Transient receptor potential (TRP) channels, in particular, are a large and diverse family of ion channel, which can sense a wide range of external and internal stimuli and trigger downstream physiological responses ^[1]. TRP channels located mostly on the plasma membrane of numerous animal cell types. Most of these are grouped into two broad groups: Group 1 includes TRPC (canonical), TRPV (vanilloid), TRPVL (vanilloid-like), TRPM (melastatin), TRPS (soromelastatin), TRPN (NO-mechano-potential, NOMP), and TRPA (ankyrin). Group 2 consists of TRPP (polycystic) and TRPML (mucolipin) ^[2]. They are a class of ion channels found in numerous tissues and cell types and are permeable to a wide range of cations such as Ca²⁺, Mg²⁺, Na⁺, K⁺, and others ^[3].

All TRP channel subunits are predicted to contain 6 transmembrane spanning domains (S1-S6). The pore region is formed by a stretch of hydrophobic residues between S5 and S6. With the exception of the Na⁺-selective TRPM4 and TRPM5, and the Ca²⁺-selective TRPV5 and TRPV6, all TRPs are non-selective cation channels ^[4]. The cytoplasmic termini of TRP subunits contain different structural and functional domains that vary between subfamilies ^[4]. A 25-amino acid motif, defined as TRP domain, located C-terminal to the sixth transmembrane segment is loosely conserved in almost all TRP channels, except TRPA1 and the distantly related TRPMLs and TRPPs ^[5]. A stretch of 6-amino acids within the TRP domain defined as TRP box is highly conserved among TRPCs (EWKFAR), but less conserved in TRPMs and TRPVs. The primary structure of TRPA1 lacks the canonical TRP box motif, but recent cryo-EM analysis revealed a domain adjacent to S6 that is topologically similar to the TRP domain in TRPV1 ^[6]. Another feature shared by TRPCs, TRPVs, and TRPA1 are N-terminal ankyrin repeat domains. Ankyrin repeats which generally function as a scaffold for protein-protein interactions may contribute to TRP channel regulation and subunit assembly ^{[7, 8, 9]}. Other structural features include coiled-coil domains, calmodulin and Ca²⁺ binding motifs, C-terminal PDZ-binding domains (TRPC4/C5), serine/threonine kinases (TRPM6/M7), and a Nudix hydrolase domain (TRPM2) ^[4]. While ankyrin repeats (except TRPM) and TRP domains are common to TRP subfamilies (TRPCs, TRPVs, TRPMs, and TRPA1) that are more closely related to Drosophila TRP, a large extracellular loop between S1 and S2 is a common feature of the two more distantly related TRP subfamilies, TRPMLs and TRPPs ^[10].

TRPV4 (PDB: 8FC8): Residue Q550, N474, T527 (Red Sphere in the 3D structure viewer） forms binding pocket for binding with GSK279, GSK101, RhoA and 4α–PDD ^[11]. TRPA1 antagonist has four binding sites, which contained the F909T46 and T874V47 mutants were referred to as the binding site 1. Similarly, the M911A mutant,48 due to the proximity to F909, was referred to as binding site 2, and the N855S mutant42 and G238K/N249S/K270N mutant49 were referred to as binding sites 4 and 3, respectively.^[12]. Nine unconserved amino acid residues in TM5 of the channel (L867, V875, L894, 175 P897, S900, I905, V942, S943, I946) are responsible for these species-specific effects, 176 without being the actual binding site for menthol. Instead, mutating two residues in TM5 177 (S873 and T874 in human; S876 and T877 in mouse) abolishes or strongly reduces menthol, 178 thymol and carvacrol sensitivity in both human and mouse TRPA1 ^[13]. The inhibitor HC067047 binds to a pocket conformed by residues from S2–S3 linker (xTRPV4‐D542), S4 (xTRPV4‐M583 and Y587) and S5 (xTRPV4‐D609 and F613). ^[14].

Commonly used blockers such as Ruthenium red or 2-APB (2-Aminoethoxydiphenyl borate) are highly nonspecific ^[21].

The residues of T550 in S4 and Y511 in S3 form binding site of capsaicin with TRPV1 ^[15]. The bilayer composition is known to be important in the regulation of TRPV1 with experimental evidence recording the effect of cholesterol ^[16]. and PIP2 ^[17]. Thus, in this respect, a limitation of this study is the use of a simple single-component model bilayer, which fails to consider the influence of membrane asymmetry, its rich composition, and thus, overall, the complexity of the real cell membrane. Likewise, we have used a truncated model that facilitated extensive sampling, prohibited otherwise if we had employed the full-length protein. However, it is known that residues in the N- and C-termini are required for capsaicin and RTX activation of TRPV1 ^[18]. Therefore, further simulations of capsaicin interactions with the full-length protein will be needed if a complete picture of the mechanism of activation is desirable. The intracellular domain of TRPV channels is a highly conserved region adjacent to the internal gate and essential for tetramerization and allosteric activation ^[19]. Allosteric modulators of TRPV1 activity are a class of non-competitive antagonists, which interfere with the allosteric mechanism that gates the channel, while uncompetitive antagonists act as open channel blockers preferentially binding to over-activated channels with minimal interaction with the physiologically working channels. The atomic detailed information provided in this study constitutes a fundamental step to understand TRPV1 chemical modulation at least for those compounds similar to capsaicin, which is at the core of TRP channel drug design ^[20].

Y745 (Black Sphere in the 3D structure viewer) at the menthol binding site is critical for inhibition mediated by SKF96365 of cold- and voltage-activated TRPM8 currents ^[22]. Thermodynamic mutant cycle analysis shows that E652 in TRPV1 outer pore specifically interacts with R12 and K22 in s-RhTx ^[23].