1.A.4 The Transient Receptor Potential Ca²⁺ 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). 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. 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). However, the precise mechanisms leading to TRPC activation remain unclear. TRPC channels regulate nicotine-dependent behavior (Feng et al., 2006).

The mammalian TRP superfamily of cation channels contains 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 have been proposed, bringing the total number of TRP-related proteins to around 30 (Earley S, Reading S, Brayden JE, 2007; PMID# 21204506). 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 (Earley et al., 2007).

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).

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.

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).

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 et al. (Brauchi and Orio, 2011).

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.

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.

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).

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).

The generalized transport reaction catalyzed by TRP-CC family members is:

Ca²⁺ (out) ⇌ Ca²⁺ (in)

or

C⁺ and Ca²⁺ (out) ⇌ C⁺ and Ca²⁺ (in).