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date: 25 May 2019

Chloride Channels in Nociceptors

Abstract and Keywords

Pain may be induced by activation of various ion channels expressed in primary afferent neurons. These channels function as molecular sensors that detect noxious chemical, temperature, or tactile stimuli and transduce them into nociceptor electrical signals. Transient receptor potential channels are good examples because they are activated by chemicals, heat, cold, and acid in nociceptors. Anion channels were little studied in nociception because of the notion that anion channels might induce hyperpolarization of nociceptors on opening. In contrast, opening of Cl- channels in dorsal root ganglion (DRG) neurons depolarizes sensory neurons, resulting in excitation of nociceptors, thereby inducing pain. Anoctamin 1(ANO1)/TMEM16A is a Ca2+-activated Cl- channel expressed mainly in small DRG neurons, suggesting a nociception role. ANO1 is a heat sensor that detects heat over 44°C. Ano1-deficient mice elicit less nocifensive behaviors to hot temperatures. In addition, mechanical allodynia and hyperalgesia induced by inflammation or nerve injury are alleviated in Ano1-/- mice. More important, Ano1 transcripts are increased in chronic pain models. Bestrophin 1 (Best1) is another Ca2+-activated Cl- channel expressed in nociceptors. Best1 is increased in axotomized DRG neurons. The role of Best1 in nociception is not clear. GABAA receptors are in the central process of DRG neurons; GABA depolarizes the primary afferents. This depolarization consists of primary afferent depolarization essential for inhibiting nociceptive input to second-order neurons in the spinal cord, regulating pain signals to the brain. Thus, although Cl- channels in nociceptors are not as numerous as TRP channels, their role in nociception is distinct and significant.

Keywords: nociception, anoctamin 1, heat sensor, bestrophin 1, GABAA receptor, primary afferent depolarization

Introduction

Nociceptors are primary afferent neurons that deliver tissue damage signals from the periphery, such as skin, muscle, joint, or visceral organs, to the spinal cord. Nociceptors sense various harmful chemical, thermal, and mechanical stimuli and transmit the neural signals to higher order neurons in the spinal cord. In order to detect the noxious stimuli, the nociceptors are equipped with a wide variety of transduction channels. These channels are molecular sensors that detect specific stimuli and depolarize the nociceptor membrane. The depolarization of the nerve terminals generates receptor potentials that are converted to action potentials via the action of voltage-gated ion channels (Dubin & Patapoutian, 2010). Voltage-gated sodium, calcium, and potassium channels underlie action potential firing, synaptic transmission, and stabilization of the resting membrane potential and have therefore been considered as therapeutic targets (Waxman & Zamponi, 2014). Besides the voltage-gated channels, transient receptor potential (TRP) channels form a big family with diverse physiological functions, including pain transduction (Y. Lee, Lee, & Oh, 2005). TRP channel vanilloid 1 (TRPV1) and TRP cation channel subfamily A1 (TRPA1) are good examples because of their roles in nociception. Both are highly expressed in nociceptors and respond to natural irritants such as capsaicin in hot peppers and allyl isothiocyanates in wasabi and noxious heat and cold, respectively. In addition, because the two TRP channels mediate downstream signals of bradykinin (BK), they are considered molecular sensors for detecting pain-inducing stimuli (Dubin & Patapoutian, 2010). Thus, the majority of these ion channels involved in nociception are cation channels that depolarize the nerve terminals on stimulation.

Compared to cation channels, Cl- channels were not studied enthusiastically for their roles in nociception. This probably stems from the notion that the stimulation of Cl- channels contributes to hyperpolarization in nociceptors. In dorsal root ganglia (DRG) neurons, however, the opening of Cl- channels actually depolarizes the peripheral nerves (Carlton, Zhou, & Coggeshall, 1999; Price, Cervero, Gold, Hammond, & Prescott, 2009).

Transmembrane Cl- Concentration Gradients

The Cl- concentration gradient across the cell membrane determines the direction of Cl- flow in DRG neurons. The transmembrane Cl- concentration gradient is maintained mainly by NKCC1 (Na-K-2Cl cotransporter 1) and KCC2 (K-Cl cotransporter 2) (Payne, Rivera, Voipio, & Kaila, 2003; Price et al., 2009). NKCC1 transports Cl- into the cell, whereas KCC2 drives Cl- out of the cell. These cation–chloride cotransporters act without using adenosine triphosphate (ATP) because they are fueled by the concentration gradients of Na+ or K+. These cotransporters show cell type–specific expression patterns under the control of transcriptional regulation and alternative splicing during development (Blaesse, Airaksinen, Rivera, & Kaila, 2009). In DRG neurons, NKCC1 establishes intracellular Cl- accumulation higher than the electrochemical equilibrium, resulting in an outflow of Cl-. On the other hand, mature central nervous system (CNS) neurons maintain low intracellular Cl- concentrations by Cl- extrusion through KCC2, resulting in Cl- influx (Alvarez-Leefmans, 2009). The expression of NKCC1 is high in DRG neurons (Alvarez-Leefmans et al., 2001; Price, Hargreaves, & Cervero, 2006), especially in small and medium-size DRG and trigeminal ganglion neurons that express TRPV1, a well-known marker for nociceptors (Mao et al., 2012; Price et al., 2006). The expression of KCC2 in DRG neurons was also reported previously (Gilbert et al., 2007; Sung, Kirby, McDonald, Lovinger, & Delpire, 2000). In contrast, other studies reported that the expression of the K-Cl cotransporter is barely detectable in DRG neurons (Coull et al., 2003; Mao et al., 2012; Morales-Aza, Chillingworth, Payne, & Donaldson, 2004; Toyoda et al., 2005).

The measurement of intracellular Cl- concentration ([Cl-]i) was attempted in several laboratories using different techniques. In the early days, Alvarez-Leefmans and colleagues measured the intracellular Cl- activity of adult frog’s DRG neurons using Cl--selective microelectrodes. They estimated the Cl- activity for 23.6 mM (Alvarez-Leefmans, Gamino, Giraldez, & Nogueron, 1988). On measuring the reversal potential of GABA receptor, the [Cl-]i of cat DRG neurons was estimated to be 53 mM (Gallagher, Higashi, & Nishi, 1978). [Cl-]i measured with Cl--sensitive fluorescent dyes was 31 or 44.2 mM in rat DRG neurons (Alvarez-Leefmans, 2009; Kaneko, Putzier, Frings, & Gensch, 2002). The Alvarez-Leefmans group also estimated [Cl-]i in DRG neurons positive for IB4, a marker for small and nonpeptidergic DRG neurons. With a Cl--sensitive dye and changing the extracellular Cl- concentration, they found that DRG neurons contained 51 and 54 mM Cl- in IB4+ and IB4- neurons, respectively (Mao et al., 2012). Thus, [Cl-]i is relatively high in nociceptors.

The Cl- accumulation in DRG neurons is not static but dynamically modulated by changes in the expression of cotransporters or their functional activities via phosphorylation and membrane trafficking (Price et al., 2009). NKCC1 is phosphorylated at three sites in the N-terminal cytosolic domain, which is critical for NKCC1 function (Darman & Forbush, 2002). For example, treatment of inflammatory mediators enhances NKCC1 expression with increased phosphorylation and attenuates KCC2 expression, which suggests augmented excitatory Cl- current in sensory neurons, contributing to inflammatory hyperalgesia (Funk et al., 2008). Altered KCC2 and NKCC1 expressions were also observed in arthritis (Morales-Aza et al., 2004). Intracolonic capsaicin treatment induced phosphorylation and trafficking of NKCC1 in a visceral hyperalgesia model (Galan & Cervero, 2005). Taken together, to modulate neuronal excitabilities, activities of NKCC1 and KCC2 were tightly regulated via changes in their expression or function, which facilitated the development and maintenance of a persistent pain state.

Anoctamin 1 in Nociception

Many Cl- channels are known to be present in primary afferents, thereby regulate nociceptive pathways. Among these are anoctamin 1, bestrophin 1, and GABA receptor. However the functions of these Cl- channels are different as their expression loci are different. Depending on their expression in peripheral or central nerve terminal of primary afferents, the activation of these Cl- channels can excite or inhibit the nociceptive conduction pathway.

Calcium-Activated Cl- Channels

The Ca2+-dependent outward currents were first found in Xenopus laevis oocytes (Barish, 1983; Miledi, 1982). Transient outward currents are elicited by depolarization, amplified in the presence of external Ca2+ ion and abolished by Cl- depletion in the media. As a result, it was considered that these outward currents are carried by Cl-, which is activated by Ca2+. Since then, diverse functional and biophysical properties of Ca2+-activated Cl- channels (CaCCs) have been characterized in various cells across the species, from invertebrates to humans (Fuller, 2002). As their prime function, CaCCs are involved in fluid and electrolyte secretion in exocrine glands, such as the lacrimal gland, the parotid gland, the salivary gland, the pancreas, airway, and the intestines (Arreola, Melvin, & Begenisich, 1996; Evans & Marty, 1986; Marty, Tan, & Trautmann, 1984; Melvin, Arreola, Nehrke, & Begenisich, 2002; Tarran et al., 2002; Winpenny, Harris, Hollingsworth, Argent, & Gray, 1998). In addition, CaCCs regulate smooth muscle contraction in the airway (Janssen & Sims, 1995); blood pressure control in the vasculature (Large & Wang, 1996); and cardiac excitability (Zygmunt & Gibbons, 1992). CaCCs also contribute to neuronal excitability in sensory systems, such as vision (Lalonde, Kelly, & Barnes, 2008); taste (Taylor & Roper, 1994); olfaction (Reisert, Bauer, Yau, & Frings, 2003; Reuter, Zierold, Schroder, & Frings, 1998); and somatosensation (Frings, Reuter, & Kleene, 2000; Mayer, 1985).

The CaCCs are sensitive to intracellular Ca2+ so that they are well activated by relatively small intracellular Ca2+, less than 1.0 μM (Machaca, Qu, Kuruma, Hartzell, & McCarty, 2002). In addition, the activation of CaCCs by Ca2+ is voltage dependent. A small amount of Ca2+ can activate more channels at depolarization compared to those at hyperpolarization. Thus, the current–voltage relationship is outwardly rectifying. Among the halides, CaCCs in Xenopus oocytes elicit the permeability sequence I- > Br- > Cl- > F- (Qu & Hartzell, 2000). More important, because intracellular Ca2+ releasing from the endoplasmic reticulum (ER) activates CaCCs, many physiological ligands, such as ATP, acetylcholine, endothelin 1, histamine, and angiotensin II, are known to activate CaCCs (Guibert, Marthan, & Savineau, 1997; M. G. Lee, Zeng, & Muallem, 1997; Zholos et al., 2005). A few electrophysiological studies reported the presence of CaCCs in DRG neurons of different species (Bader, Bertrand, & Schlichter, 1987; Currie, Wootton, & Scott, 1995; Schlichter, Bader, Bertrand, Dubois-Dauphin, & Bernheim, 1989; Scott, McGuirk, & Dolphin, 1988). CaCCs are found in a subset of medium-size DRG neurons of mice (Andre et al., 2003). A subset of trigeminal ganglion neurons from the quail also elicited CaCC currents (Bader et al., 1987; Schlichter et al., 1989). Boudes and colleagues found augmented CaCC currents in DRG neurons after axotomy (Boudes et al., 2009). These reports thus hint at the involvement of CaCCs in nociception and other somatosensory sensation.

Anoctamin 1 as a CaCC

Because of the vast physiological functions of CaCCs, a vigorous search for the molecular identity was made by many researchers (Hartzell, 2008). In 2008, anoctamin 1 (ANO1), also known as TMEM16A, was identified as a CaCC (Caputo et al., 2008; Schroeder, Cheng, Jan, & Jan, 2008; Y. D. Yang et al., 2008). ANO1 is evolutionarily conserved from nematode to humans. The anoctamin gene family has 10 homologs (ANO1 to ANO10) in the vertebrates. ANO1 shows a typical activation pattern similar to those of native CaCCs, which includes Ca2+-dependent activation, voltage-dependent Ca2+ affinity, outwardly rectifying I-V curve, activation by endogenous G protein–coupled receptor (GPCR) ligands, and blockage by CaCC blockers (Y. D. Yang et al., 2008). ANO1 is broadly distributed to tissues such as the salivary gland, intestinal epithelia, smooth muscle, and sensory neurons (Pedemonte & Galietta, 2014). Thus, ANO1 appears to be involved in various physiological functions, including fluid secretion, gut motility, vascular smooth muscle contraction, and nociception. Moreover, ANO1 was found to be amplified in various cancers, including head and neck squamous cell carcinoma (Ayoub et al., 2010); breast cancer (Britschgi et al., 2013); and oral cancer (X. Huang, Gollin, Raja, & Godfrey, 2002), suggesting a role in tumorigenesis.

Anoctamin 1 is a Heat Sensor in Nociceptors

As CaCCs were observed in DRG neurons, the expression of ANO1 in DRG neurons was also studied (Cho et al., 2012). ANO1 is expressed mostly in small DRG neurons. A large portion (78%) of ANO1-positive DRG neurons are also positive for TRPV1. In addition, 58% of ANO1-positive neurons are co-localized with IB4 (Cho et al., 2012). In contrast, a small portion (22%) of ANO1+ neurons is co-localized with neurofilament M, a marker for myelinated fibers. Thus, ANO1 appears to be expressed largely in small sensory neurons, suggesting a possible link to nociception (Cho et al., 2012; Y. D. Yang et al., 2008).

Surprisingly, ANO1 was found to be activated by noxious heat. When heat was applied to the bath of ANO1-expressing human embryonic kidney (HEK) cells, robust currents were observed (Cho et al., 2012). The threshold temperature for ANO1 activation was estimated as 44°C, which is slightly over the threshold temperature (43°C) for pain in humans (LaMotte & Campbell, 1978). The temperature threshold of ANO1 is very similar to that of TRPV1 (Tominaga & Julius, 2000) and DRG neurons (Cesare & McNaughton, 1996). This heat-mediated activation is the direct effect of the ANO1 channel, rather than through other intracellular mediators, because this heat activation of ANO1 was still observed in isolated inside-out membrane patch or under intracellular Ca2+ free conditions (Cho et al., 2012). This heat-evoked activation was synergistic with Ca2+ and voltage. The temperature thresholds for ANO1 activation become lower with an increase in intracellular Ca2+. Namely, when the concentration of intracellular Ca2+ rises to 0.5 μM, body temperature can activate ANO1. This is important for nociception. As intracellular Ca2+ rises in inflammatory conditions, ANO1 can be activated at body temperature. Because ANO1 is expressed in small DRG neurons and is activated by heat over 44°C, its role in mediating acute thermal pain is expected. Indeed, various approaches to functional perturbation, such as ANO1 blockers, Ano1 knockdown with small interfering RNA treatment, and Ano1 conditional knockout (KO) in DRG neurons, showed a significant reduction in nocifensive behaviors in response to acute heat (Cho & Oh, 2013; Cho et al., 2012). Thus, ANO1 appears to mediate acute thermal pain (Figure 1).

Chloride Channels in NociceptorsClick to view larger

Figure 1. Chloride channels in nociceptors. ANO1 and Best 1 are thought to express in peripheral terminals of nociceptors, where they depolarize the cell membrane because of high Cl- concentration. ANO3 is also expressed in nociceptors. However, its function as a channel is in question. In central terminals of nociceptors, gamma-aminobutyric acid A (GABAA) receptors are present and also depolarize the cell membrane, which is known to inhibit synaptic transmission to second-order neurons in the dorsal horn of the spinal cord, probably because of inducing the desensitization of Cav channels.

Anoctamin 1 as a Pain Amplifier

In addition to acute thermal pain, ANO1 has the potential to contribute to chronic pain caused by various pathologic conditions. Genetic ablation of Ano1 in DRG neurons drastically reduced carrageenan-induced mechanical allodynia as well as thermal hyperalgesia (B. Lee et al., 2014). In addition, the genetic deletion of Ano1 also reduced BK- and formalin-induced nocifensive behaviors. The genetic ablation of Ano1 also alleviated neuropathic pain induced by a nerve injury (spared nerve injury model) (B. Lee et al., 2014). Liu and colleagues found that BK-induced nocifensive behaviors were significantly reduced by the knockdown of Ano1 (Liu et al., 2010). Formalin-induced pain behavior was alleviated by the treatment of ANO1 antagonists niflumic acid and CaCCinh-A01 (Garcia, Martinez-Rojas, Rocha-Gonzalez, Granados-Soto, & Murbartian, 2014). Interestingly, these blockers were effective in antinociceptive action when applied intrathecally (Garcia et al., 2014). Others also found that the intrathecal injection of T16Ainh-A01 and niflumic acid reduced tactile allodynia and thermal hyperalgesia induced by spinal nerve ligation (Pineda-Farias et al., 2015). These results clearly suggest that ANO1 in the central terminal contributes to the development or maintenance of chronic pain. However, how ANO1 in the central terminals of primary afferents contributes to inflammation-induced hyperalgesia is not known.

Upregulation of Ano1 in a Chronic Pain Condition

As many genes such as Trpv1 are dysregulated during chronic pain conditions (Hudson et al., 2001; Ji, Samad, Jin, Schmoll, & Woolf, 2002), Ano1 gene transcripts are also upregulated during chronic pain conditions. For example, formalin injection to the paws significantly increased the ANO1 expression in DRGs, which was reversed by the intrathecal injection of an ANO1 inhibitor, CaCCinh-A01 (Garcia et al., 2014). Similar results were also observed in a neuropathic pain model in rats. The ligation of spinal nerves upregulated the protein as well as the messenger RNA (mRNA) level of Ano1 in DRGs and in the spinal cord (Pineda-Farias et al., 2015). In addition, the nerve injury–induced upregulation of Ano1 was blocked by the intrathecal injection of T16Ainh-A01 or niflumic acid (Pineda-Farias et al., 2015). In an orofacial pain model, Yamagata and colleagues tested the effect of estrogen on capsaicin-induced nocifensive behaviors (Yamagata et al., 2016). In this model, a high dose of estrogen augmented the capsaicin-evoked nocifensive behaviors. Here, the estrogen treatment upregulated the Ano1 mRNA level in trigeminal ganglia. Thus, it is clear that nociceptive conditions upregulate Ano1 expression in sensory neurons. However, how the chronic pain conditions evoke the upregulation of Ano1 is not known.

Upstream Signals to ANO1 in Nociceptors

TRPV1. As ANO1 is activated by intracellular Ca2+, ANO1 function may be regulated by molecules that influence intracellular Ca2+ concentration. ANO1 is highly co-localized with TRPV1 in small DRG and trigeminal ganglion neurons (Cho et al., 2012; Kanazawa & Matsumoto, 2014). Thus, it is plausible to think that TRPV1 activation might lead to ANO1 activation. Indeed, two groups concurrently reported that an ANO1-specific blocker, T16Ainh-A01, significantly reduced capsaicin-evoked TRPV1 inward current or excitation of DRG neurons (Deba & Bessac, 2015; Takayama, Uta, Furue, & Tominaga, 2015). These results suggest a functional link between the two channels. Namely, the activation of TRPV1 by capsaicin caused a rise in intracellular Ca2+, which gates ANO1, leading to the augmentation of the capsaicin-induced depolarization of DRG neurons. Therefore, ANO1 can augment the excitatory action of TRPV1 on DRG neurons. This functional link between the two channels is largely based on the physical interaction between the two proteins. Indeed, Tominaga’s group demonstrated the physical interaction between ANO1 and TRPV1 (Takayama et al., 2015). Their interaction may also explain the effects of estrogen regulation on capsaicin-induced trigeminal pain. Trigeminal pain in women is likely to be exacerbated by a high level of estrogen (Yamagata et al., 2016). Thus, estrogen appears to augment nociception. In fact, ANO1 and TRPV1 expression are induced by estrogen, which further supports the functional link between ANO1 and TRPV1.

Other TRP channels are also functionally associated with ANO1 in physiological systems other than nociception. For example, TRPV4 works synergistically with ANO1 in salivary and lacrimal glands for fluid secretion (Derouiche, Takayama, Murakami, & Tominaga, 2018). TRPC1-mediated Ca2+ entry regulated ANO1 in salivary gland (Y. Sun, Birnbaumer, & Singh, 2015). TRPV6 coupled with ANO1 in epithelial principal cells of epididymis for Ca2+ homeostasis regulation (Gao da et al., 2016).

Bradykinin. Bradykinin, a potent endogenous algogenic substance, shows two typical types of nociceptive response: acute inflammatory pain and secondary hyperalgesic/allodynic responses (Dray & Perkins, 1993). Liu and his colleagues sequentially traced BK-induced acute nociceptive signaling (Liu et al., 2010). BK inhibits M-type K+ channels and activates ANO1 simultaneously via the phospholipase C (PLC)/inositol trisphosphate (IP3)–Ca2+ pathway. The removal of extracellular Ca2+ or the treatment with TRP channel blockers failed to abolish spontaneous and acute nocifensive responses to BK (Chuang et al., 2001; Wang et al., 2008). Previously, voltage-gated Ca2+ channels were thought to be coupled to CaCC currents in medium-to-large DRG neurons (Andre et al., 2003; Boudes & Scamps, 2012). However, opening of voltage-gated Ca2+ channels failed to activate ANO1 (Jin et al., 2013). Consistent with this, voltage-gated Ca2+ channels hardly contributed to the BK-induced Ca2+ increase (Liu et al., 2010). Thus, it is unlikely that a global Ca2+ rise via voltage-gated Ca2+ channels activates ANO1. Instead, local Ca2+ signals such as the GPCR-induced Ca2+ release from the ER might provide an optimal microenvironment for stimulating ANO1. ANO1 and IP3 receptor (IP3R) are located in close proximity (within 30 nm) in small DRG neurons (Jin et al., 2013). In addition, ANO1–GPCRs and ANO1–IP3R exist in close proximity in lipid rafts, suggesting their functional association in DRGs (Jin et al., 2013). Such a tight coupling enriches Ca2+ released near ANO1, which enables overcoming the low Ca2+ sensitivity of ANO1 in the micromolar range at physiological conditions.

Other Anoctamins in Nociception

Anoctamin 2 (ANO2; TMEM16B) is also considered a CaCC. ANO2 is activated by Ca2+, displaying distinct biophysical properties, such as the fast activation/deactivation kinetics and lower sensitivity to Ca2+ compared to those of ANO1 (Pifferi, Dibattista, & Menini, 2009; Schroeder et al., 2008; Scudieri, Sondo, Ferrera, & Galietta, 2012). Like ANO1, ANO2 is also activated by heat (Cho et al., 2012). However, the role of ANO2 in nociception is doubtful because it shows very low expression in DRG (Cho et al., 2012).

ANO3 (TMEM16C) is not considered a channel because its overexpression in a heterologous system failed to elicit any current (Duran, Qu, Osunkoya, Cui, & Hartzell, 2012; Tian, Schreiber, & Kunzelmann, 2012). However, because ANO3 is expressed in DRG and the spinal cord with a preference to IB4+, nonpeptidergic nociceptors, a role in nociception was suspected (F. Huang et al., 2013). Ano3 knockout rats displayed a significant increase in thermal and mechanical sensitivity with increased DRG neuronal excitability. Thus, ANO3 appears to suppress nociception in vivo. One possible molecular mechanism underlying the ANO3-induced inhibition of pain is its interaction with Slack, a Na+-dependent K+ channel. ANO3 and Slack channels are coexpressed in small DRG neurons. ANO3 interacts physically with the Slack channel (F. Huang et al., 2013). In addition, Slack-mediated K+ currents were reduced in DRG neurons isolated from Ano3 knockout rats (F. Huang et al., 2013). Therefore, ANO3 is found to promote Slack channel activity in DRG neurons, suggesting an indirect role in pain processing (F. Huang et al., 2013) (Figure 1).

Anoctamin 6 (ANO6; TMEM16F) functions as a Ca2+-dependent phospholipid scramblase, an enzyme that disrupts polarized phospholipids in membrane. Mutations in Ano6 are found in patients with a rare bleeding disorder, Scott syndrome (Castoldi, Collins, Williamson, & Bevers, 2011; Kunzelmann et al., 2014; H. Yang et al., 2012). Interestingly, ANO6 is highly expressed in microglia in the spinal cord. Microglia-specific Ano6 ablation inhibited mechanical hyperalgesia in neuropathic pain model, which was presumed to be associated with impaired microglial phagocytosis and motility (Batti et al., 2016). This provides a new regulatory role for ANO6 in microglial scavenger activity during nerve injury.

Bestrophin 1

Bestrophin 1 (Best 1) is present in the retinal pigment epithelium (Marmorstein et al., 2000; Petrukhin et al., 1998), whose mutation causes Best vitelliform macular dystrophy, a retinopathy with the degeneration of retinal pigmental epithelium (Marquardt et al., 1998; Petrukhin et al., 1998). Best 1 and 2 are activated by intracellular Ca2+ (Park et al., 2009; H. Sun, Tsunenari, Yau, & Nathans, 2002). In particular, Best 1 works as a glutamate-permeable CaCC in glia (Woo et al., 2012). Best 1 and Best 3 are also expressed in DRG neurons (Boudes et al., 2009). After axotomy of primary afferents, the mRNA level of Best1 is increased, but not Ano1 and Ano2 (Boudes et al., 2009). In contrast, this was not found in the spinal nerve ligation model of neuropathic pain (Pineda-Farias et al., 2015). In the DRG neurons of rat neuropathic pain models induced by L5/L6 spinal nerve ligation, the mRNA level of Best1 was not increased when compared to sham-operated rats. In this model, however, the Ano1 transcript and protein levels were increased in the neuropathic group compared to that of the sham-operated group (Pineda-Farias et al., 2015).

GABAA Receptor and Primary Afferent Depolarization

Numerous studies have reported the presence of gamma-aminobutyric acid A (GABAA) receptor, a Cl- channel, in DRG neurons. Earlier studies found that the application of GABA depolarized the cell bodies and peripheral and central processes of primary afferent fibers (Ault & Hildebrand, 1994; Bhisitkul, Kocsis, Gordon, & Waxman, 1990; Carlton et al., 1999; Gallagher et al., 1978; Labrakakis, Tong, Weissman, Torsney, & MacDermott, 2003; Nishi, Minota, & Karczmar, 1974). GABA depolarizes both A- and C-afferent fibers (Desarmenien, Feltz, Occhipinti, Santangelo, & Schlichter, 1984). The GABA-induced depolarization of primary sensory neurons confirms that the opening of Cl- channels cause Cl- egress because of higher intracellular Cl- concentration than the electrochemical equilibrium.

The depolarization of the central process of primary afferents was called primary afferent depolarization (PAD). It is well known that GABA induces PAD. It is also known that GABA inhibits the primary afferent inputs to the spinal cord (Alvarez-Leefmans, 2009). GABAA receptors are present in the presynaptic membrane of the central process of primary afferents (Bohlhalter, Weinmann, Mohler, & Fritschy, 1996; Labrakakis et al., 2003; Witschi et al., 2011). How the GABA-induced PAD inhibits nociceptive inputs is largely unknown. One of the leading theories is the desensitization of voltage-gated channels in the primary afferents that are necessary for the influx of Ca2+ to the axon terminals for releasing vesicles (Alvarez-Leefmans, 2009). When GABAA receptors are stimulated, they depolarize the membrane of the central process, which in turn desensitizes the voltage-gated Ca2+ channels, whose activation is essential for neurotransmitter release to the postsynaptic membrane. The source of GABA for presynaptic inhibition is thought to be inhibitory interneurons in the spinal cord (Alvarez-Leefmans, 2009). Therefore, the PAD induced by GABA achieves the inhibitory effect on the afferent inputs to the second-order neurons in the spinal cord (Figure 1).

Conclusion

A few Cl- channels are present in nociceptors. These Cl- channels can depolarize primary afferent fibers due to the efflux of Cl-, leading to the excitation of nociceptors. Among these channels, the role of ANO1 in nociception is well described. Present in small DRG neurons, ANO1 senses heat and thereby contributes to acute thermal pain. ANO1 also contributes to allodynia or hyperalgesia induced by inflammation or nerve injury. The transcript of Ano1 is increased by these chronic pain conditions. GABAA receptors are also present in primary afferents, including nociceptors. However, their actions are more or less limited to the central process of primary afferents. GABAA receptors provoke the primary afferent depolarization, which results in the inhibition of nociceptive inputs to the spinal cord. Best1 is another CaCC in sensory nerves. Its role in nociception is not clear as yet.

Acknowledgment

This study was supported by the National Research Foundation of Korea (NRF) (2011-0018358).

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