**1. Introduction**

The ability to sense elements of the natural environment (including temperature, pH, pressure, light, and noxious compounds) is critical for survival. Detection and response to environmental agents and stimuli are frequently mediated by receptor-type plasma membrane proteins, particularly ion channels that show versatile function in a range of organisms from prokaryotes to eukaryotes. Relative to G-protein coupled receptors (GPCRs), ion channels can directly impact neural excitation by both sensing natural stimuli and converting these signals into electrical changes to affect the polarization state of the plasma membrane.

In this review, we focus on several transient receptor potential (TRP) channels that are specifically activated by natural compounds and largely localize to primary sensory neurons. There are three types of nerves in primary sensory neurons, including Aβ-, Aδ-, and C-fibers. Aβ-fibers are myelinated afferent nerves that respond to innocuous mechanical stimuli. Aδ-fibers are also myelinated nerves, but alternatively this nervous pathway responds to rapid noxious stimuli. C-fibers are nonmyelinated nerves involved in slow pain [1]. The TRP channel superfamily comprises six subfamilies: TRP vanilloid (TRPV), canonical (TRPC), mucolipin (TRPML), polycystin (TRPP), ankyrin (TRPA), and melastatin (TRPM). Several TRP channels are expressed in small-size dorsal root ganglion (DRG) and trigeminal ganglion (TG) neurons (C fibers and Aδ fibers) [2]. While TRPV1 and TRPA1 are considered

to be the major receptors of this superfamily involved in nociception [3]. In particular, TRPV1 and sensitized TRPA1 are activated by heat and cold, respectively, and as such are important for detection of noxious temperature changes. Recently, TRPM3 involvement in heat sensation was also reported in mice [4].

The calcium-activated chloride channel, anoctamin 1 (ANO1, also known as TMEM16A) [5–7], was recently reported to be directly activated in DRG neurons by extremely rapid temperature changes that reach noxious ranges [8,9]. ANO1 can also be activated immediately downstream of Gq protein-coupled receptors (GqPCRs), including the bradykinin receptor, as evidenced by direct interaction of ANO1 with inositol trisphosphate (IP3) receptors on endoplasmic reticulum membranes [10]. Chloride channels typically function in neuronal suppression in the central nervous system, in part because intracellular chloride concentrations are maintained at low levels by the potassium–chloride co-transporter type 2 (KCC2). However, in DRG neurons, KCC2 expression is either absent or very low, whereas expression of the sodium–potassium–chloride co-transporter type 1 (which is an important molecule in the chloride intake pathway) is high [11]. Thus, chloride efflux through ANO1 activation is a key pathway for generation of neuronal excitation in many primary sensory neurons.

Here, we summarize the physiological significance of TRP and ANO1 channels. First, we describe current understanding of representative ion channels, namely TRPA1, TRPV1, and ANO1 (Part 1). Second, we discuss the multiple functions of TRP channels and ANO1 (Part 2). Finally, we propose the significance of those functions in clinical situations, including headache, migraine, and fungus infection (Parts 3 and 4).

## **2. Basic Understanding of Ion Channels in Primary Sensory Neurons**

#### *2.1. TRPA1*

#### 2.1.1. TRPA1 Activation by Natural Ligands

TRPA1 is activated by many natural ligands such as allyl isothiocyanate (AITC), tetrahydrocannabinol, cinnamaldehyde, allicin, diallyl sulfide, carvacrol, eugenol, gingerol, methyl salicylate, capsiate, thymol, propofol, 1,4-cineole, oleocanthal, and carbon dioxide, and by membrane extension and intracellular alkalization [12–26]. Moreover, TRPA1 is activated by calcium [27]. The magnitude of TRPA1 currents gradually increases during application of a given agonist, as observed for the lagging peak current induced after application of -eudesmol from hops to HEK293 cells expressing human TRPA1 [28]. Although the precise mechanism of TRPA1 activation remains unclear, covalent protein modification is involved. Carbons of AITC and *N*-methyl maleimide covalently bind to cysteine in the N-terminus of TRPA1 to enhance channel activation, whereas C-terminal lysine and arginine are important for AITC-mediated activation [29–31]. Menthol also has agonistic effects on human and mouse TRPA1, although the effects are bimodal [32,33]. The agonistic and antagonistic effects on mouse TRPA1 involves serine 876 and threonine 877 in the transmembrane (TM)-5 region [33]. Interestingly, G878 is also important for TRPA1-mediated cold sensitivity in rodents [34]. Rodent TRPA1 can be activated by cold stimulation and is involved in cold hyperalgesia after application of complete Freund's adjuvant (CFA) [35,36]. Although human TRPA1 does not show a cold response, it nonetheless responds to cold at approximately 18 ◦C if oxidization with dehydroxylation at proline 394 occurs [37].

## 2.1.2. TRPA1 in Pathological Conditions

TRPA1 activation induces hyperalgesia during inflammation because inflammatory factors (such as bradykinin released by tissue injury) activate and sensitize TRPA1 in DRG neurons. In this pathway, protein kinase A (PKA) and phospholipase C (PLC) are important for TRPA1 sensitization [38]. Adenosine triphosphate (ATP) is another important inducer in inflammatory pain. Pain sensations

are enhanced via a similar pathway through activation of purinergic P2Y receptors expressed in DRG neurons. Moreover, P2X receptors are involved in neuropathic pain via phospholipase A2 (PLA2) signaling [39], which activates protein kinase C (PKC), and in turn sensitizes TRPA1 [40].

TRPA1 expression increases after application of nerve growth factor (NGF) and is inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580. In DRG neurons, NGF released from inflamed tissue phosphorylates p38, which subsequently enhances TRPA1 expression [36]. Thus, the NGF–p38 MAPK–TRPA1 axis is one of the pathways that exacerbates TRPA1-mediated pain sensation in DRG neurons. For example, gastric distension-induced visceral pain relies on activation of both TRPA1 and p38 [41]. TRPA1 localization can be modified by pathological stimulation. TRPA1 localization to the plasma membrane is enhanced in forskolin-treated DRG neurons [42]. Further, in mice, full-length TRPA1 positively translocates to the plasma membrane by co-expression of a TRPA1 splicing variant [43]. There are two TRPA1 splicing variants: TRPA1a is the full-length protein whereas TRPA1b lacks exon 20, which encodes part of TM2 and the intracellular domain between TM2 and TM3. TRPA1b has no ion channel activity but instead enhances TRPA1a translocation to the plasma membrane. One-day of CFA treatment or partial sciatic nerve ligation (PSL) causes inflammatory and neuropathic pain, respectively. In both cases, TRPA1a expression levels increase transiently. Interestingly, TRPA1b expression levels significantly increase while TRPA1a expression reduces to basal levels at five days after CFA treatment or PSL. As such, up-regulation of TRPA1a translocation via TRPA1b overexpression causes a continuous pathological condition.

As with CFA, lipopolysaccharide (LPS) is also often used to induce the inflammatory condition. LPS can activate Toll-like receptor (TLR)-4, and cause subsequent release of multiple cytokines from immune cells [44]. The cytokine, tumor necrosis factor-alpha (TNF-α), enhances AITC-induced calcium increases in nodose and jugular ganglion neurons from rats [45]. However, a recent report suggested that LPS-induced calcium increases in nodose ganglion neurons from mice do not depend on TLR4, even although the responses are reduced in TRPA1 knockout mice [46]. Altogether, these results suggest that LPS directly activates TRPA1. Further, LPS increases single channel activity in TRPA1-expressing CHO cells. Ultimately, this novel relationship between bacteria and primary sensory nerves suggests that TRPA1 antagonists could be valuable for reducing pain induced by bacterial infections.

#### 2.1.3. TRPA1 Activation by Reactive Oxygen Species and Hypoxia

In addition to thermal stimuli and environmental agents, TRPA1 is activated by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) [47–49]. Responses to certain pathological conditions involving increased ROS synthesis (such as dysesthesia in ischemia and reperfusion of blood flow) are dependent on TRPA1 activity in mice [50]. Pain-related behavior due to dysesthesia is reduced by activation of prolyl hydroxylase (PHD)-2 involving hydroxylation at proline. Under normoxic conditions, TRPA1 steady status activity is maintained by PHD-mediated hydroxylation of proline 394, but under hypoxic conditions hydroxylation is inhibited and H2O2-induced TRPA1 activity is enhanced [50]. In contrast, high concentrations of oxygen also activate TRPA1 by directly modifying TRPA1 cysteines [51]. Collectively, these two functions allow TRPA1 to act as an oxygen sensor under both hypoxic and hyperoxic conditions.

Side effects of the anti-cancer agent oxaliplatin include induction of various dysesthesias, including peripheral nerve disorder and cold hyperalgesia. These dysesthesias are associated with enhanced TRPA1 expression in DRG and PHD-induced modification of TRPA1 [52–54]. Moreover, the oxaliplatin degradation product, oxalate, inhibits PHD and subsequently TRPA1 dehydroxylation, and also promotes cold hypersensitivity upon activation of TRPA1 in response to ROS production by mitochondria [37,55]. Mechanical allodynia associated with oxaliplatin treatment can be inhibited by the TRPA1 antagonist, ADM\_09 [56]. Together, these results clearly indicate the importance of the relationship between TRPA1 and the PHD cascade, and also that TRPA1 could be targeted as part of treatment for dysesthesia induced by ischemia and hypoxia, as well as drug-induced cold and mechanical hyperalgesia.
