1.A.4 The Transient Receptor Potential Ca2+/Cation Channel (TRP-CC) Family
TRP (transient receptor potential) channels represent a superfamily of cation channels conserved from worms to humans (Vennekens et al. 2012). They comprise seven subfamilies (TRPC, TRPV, TRPM, TRPN, TRPA, TRPP, and TRPML) that can be consider to be in two classes, group I (TRPC/V/M/N/A) and TRPML (TRP MucoLipin) and TRPP (TRP Polycystin) making up group II (Fine et al. 2019). According to Latorre et al. (2009), TRP channels can be grouped into seven subfamilies based on their amino acid sequence homology: (1) the canonical or classic TRPs, (2) the vanilloid receptor TRPs, (3) the melastatin or long TRPs, (4) ankyrin (whose only member is the transmembrane protein 1 [TRPA1]), (5) TRPN after the nonmechanoreceptor potential C (nonpC), and the more distant cousins, the (6) polycystins and (7) mucolipins. Members of the VIC (1.A.1), RIR-CaC (2.A.3) and TRP-CC (1.A.4). Families have similar transmembrane domain structures, but very different cytosolic doman structures (Mio et al. 2008). Because of their role as cellular sensors, polymodal activation and gating properties, many TRP channels are activated by a variety of different stimuli and function as signal integrators (Latorre et al., 2009; Montell, 2005; Ramsey et al., 2006). These mammalian proteins have been tabulated revealing their accepted designations, activators and inhibitors, putative interacting proteins and proposed functions (Clapham, 2007). The founding members of the TRP superfamily are the TRPC (TRP canonical) channels, which can be activated following the stimulation of phospholipase C and/or depletion of internal calcium stores (Montell, 2005). TRPC channels regulate nicotine-dependent behavior (Feng et al., 2006). The intrinsic assembly domains that assure tetrameric TRP channel formation have been reviewed (Schindl and Romanin 2007). They also function by depolarising the membrane potential, which triggers the activation of voltage-gated Ca2+ channels. Structural differences in the extracellular portions, transmembrane domains and the cytoplasmic domains of TRPC channels have been reviewed (Guo and Chen 2019). Cryo-EM has led to an explosion of TRP structures in the last few years (Cao 2020). These structures have confirmed that TRP channels assemble as tetramers and resemble voltage-gated ion channels in their overall architecture. However, each TRP subtype is endowed with a unique set of soluble domains that may confer diverse regulatory mechanisms. TRP channel structures have revealed sites and mechanisms of action of numerous synthetic and natural compounds, as well as those for endogenous ligands such as lipids, Ca2+, and calmodulin (Cao 2020). The role of TRP channels on taste and pain perception has been reviewed (Aroke et al. 2020). Genomic variability in the TRPV1 gene has been associated with alterations in various pain conditions, and polymorphisms of the TRPV1 gene have been associated with alterations in salty taste sensitivity and salt preference (Aroke et al. 2020). The involvement of TRPM channels in human diseases has been reviewed (Jimenez et al. 2020). Interfacial binding sites for cholesterol are present on TRP ion channels (Lee 2019). It has been proposed that there is a close relationship between cholesterol binding and TRP channel function (Méndez-Reséndiz et al. 2020). The endogenous and pharmacological ligand-binding sites of TRP channels and their regulatory mechanisms have been reviewed (Zhao et al. 2021).
Alonso-Carbajo et al. 2017 reviewed the functions of these proteins in human vascular smooth muscle and cardiac striated muscle. The mammalian TRP superfamily includes at least 22 genes grouped into three major subfamilies based on sequence homology: TRPV (vanilloid), TRPC (canonical), and TRPM (melastatin). Three additional subfamilies (the 'distant TRPs'), TRPP (polycystin), TRPML (mucolipin), and TRPA bring the total number of TRP-related proteins to around 30. TRP proteins are six transmembrane-domain polypeptide subunits, and four subunits assemble in the plasma membrane to form functional channels. All TRP channels are cation permeable, and most are not selective for monovalent or divalent ions. However, TRPV5 and TRPV6, display specificity for Ca2+ ions, and TRPM4 and TRPM5 are highly selective for monovalent cations and impermeant to Ca2+. TRP channels are activated by stimuli including changes in pressure, temperature, osmolarity, and intracellular Ca2+. Fatty acids and receptor-dependent vasoconstrictor agonists also activate vascular TRP channels. Most channels assemble from four identical TRP subunits, but when multiple TRP subunits are coexpressed, heteromeric channels can form (Alonso-Carbajo et al. 2017). Over 75 structures of these proteins have been solved by cryo-EM (Madej and Ziegler 2018). They reveal a lack of an apparent general mechanism underlying channel opening and closing. Similarly, the structures reveal a surprising diversity in which chemical ligands bind TRP channels (Hilton et al. 2019). TRP channels in zhikong scallops (Chlamys farreri) are functional diversity (Peng et al. 2021).
The mammalian TRPM gene family can be subdivided into distinct categories of cation channels that are either highly permeable for Ca2+ (TRPM3/6/7), nonselective (TRPM2/8), or Ca2+ impermeable (TRPM4/5). TRPM6/7 are fused to alpha-kinase domains, whereas TRPM2 is linked to an ADP-ribose phosphohydrolase (Nudix domain). Phylogenetic evidence suggests that Nudix-linked channels represent an ancestral type of TRPM that is present in various phyla, ranging from protists to humans (Mederos y Schnitzler et al., 2008). The pore-forming segments of invertebrate TRPM2-like proteins display high sequence similarity to those of Ca2+-selective TRPMs. Restoration of only two 'ancient' pore residues in human TRPM2 (Q981E/P983Y) increased (4-fold) its permeability for Ca2+. Conversely, introduction of a 'modern' sequence motif into mouse TRPM7 (E1047Q/Y1049P) resulted in the loss of Ca2+ permeation and a linear TRPM2-like current-voltage relationship (Mederos y Schnitzler et al., 2008). A cooperative knock-on mechanism underpins Ca2+-selective cation permeation in TRPV channels (Ives et al. 2023).
Volatile anaesthetics (VAs) are the most widely used compounds to induce reversible loss of consciousness and maintain general anaesthesia during surgical interventions. VAs depress central nervous system functions mainly through modulation of ion channels in the neuronal membrane, including 2-pore-domain K+ channels, GABA, NMDA receptors and nociceptive and thermosensitive TRP channels expressed in the peripheral nervous system, including TRPV1, TRPA1, TRPM3 and TRPM8 (Kelemen et al. 2020). Comparison of structures determined of many Trp channels in the absence or presence of activating stimuli revealed similar constrictions in the central ion permeation pathway near the intracellular end of the S6 helices, pointing to a conserved cytoplasmic gate and suggesting that most available Trp channel structures represent non-conducting states (Huffer et al. 2020). Organisms consuming plants use TRP channel agonists as defense mechanisms (Gandhi et al. 2021). Since the expression pattern and ligand sensitivity of TRP channels varies between species, this presents an intriguing evolutionary adaptation to their specific habitat and life cycles. TRP channel levels influence symptoms of patients with pancreatic adenocarcinoma (Chelaru et al. 2022). The blockade of zinc translocation via the inhibition of the TRPC and TRPM channels was shown to be neuroprotective in brain disease (Hong et al. 2023).
The TRP-CC family includes a variety of channel/sensors that respond to temperature, touch, pain, osmolarity, pheromones, taste, and other stimuli (Clapham, 2003). It has also been called the store-operated calcium channel (SOC) family. These proteins are the prinicipal components in mechanosensitive channels in vertebrate hair cells (TRPA1; 1.A.4.6.1) and stretch-activated channels in various vertebrate cell types (TRPC1; 1.A.4.1.3) (Barritt and Rychkov, 2005). TRPA1 and TRPC1 may use different mechanisms of activation. (a) The functional TRPA1 channel is probably a tetramer that is composed of four identical TRPA1 polypeptide chains or a mixture of TRPA1 and another channel polypeptide. Each TRPA1 polypeptide has 17 ankyrin repeats at the cytoplasmic amino terminus. It is proposed that these are coupled to motor proteins or other regulatory proteins on the cytoplasmic face of the plasma membrane (Barritt and Rychkov, 2005). In response to the deflection of the mechanosensitive cilia bundle induced by sound, tension on the ankyrin repeat domains or changes in protein-protein interactions are altered and the channel opens to admit Ca2+ and other cations. (b) The functional TRPC1 channel is probably a tetramer that is composed of four identical TRPC1 polypeptides or a mixture of TRPC1 polypeptides and another polypeptide. Although each TRPC1 polypeptide contains 3 or 4 ankyrin domains at the N terminus, it is proposed that these are not directly involved in channel gating. In response to a stimulus, such as stretching of the membrane by an increase in the volume of the cell, the channel opens and admits Ca2+. It is possible that release of Ca2+ from the endoplasmic reticulum that is induced by thapsigargin also acts as a stimulus, which alters cell volume and therefore can activate TRPC1 through changes in tension of the phospholipid bilayer. The activation of TRP channels by polyunsaturated fatty acids has been examined, and residues involved have been identified (Riehle et al. 2018).
Prototypical members of the TRP-CC family include the Drosophila retinal proteins TRP and TRPL (Montell and Rubin, 1989; Hardie and Minke, 1993). The 81 aas integral membrane INAF-B protein forms a complex with TRP channels, and they stabilize each other (Cheng and Nash, 2007). SOC members of the family mediate the entry of extracellular Ca2+ into cells in response to depletion of intracellular Ca2+ stores (Clapham, 1996) and agonist stimulated production of inositol-1,4,5 trisphosphate (IP3). One member of the TRP-CC family, mammalian Htrp3, has been shown to form a tight complex with the IP3 receptor (TC #1.A.3.2.1). This interaction is apparently required for IP3 to stimulate Ca2+ release via Htrp3. The vanilloid receptor subtype 1 (VR1), which is the receptor for capsaicin (the 'hot' ingredient in chili peppers) and serves as a heat-activated ion channel in the pain pathway (Caterina et al., 1997), is also a member of this family, and is activated by cannabinoids (i.e., anandamide) and certain inflammatory metabolites of arachidonate such as prostaglandin E2 (Olah et al., 2001). The stretch-inhibitable non-selective cation channel (SIC) is identical to the vanilloid receptor throughout all of its first 700 residues, but it exhibits a different sequence in its last 100 residues. VR1 and SIC transport monovalent cations as well as Ca2+. VR1 is about 10x more permeable to Ca2+ than to monovalent ions. Ca2+ overload probably causes cell death after chronic exposure to capsaicin (McCleskey and Gold, 1999). Molecular characteristics and expression profiles of nine TRP channels in the brown planthopper, Nilaparvata lugens, have been described (Wang et al. 2021).
The proteins of the TRP-CC family exhibit the same topological organization with a probable KscA-type 3-dimensional structure (Dodier et al., 2004; Dohke et al., 2004). They consist of about 700-800 (VR1, SIC or ECaC) or 1300 (TRP proteins) amino acyl residues with six transmembrane spanners (TMSs) as well as a short hydrophobic 'loop' region between TMSs 5 and 6. This loop region may dip into the membrane and contribute to the ion permeation pathway (Hardie and Minke, 1993). An aspartate residue in the P-loop may form a ring of negative charges that modulate pore properties including ion selectivity and inhibitory characteristics (García-Martínez et al., 2000). VR1 forms homotetramers. In these respects, members of the TRP-CC family resemble those of the VIC family. When one member of the TRP-CC family, the IGF-regulated Ca2+ channel of Mus musculus (TC #1.A.4.2.4), was PSI-BLASTED, it retrieved a partial sequence of a Zea mays K+ channel protein (887 aas; gbY07632) that is clearly a member of the VIC family. The two homologous protein segments of 150 residues were 28% identical, 42% similar with a PSI-BLAST score (without iterations) of 2e6. This observation further suggests a common origin for certain domains in the TRP-CC and VIC families.
All members of the vanilloid family of TRP channels (TRPV) possess an N-terminal ankyrin repeat domain (ARD), which regulates calcium uptake and homeostasis. It is essential for channel assembly and regulation. The 1.7 Å crystal structure of the TRPV6-ARD revealed conserved structural elements unique to the ARDs of TRPV proteins. First, a large twist between the fourth and fifth repeats is induced by residues conserved in all TRPV ARDs. Second, the third finger loop is the most variable region in sequence, length and conformation. In TRPV6, a number of putative regulatory phosphorylation sites map to the base of this third finger. The TRPV6-ARD does not assemble as a tetramer and is monomeric in solution (Phelps et al., 2008). Voltage sensing in thermo-TRP channels has been reviewed by Brauchi and Orio, 2011. TRPV were named after the first identified member TRPV1, that is sensitive to the vanillylamide capsaicin. Six TRPV channel subtypes (TRPV1-6) are subdivided into the thermoTRPV (TRPV1-4) and the Ca2+-selective TRPV channels (TRPV5, TRPV6) (Seebohm and Schreiber 2021). They are not primarily voltage gated but have distinct properties and react to several endogenous ligands as well as different gating stimuli such as heat, pH, mechanical stress, and osmotic changes. Their physiological functions are diverse and tissue specific. They serve as sensors for different pain stimuli (heat, pressure, pH) and contribute to the homeostasis of electrolytes, the maintenance of barrier functions and the development of macrophages. Different aspects of their structures, different gating stimuli, expression patterns, physiological-pathological roles and modulating mechanisms of synthetic, natural and endogenous ligands have been reviewed (Seebohm and Schreiber 2021).
The transient receptor potential (TRP) family of ion channels participate in many signaling pathways. TRPV1 functions as a molecular integrator of noxious stimuli, including heat, low pH, and chemical ligands. The 19-A structure of TRPV1 determined by using single-particle electron cryomicroscopy exhibits fourfold symmetry and comprises two distinct regions: a large open basket-like domain, likely corresponding to the cytoplasmic N- and C-terminal portions, and a more compact domain, corresponding to the transmembrane portion (Moiseenkova et al., 2008). The assignment of transmembrane and cytoplasmic regions was supported by fitting crystal structures of the structurally homologous Kv1.2 channel and isolated TRPV1 ankyrin repeats into the TRPV1 structure. TRP channels assemble as tetramers and resemble voltage-gated ion channels in their overall architecture. But beyond the relatively conserved transmembrane core embedded within the lipid bilayer, each TRP subtype appears to be endowed with a unique set of soluble domains that may confer diverse regulatory mechanisms. TRP channel structures reveal sites and mechanisms of action of numerous synthetic and natural compounds, as well as those for endogenous ligands such as lipids, Ca2+, and calmodulin (Cao 2020). Ca2+-selective TRPV channel permeation occurs by a three-binding site knock-on mechanism, whereas a two-binding site knock-on mechanism is observed in non-selective TRPV channels (Ives et al. 2023). Each of the ion binding sites displays greater affinity for Ca2+ over Na+. As such, coupling to an extra binding site in the Ca2+-selective TRPV channels underpins their increased selectivity for Ca2+ over Na+ ions. Furthermore, analysis of all available TRPV channel structures shows that the selectivity filter entrance region is wider for the non-selective TRPV channels, slightly destabilizing ion binding at this site, which is likely to underlie mechanistic decoupling.
Most local anaesthetics used clinically are relatively hydrophobic molecules that gain access to their blocking site on the sodium channel by diffusing into or through the cell membrane. These anaesthetics block sodium channels and the excitability of neurons. Binshtok et al. (2007) tested the possibility that the excitability of primary sensory nociceptor (pain-sensing) neurons could be blocked by introducing the charged, membrane-impermeant lidocaine derivative QX-314 through the pore of the noxious-heat-sensitive TRPV1 channel (TC #1.A.4.2.1). They found that charged sodium-channel blockers can be targeted into nociceptors by the application of TRPV1 agonists to produce a pain-specific local anaesthesia. QX-314 applied externally had no effect on the activity of sodium channels in small sensory neurons when applied alone, but when applied in the presence of the TRPV1 agonist capsaicin, QX-314 blocked sodium channels and inhibited excitability (Binshtok et al., 2007).
The amino termini of TRP-CC proteins normally contain a proline-rich region and one or more ankyrin domains. VR1, for example, exhibits three such repeat domains in its amino terminal hydrophilic segment (432 amino acids). It also has a hydrophilic C-terminus that lacks recognizable motifs. The sequence similarity between VR1 and other TRP-CC family proteins is within and adjacent to the sixth TMS, including the hydrophobic 'loop' region. Unlike other TRP-CC family members, VR1 is not a SOC. Mammals appear to have multiple VR1 homologues. Fingerprint residues in TRP channels have been identified (Cabezas-Bratesco et al. 2022), revealing a conserved set of residues. This fingerprint is composed of twelve residues localized at equivalent three-dimensional positions in TRP channels from the different subtypes. These amino acids are arranged in three groups, connected by a set of aromatics located at the core of the transmembrane structure. Differences in the connectivities between these different groups of residues harbor the apparent differences in coupling strategies used by TRP subgroups (Cabezas-Bratesco et al. 2022).
One member of the TRP-CC family, TRP-PLIK (1862 aas; AF346629), has been implicated in the regulation of cell division. It has an N-terminal TRP-CC-like sequence and a C-terminal protein kinase-like sequence. It was shown to autophosphorylate and exhibits an ATP phosphorylation-dependent, non-selective, Ca2+-permeable, outward rectifying conductance (Runnels et al., 2001). Another long homologue, Melastatin, is associated with melanocytic tumor progression whereas another homologue, MTR1, is associated with Beckwith-Wiedemann syndrome and a predisposition for neoplasia. Each of these proteins may be present in the cell as several splice variants.
The rabbit kidney epithelial Ca2+ channel, ECaC, is a Ca2+-selective cation channel with monovalent cation transport activity sensitive to strong inhibition by low concentrations of Ca2+ or Mg2+. ECaC is >100 x more permeable to Ca2+ than Na+. Mutation of D542 to alanine (D542A) (not present in the TRP-CC homologue) abolishes Ca2+ permeation and divalent cation inhibition of monovalent cation permeation. The mutation does not inhibit the latter transport activity. The D542K mutation generates a nonfunctional channel. Thus, a single residue determines the characteristic cation selectivity of ECaC.
The ability to detect variations in humidity is critical for many animals. Birds, reptiles and insects all show preferences for specific humidities that influence their mating, reproduction and geographic distribution. Because of their large surface area to volume ratio, insects are particularly sensitive to humidity, and its detection can influence their survival. Two types of hygroreceptors exist in insects: one responds to an increase (moist receptor) and the other to a reduction (dry receptor) in humidity. Although previous data indicated that mechanosensation might contribute to hygrosensation, the cellular basis of hygrosensation and the genes involved in detecting humidity remain unknown. To understand better the molecular bases of humidity sensing,(Liu et al., 2007b) investigated several genes encoding channels associated with mechanosensation, thermosensing or water transport. They identified two Drosophila melanogaster transient receptor potential channels needed for sensing humidity: CG31284, named water witch (wtrw), which is required to detect moist air, and nanchung (nan), which is involved in detecting dry air. Neurons associated with specialized sensory hairs in the third segment of the antenna express these channels. Neurons expressing wtrw and nan project to central nervous system regions associated with mechanosensation (Liu et al., 2007b). The six TRP channels of dinoflagelates do not appear to be mechanoreceptors but rather are components of a mechanotransduction signaling pathway and may be activated via a PLC-dependent mechanism (Lindström et al. 2017).
TRP channels are calcium-permeable nonselective cation channels with six TMS domains and a putative pore loop between TMSs 5 and 6 (Hu et al., 2012). About 28 mammalian TRP channels have been identified, with different numbers of splicing variants for each channel gene. TRP channels have been classified into six different subgroups, including TRPV (1-6), TRPM (1-8), TRPC (1-7), TRPA1, TRPP (1-3), and TRPML (1-3), according to their sequence similarities. In general, TRP channels are involved in calcium handling (e.g., intracellular calcium mobilization and calcium reabsorption) and a broad range of sensory modalities, including pain, temperature, taste, etc. TRP channelopathies are part of important mechanisms in a variety of diseases such as neurodegenerative disorders, diabetes mellitus, inflammatory bowel diseases, epilepsy, cancer, etc. Several members of the TRP family, TRPV1-4, TRPM8, and TRPA1, also called 'ThermoTRPs,' are involved in the detection of temperature changes, thus acting as the molecular thermometers of our body. They are also polymodal nociceptors that integrate painful stimuli such as noxious temperatures and chemical insults. For example, the TRPV1 channel mediates thermal hyperalgesia and pain induced by capsaicin and acid. TRPA1 is a nociceptor that integrates many noxious environmental stimuli including oxidants and electrophilic agents. Gene deletion animals have been created to study the role of TRP channels in pain and nociception; involvement of TRPV1, TRPV3, TRPV4, and TRPA1 in nociception has been confirmed (Hu et al., 2012).
A class of ion channels that belongs to the transient receptor potential (TRP) superfamily and is present in specialized neurons are temperature detectors. These channels are classified into subfamilies, namely canonical (TRPC), melastatin (TRPM), ankyrin (TRPA), and vanilloid (TRPV). Some of these channels are activated by heat (TRPM2/4/5, TRPV1-4), while others by cold (TRPA1, TRPC5, and TRPM8) (Baez et al. 2014). These channels resemble voltage-dependent K+ channels, with their subunits containing six transmembrane segments that form tetramers. Thermal TRP channels are polymodal receptors that can be activated by temperature, voltage, pH, lipids, and agonists. Their high temperature sensitivity is due to a large enthalpy change ( approximately 100 kcal/mol), which is about five times the enthalpy change in voltage-dependent gating. Pi-helices in TRP channels probably function in gating (Zubcevic and Lee 2019).
TRPV cation channels are polymodal sensors involved in a variety of physiological processes. TRPV2 is regulated by temperature, ligands such as probenecid and cannabinoids, and lipids. It may play a role in somatosensation, osmosensation and innate immunity. Zubcevic et al. 2016 presented the atomic model of rabbit TRPV2 in its putative desensitized state, as determined by cryo-EM at 4 A resolution. TMS6 (S6), which is involved in gate opening, adopts a conformation different from the one observed in TRPV1. Structural comparisons of TRPV1 and TRPV2 indicate that a rotation of the ankyrin-repeat domain is coupled to pore opening via the TRP domain, and this pore opening can be modulated by rearrangements in the secondary structure of S6.
Plasma membrane ion channels, and in particular TRPC channels, need a specific membrane environment and association with scaffolding, signaling, and cytoskeleton proteins in order to play their important functional roles. TRPC proteins are incorporated into macromolecular complexes including Ca2+ signaling proteins and proteins involved in vesicle trafficking, cytoskeletal interactions, and scaffolding. Association of TRPC with calmodulin (CaM), IP3R, PMCA, Gq/11, RhoA, and a variety of scaffolding proteins has been demonstrated. The interactions between TRPC channels and adaptor proteins determines their modes of regulation as well as their cellular localizations and functions. Adaptor proteins are involved in assembling Ca2+signaling complexes, in the correct sub-cellular localization of protein partners, and in the regulation of TRPC channelosome.The S4 - S5 linker is the gear box of TRP channel gating, and many pathogenic mutations occur in this region (Hofmann et al. 2017). High resolution structures are known for TRPV1, TRPV2, TRPV6, TRPA1, and TRPP2 (Hofmann et al. 2017).
Mechanosensory transduction for senses such as proprioception, touch, balance, acceleration, hearing and pain relies on mechanotransduction channels, which convert mechanical stimuli into electrical signals in specialized sensory cells. There are two major models. One is the membrane-tension model: force applied to the membrane generates a change in membrane tension that is sufficient to gate the channel, as in bacterial MscL channels (TC# 1.A.22) and certain eukaryotic potassium channels (TC# 1.A.1). The other is the tether model: force is transmitted via a tether to gate the channel. The transient receptor potential (TRP) channel NOMPC is important for mechanosensation-related behaviours such as locomotion, touch and sound sensation across different species including Caenorhabditis elegans, Drosophila and zebrafish. NOMPC is the founding member of the TRPN subfamily, and is thought to be gated by tethering of its ankyrin repeat domain to microtubules of the cytoskeleton (Jin et al. 2017).
The generalized transport reaction catalyzed by TRP-CC family members is:
Ca2+ (out) ⇌ Ca2+ (in)
C+ and Ca2+ (out) ⇌ C+ and Ca2+ (in).