*2.1. TRP Channels Involved in Pain Sensation*

Transient receptor potential (TRP) channels are expressed in a variety of tissues throughout the body, such as skin, kidney, bladder, vascular smooth muscle cells and the nervous system [9]. The TRP channel family consists of six sub-families: TRPA (Ankyrin), TRPC (canonical), TRPM (melastatin), TRPML (mucolipin), TRPP (polycystin), and TRPV (vanniloid), encoded by a total of 28 genes [10]. The latter five can be further divided into subtypes: TRPC1-7, TRPM1-8, TRPML1-3,

TRPP1-3, and TRPV1-6 [11]. The broad variety of TRP channels allows sensing both noxious and innocuous signals [12]. Thus far, TRPV1-4 [12] and TRPM3 [13] channels have been implicated in the sensation of noxious heat. TRPA1, TRPC5 and TRPM8 channels have been suggested to detect noxious cold temperatures [14]. In addition, both TRPA1 and TRPV4 subtypes are thought to be involved in the detection of noxious mechanical stimuli [12], while TRPA1, TRPV1, TRPV3, TRPV4, TRPM8, and TRPC3 may contribute to the sensation of itch [15,16].

The aforementioned TRP channel subtypes are expressed in different types of peripheral sensory neurons such as dorsal root ganglion (DRG) and trigeminal ganglion (TG) neurons. TRPV1 channels and TRPM8 channels are mainly expressed on separate sets of neurons. TRPV1 channels can be found in C-fibers, whereas TRPM8 channels can be found on both fiber types transferring noxious signals (A*δ*- and C-fibers) [17]. Nevertheless, coexpression of TRPM8 and TRPV1 in one DRG neuron has been reported as well [18,19]. TRPA1 and TRPV1, in contrast, are mostly coexpressed in sensory neurons [17,19]. TRPV2 channels have been detected in A*δ*-fibers [12]. TRPV3 channels can be found on keratinocytes rather than sensory neurons [20], and TRPV4 channels are expressed in variety of tissues including the CNS and peripheral neurons [21]. TRPM3 channels are expressed in a large subset of DRG and trigeminal ganglion neurons. TRPM3 mRNA can be detected in approximately 80% of these sensory neurons at a level that is comparable to that of TRPA1 and TRPV1. However, only small-diameter neurons produce currents mediated by TRPM3 [22].

The role of TRPV1 channels as sensors of noxious heat is well established [23]. In general, TRP channels resemble the structure of voltage-gated K<sup>+</sup> (K*V*) channels. Each channel is made up of four subunits, each having six membrane spanning domains. Similar to K*<sup>V</sup>* channels, transmembrane domains 5 and 6 comprise the channel pore, whereas transmembrane domains 1–4 resemble a voltage sensor. Both N- and C-termini are found on the intracellular side [14] and harbor a number of regulatory domains. Channel trafficking and assembly is regulated by six so-called ankyrin repeats located at the N-terminus [24,25]. TRPV1 channels are not just activated by noxious heat, but also by voltage, binding of vanilloids, such as capsaicin, or high concentrations of H<sup>+</sup> ions [10]. As compared to K*<sup>V</sup>* channels, TRPV1 channels display a rather weak voltage sensitivity [26], which can be explained by the fact that the voltage-sensing transmembrane domains 1–4 remain fairly static during activation [26–28]. In addition, transmembrane domain 4 of TRPV1 channels contains a lower number of positively charged amino acids as compared to K*<sup>V</sup>* channels. Hence, an additional voltage-sensing segment might be required for TRP channels [14]. The gating in response to heat is regulated by the so-called TRP domain. However, this process remains incompletely understood [29]. The TRP domain spans 25 amino acid residues and is located immediately adjacent to transmembrane domain 6. It contains the TRP box, a stretch of conserved amino acid residues (WKFQR), which is a hallmark of TRP channels. The TRP domain is thought to be involved in a number of processes, like PIP2 binding or channel assembly, but the exact mechanism still needs to be fully elucidated [24]. As mentioned before, PIP2 is thought to regulate TRPV1 channel function, however it is still under debate if PIP2 is a positive or a negative regulator [30]. Cryo-EM studies in nanodiscs revealed the position of PIP2 in proximity to the vanilloid binding site. Binding of a vanilloid displaces a part of the PIP2 molecule, which reaches into the vanilloid binding pocket. The removal of the phosphoinositide is thought to lead to channel gating [26]. Such an effect would rather point towards a negative regulatory effect of PIP2.

The threshold value for classifying a thermal response as noxious was determined to be 43 ◦C [2]. TRPV1 channels activate at temperatures that exceed 43 ◦C, TRPV2 activate at even higher temperatures (>52 ◦C), whereas TRPV3 and TRPV4 channels gate in a temperature range between 26 ◦C and 34 ◦C [20]. Similar to TRPV1 channels, heterologously expressed TRPM3 channels activate at a temperature exceeding 40 ◦C [22]. Interestingly, mice lacking TRPV1 channels display a delayed nocifensive response only at temperatures exceeding 50 ◦C [31,32]. However, as compared to TRPV1 channels, the role of the other TRPV channels linked to the detection of noxious signals remains incompletely understood [12]. The role of TRPV2 to TRPV4 channels in detecting noxious signals remains debated [13], since both TRPV2 knock-out [33] and TRPV3/TRPV4 double knock-out [34]

animals retain normal thermal and mechanical sensation. The nocifensive response times of TRPM3 knock-out mice is prolonged at temperatures exceeding 52 ◦C [22]. Mice lacking both TRPV1 and TRPM3 show a significantly increased nocifensive response time already at 45 ◦C. However, some sensory neurons still produce currents in response to heat. Only a triple knock-out of TRPV1, TRPM3 and, interestingly, TRPA1 leads to a complete heat-insensitivity of sensory neurons. Furthermore, these mice were completely heat insensitive in behavioral tests [35].

The detection of both noxious and innocuous cold signals is suggested to involve TRPM8 and TRPA1 channels [23]. TRPM8 channels gate at temperatures below 25 ◦C [20]. Knock-out animals of TRPM8 channels lose the ability to detect cool temperatures, but retain the ability to detect noxious cold signals below 15 ◦C [13]. Hence, the role of TRPM8 channels as cold sensors is well established, but an additional set of ion channels needs to be involved in detecting noxious cold temperatures. TRPA1 channels are thought to be involved, but their role remains controversial [20]. Rodent TRPA1 channels were found to be gated by noxious cold temperatures, however, that function is lost in primate TRPA1 channels [36]. By contrast, human TRPA1 channels, reconstituted in lipid bilayers, were found to be activated by noxious cold temperatures [37]. In addition to these conflicting results, TRPA1 channels are usually expressed on the same set of neurons as TRPV1 channels, which appears counterintuitive [17]. Furthermore, animal studies involving TRPA1 knock-out mice point towards an insignificant role in the detection of noxious cold temperatures [20]. While their role in the detection of mechanical stimuli remains controversial as well, their contribution to the detection of noxious chemical signals is well established [12]. A large number of structurally unrelated electrophilic compounds can gate TRPA1 channels [13]. These compounds covalently modify one or more of the 31 cysteine residues, which causes channel opening [38].
