscopic parameters and reduced weight loss during colitis.⁵⁷⁵ In addition, Peiris et al. observed that the use of TRPM8 agonist icilin for irritable bowel syndrome resulted in reduced release of inflammatory cytokines IL-1β, IL-6, and TNF-α in patient biopsies.⁵⁸⁰ These results suggest that specific activation of TRPM8 may alleviate experimental colitis and irritable bowel syndrome and may be a promising additional therapy for the treatment of IBD.

Other medicinal TRP uses

TRPV1, TRPA1, and TRPM8 are currently being studied in clinical trials, but clinical studies of other channels should not be overlooked. A TRPV4 inhibitor, GSK2798745 was launched by GlaxoSmithKline as a clinical candidate.⁵⁸¹ In 2019, GSK2798745 underwent its first clinical study (ClinicalTrials.gov: NCT02119260). Positive results were obtained; GSK2798745 was well tolerated in healthy volunteers and in patients with stable heart failure, which allowed further evaluation in long-term clinical studies of heart failure and in other indications.⁵⁸²

Preclinical studies have provided a certain rationale to support the development of TRPV3 antagonists for the treatment of inflammatory skin diseases, pruritus and pain.⁵⁸³ However, to date, only one TRPV3 inhibitor (GRC15300) has entered clinical trial targeting osteoarthritis and neuropathic pain.⁵⁸⁴ Moreover, by the end of 2013, these trials were discontinued. The Sanofi–Glimark agreement was terminated in 2014, and no further developments have been reported since then.⁵⁸⁴ Although numerous TRP channel drugs have entered clinical trials, only a few have been approved by the US Food and Drug Administration (FDA) (Table 4). In summary, researchers have made progress in understanding the role of TRPs in health and diseases. TRPs show promise as therapeutic targets for the treatment of inflammation, pain, itching, and other conditions.