1.1 TRPA1 gene and protein structure
Transient receptor potential ankyrin or TRPA1 is the only member of the TRPA subfamily(Clapham, Montell et al., 2003). It was first cloned in 1999 by Jacquemar et al ., from lung fibroblasts(Jaquemar, Schenker et al., 1999). TRPA1 gene comprises of 73635 bases, 29 exons and is present on chromosome 8th in the humans. Homologous genes of TRPA1 has been identified in both mammalian species like dog, non-human primates, cattle, pigs and non-mammalian species including birds, fishes, nematodes etc.(Talavera et al., 2020). In mammals, there is only one homologue of the TRPA1 gene but non-mammalian species do have more than one homologue of the TRPA1, for example, Drosophila has 4 homologues of TRPA1 gene and Zebrafish has 2 homologues of the TRPA1 gene(Nilius, Appendino et al., 2012). The TRPA1 protein consists of about 1100 amino acids with slight variation from one species to another (human TRPA1-1119 amino acids, mouse- 1115 amino acids and rat-1125 amino acids)(Nilius et al., 2012). The average molecular weight of TRPA1 protein is between 120kDa and 130kDa.
The TRPA1’s structural topology is very similar to other TRP proteins. TRPA1 is a homo- or hetero tetrameric non-selective cation channel. Each subunit of TRPA1 consists of six transmembrane alpha helices (S1 to S6), pre-S1 helix, linker domains connecting cytosolic domains to the transmembrane domain, β-sheets, TRP like domain and intracellular NH2 and COOH terminal (figure 1) (Cvetkov, Huynh et al., 2011). Paulsenet al . in 2015 used single particle electron cryo-microscopy (~4 Å resolution) to reveal a large part of structure of human TRPA1. A re-entrant pore loop is present between S5 and S6 helix with two restriction points or gates. The upper gate is restricted by two diagonally opposed Asp 915 to accommodate calcium ions, on the other hand, the lower gate consists of two hydrophobic seals formed by Ile957 and Val961, that constrain the entry of rehydrated cations(Paulsen et al. 2015). TRPA1 has a very long N-terminal array of ankyrin repeats, in fact, the longest ankyrin repeat domain (ARD) among the other invertebrate TRP channels, consisting of 14-18 ankyrin repeats (humans have 16 ARD whereas mouse has 14 ARD)(Gaudet, 2008; Story et al., 2003). Each ARD is 33 amino-acid long sequence, arranged as an anti-parallel helix turn helix structure. The N-terminal ankyrin repeats play a major role in the protein-protein interactions, provides elasticity to the channel structure and is involved in the membrane trafficking of the TRPA1. Indeed, ARD deletion accounts for low or no insertion of the TRPA1 in the plasma membrane(Nilius, Prenen et al., 2011). Chimeric studies have revealed that the AR domain can be divided into two parts i.e., a primary module composed of AR10 to AR15 and an enhancer module composed of AR 3 to AR8, contributing to the channel modulation in the mammals and snakes respectively(Cordero-Morales, Gracheva et al.,2012). Recently, it has been identified that the N-terminus, the pre-S1 helix and linker domain harbor the key cysteine and lysine residues (C622,C642,C666, K710), required for the channel activation by electrophilic and non-electrophilic agonists(Bahia et al., 2016; Samanta, Kiselar et al., 2018). In some of the non-mammalian species like snakes and insects, TRPA1 is activated by heat and low response has been observed to electrophilic activators(Gracheva et al., 2010; Sokabe, Tsujiuchi et al., 2008). Notably, in TRPA1, there is a pre S1 helix that connects ARD to the S1 region and is an important site where electrophilic agonist reacts with the key cysteine and lysine residues during interactions(Macpherson et al., 2007; Paulsen, Armache et al., 2015b). The TRP like domain, present right after the S6 helix at the C-terminus makes contact with non-contiguous structures including the pre-S1 helix, linker region in the N-terminus and S5-S6 linker region to regulate the allosteric modulation of the channel(Paulsen et al. 2015).