Show Summary Details

Page of

PRINTED FROM OXFORD HANDBOOKS ONLINE ( © Oxford University Press, 2018. All Rights Reserved. Under the terms of the licence agreement, an individual user may print out a PDF of a single chapter of a title in Oxford Handbooks Online for personal use (for details see Privacy Policy and Legal Notice).

date: 02 June 2020

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


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 Nociceptors

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).


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.


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


Alvarez-Leefmans, F. J. (2009). Chloride transporters in presynaptic inhibition, pain and neurogenic inflammation. In F. J. Alvarez-Leefmans & E. Delpire (Eds.), Physiology and pathology of chloride transporters and channels in the nervous system (pp. 439–470). London, England: Academic Press.Find this resource:

Alvarez-Leefmans, F. J., Gamino, S. M., Giraldez, F., & Nogueron, I. (1988). Intracellular chloride regulation in amphibian dorsal root ganglion neurones studied with ion-selective microelectrodes. The Journal of Physiology, 406, 225–246. doi:10.1152/jn.01007.2007Find this resource:

Alvarez-Leefmans, F. J., Leon-Olea, M., Mendoza-Sotelo, J., Alvarez, F. J., Anton, B., & Garduno, R. (2001). Immunolocalization of the Na(+)-K(+)-2Cl(-) cotransporter in peripheral nervous tissue of vertebrates. Neuroscience, 104(2), 569–582.Find this resource:

Andre, S., Boukhaddaoui, H., Campo, B., Al-Jumaily, M., Mayeux, V., Greuet, D., … Scamps, F. (2003). Axotomy-induced expression of calcium-activated chloride current in subpopulations of mouse dorsal root ganglion neurons. Journal of Neurophysiology, 90(6), 3764–3773. doi:10.1152/jn.00449.2003Find this resource:

Arreola, J., Melvin, J. E., & Begenisich, T. (1996). Activation of calcium-dependent chloride channels in rat parotid acinar cells. The Journal of General Physiology, 108(1), 35–47.Find this resource:

Ault, B., & Hildebrand, L. M. (1994). GABAA receptor-mediated excitation of nociceptive afferents in the rat isolated spinal cord-tail preparation. Neuropharmacology, 33(1), 109–114.Find this resource:

Ayoub, C., Wasylyk, C., Li, Y., Thomas, E., Marisa, L., Robe, A., … Wasylyk, B. (2010). ANO1 amplification and expression in HNSCC with a high propensity for future distant metastasis and its functions in HNSCC cell lines. British Journal of Cancer, 103(5), 715–726. doi:10.1038/sj.bjc.6605823Find this resource:

Bader, C. R., Bertrand, D., & Schlichter, R. (1987). Calcium-activated chloride current in cultured sensory and parasympathetic quail neurones. The Journal of Physiology, 394, 125–148.Find this resource:

Barish, M. E. (1983). A transient calcium-dependent chloride current in the immature Xenopus oocyte. The Journal of Physiology, 342, 309–325.Find this resource:

Batti, L., Sundukova, M., Murana, E., Pimpinella, S., De Castro Reis, F., Pagani, F., … Heppenstall, P. A. (2016). TMEM16F regulates spinal microglial function in neuropathic pain states. Cell Reports, 15(12), 2608–2615. doi:10.1016/j.celrep.2016.05.039Find this resource:

Bhisitkul, R. B., Kocsis, J. D., Gordon, T. R., & Waxman, S. G. (1990). Trophic influence of the distal nerve segment on GABAA receptor expression in axotomized adult sensory neurons. Experimental Neurology, 109(3), 273–278.Find this resource:

Blaesse, P., Airaksinen, M. S., Rivera, C., & Kaila, K. (2009). Cation-chloride cotransporters and neuronal function. Neuron, 61(6), 820–838. doi:10.1016/j.neuron.2009.03.003Find this resource:

Bohlhalter, S., Weinmann, O., Mohler, H., & Fritschy, J. M. (1996). Laminar compartmentalization of GABAA-receptor subtypes in the spinal cord: An immunohistochemical study. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 16(1), 283–297.Find this resource:

Boudes, M., Sar, C., Menigoz, A., Hilaire, C., Pequignot, M. O., Kozlenkov, A., … Scamps, F. (2009). Best1 is a gene regulated by nerve injury and required for Ca2+-activated Cl- current expression in axotomized sensory neurons. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 29(32), 10063–10071.Find this resource:

Boudes, M., & Scamps, F. (2012). Calcium-activated chloride current expression in axotomized sensory neurons: What for? Frontiers in Molecular Neuroscience, 5, 35. doi:10.3389/fnmol.2012.00035Find this resource:

Britschgi, A., Bill, A., Brinkhaus, H., Rothwell, C., Clay, I., Duss, S., … Bentires-Alj, M. (2013). Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling. Proceedings of the National Academy of Sciences of the United States of America, 110(11), E1026–E1034. doi:10.1073/pnas.1217072110Find this resource:

Caputo, A., Caci, E., Ferrera, L., Pedemonte, N., Barsanti, C., Sondo, E., … Galietta, L. J. (2008). TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science, 322(5901), 590–594. doi:10.1126/science.1163518Find this resource:

Carlton, S. M., Zhou, S., & Coggeshall, R. E. (1999). Peripheral GABA(A) receptors: Evidence for peripheral primary afferent depolarization. Neuroscience, 93(2), 713–722.Find this resource:

Castoldi, E., Collins, P. W., Williamson, P. L., & Bevers, E. M. (2011). Compound heterozygosity for 2 novel TMEM16F mutations in a patient with Scott syndrome. Blood, 117(16), 4399–4400. doi:10.1182/blood-2011-01-332502Find this resource:

Cesare, P., & McNaughton, P. (1996). A novel heat-activated current in nociceptive neurons and its sensitization by bradykinin. Proceedings of the National Academy of Sciences of the United States of America, 93, 15435–15439.Find this resource:

Cho, H., & Oh, U. (2013). Anoctamin 1 mediates thermal pain as a heat sensor. Current Neuropharmacology, 11(6), 641–651. doi:10.2174/1570159x113119990038Find this resource:

Cho, H., Yang, Y. D., Lee, J., Lee, B., Kim, T., Jang, Y., … Oh, U. (2012). The calcium-activated chloride channel anoctamin 1 acts as a heat sensor in nociceptive neurons. Nature Neuroscience, 15(7), 1015–1021. doi:10.1038/nn.3111Find this resource:

Chuang, H. H., Prescott, E. D., Kong, H., Shields, S., Jordt, S. E., Basbaum, A. I., … Julius, D. (2001). Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition. Nature, 411(6840), 957–962.Find this resource:

Coull, J. A., Boudreau, D., Bachand, K., Prescott, S. A., Nault, F., Sik, A., … De Koninck, Y. (2003). Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain. Nature, 424(6951), 938–942. doi:10.1038/nature01868Find this resource:

Currie, K. P., Wootton, J. F., & Scott, R. H. (1995). Activation of Ca(2+)-dependent Cl- currents in cultured rat sensory neurones by flash photolysis of DM-nitrophen. The Journal of Physiology, 482(Pt. 2), 291–307.Find this resource:

Darman, R. B., & Forbush, B. (2002). A regulatory locus of phosphorylation in the N terminus of the Na-K-Cl cotransporter, NKCC1. The Journal of Biological Chemistry, 277(40), 37542–37550. doi:10.1074/jbc.M206293200Find this resource:

Deba, F., & Bessac, B. F. (2015). Anoctamin-1 Cl(-) channels in nociception: Activation by an N-aroylaminothiazole and capsaicin and inhibition by T16A[inh]-A01. Molecular Pain, 11, 55. doi:10.1186/s12990-015-0061-yFind this resource:

Derouiche, S., Takayama, Y., Murakami, M., & Tominaga, M. (2018). TRPV4 heats up ANO1-dependent exocrine gland fluid secretion. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology, fj201700954R. doi:10.1096/fj.201700954RFind this resource:

Desarmenien, M., Feltz, P., Occhipinti, G., Santangelo, F., & Schlichter, R. (1984). Coexistence of GABAA and GABAB receptors on A delta and C primary afferents. British Journal of Pharmacology, 81(2), 327–333.Find this resource:

Dray, A., & Perkins, M. (1993). Bradykinin and inflammatory pain. Trends in Neurosciences, 16(3), 99–104.Find this resource:

Dubin, A. E., & Patapoutian, A. (2010). Nociceptors: The sensors of the pain pathway. The Journal of Clinical Investigation, 120(11), 3760–3772. doi:10.1172/jci42843Find this resource:

Duran, C., Qu, Z., Osunkoya, A. O., Cui, Y., & Hartzell, H. C. (2012). ANOs 3–7 in the anoctamin/Tmem16 Cl- channel family are intracellular proteins. American Journal of Physiology. Cell Physiology, 302(3), C482–C493. doi:10.1152/ajpcell.00140.2011Find this resource:

Evans, M. G., & Marty, A. (1986). Calcium-dependent chloride currents in isolated cells from rat lacrimal glands. The Journal of Physiology, 378, 437–460.Find this resource:

Frings, S., Reuter, D., & Kleene, S. J. (2000). Neuronal Ca2+-activated Cl- channels—Homing in on an elusive channel species. Progress in Neurobiology, 60(3), 247–289.Find this resource:

Fuller, C. M. (2002). Calcium-activated chloride channels (Vol. 53). New York, NY: Academic Press.Find this resource:

Funk, K., Woitecki, A., Franjic-Wurtz, C., Gensch, T., Mohrlen, F., & Frings, S. (2008). Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons. Molecular Pain, 4, 32. doi:10.1186/1744-8069-4-32Find this resource:

Galan, A., & Cervero, F. (2005). Painful stimuli induce in vivo phosphorylation and membrane mobilization of mouse spinal cord NKCC1 co-transporter. Neuroscience, 133(1), 245–252.Find this resource:

Gallagher, J. P., Higashi, H., & Nishi, S. (1978). Characterization and ionic basis of GABA-induced depolarizations recorded in vitro from cat primary afferent neurones. The Journal of Physiology, 275, 263–282.Find this resource:

Gao da, Y., Zhang, B. L., Leung, M. C., Au, S. C., Wong, P. Y., & Shum, W. W. (2016). Coupling of TRPV6 and TMEM16A in epithelial principal cells of the rat epididymis. The Journal of General Physiology, 148(2), 161–182. doi:10.1085/jgp.201611626Find this resource:

Garcia, G., Martinez-Rojas, V. A., Rocha-Gonzalez, H. I., Granados-Soto, V., & Murbartian, J. (2014). Evidence for the participation of Ca(2+)-activated chloride channels in formalin-induced acute and chronic nociception. Brain Research, 1579, 35–44. doi:10.1016/j.brainres.2014.07.011Find this resource:

Gilbert, D., Franjic-Wurtz, C., Funk, K., Gensch, T., Frings, S., & Mohrlen, F. (2007). Differential maturation of chloride homeostasis in primary afferent neurons of the somatosensory system. International Journal of Developmental Neuroscience: The Official Journal of the International Society for Developmental Neuroscience, 25(7), 479–489. doi:10.1016/j.ijdevneu.2007.08.001Find this resource:

Guibert, C., Marthan, R., & Savineau, J. P. (1997). Oscillatory Cl- current induced by angiotensin II in rat pulmonary arterial myocytes: Ca2+ dependence and physiological implication. Cell Calcium, 21(6), 421–429.Find this resource:

Hartzell, C. (2008). CaCl-ing channels get the last laugh. Science, 322, 534–535.Find this resource:

Huang, F., Wang, X., Ostertag, E. M., Nuwal, T., Huang, B., Jan, Y. N., … Jan, L. Y. (2013). TMEM16C facilitates Na(+)-activated K+ currents in rat sensory neurons and regulates pain processing. Nature Neuroscience, 16(9), 1284–1290. doi:10.1038/nn.3468Find this resource:

Huang, X., Gollin, S. M., Raja, S., & Godfrey, T. E. (2002). High-resolution mapping of the 11q13 amplicon and identification of a gene, TAOS1, that is amplified and overexpressed in oral cancer cells. Proceedings of the National Academy of Sciences of the United States of America, 99(17), 11369–11374.Find this resource:

Hudson, L. J., Bevan, S., Wotherspoon, G., Gentry, C., Fox, A., & Winter, J. (2001). VR1 protein expression increases in undamaged DRG neurons after partial nerve injury. The European Journal of Neuroscience, 13(11), 2105–2114.Find this resource:

Janssen, L. J., & Sims, S. M. (1995). Ca(2+)-dependent Cl- current in canine tracheal smooth muscle cells. The American Journal of Physiology, 269(1, Pt. 1), C163–C169. doi:10.1152/ajpcell.1995.269.1.C163Find this resource:

Ji, R. R., Samad, T. A., Jin, S. X., Schmoll, R., & Woolf, C. J. (2002). p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron, 36(1), 57–68.Find this resource:

Jin, X., Shah, S., Liu, Y., Zhang, H., Lees, M., Fu, Z., … Gamper, N. (2013). Activation of the Cl- channel ANO1 by localized calcium signals in nociceptive sensory neurons requires coupling with the IP3 receptor. Science Signaling, 6(290), ra73. doi:10.1126/scisignal.2004184Find this resource:

Kanazawa, T., & Matsumoto, S. (2014). Expression of transient receptor potential vanilloid 1 and anoctamin 1 in rat trigeminal ganglion neurons innervating the tongue. Brain Research Bulletin, 106, 17–20. doi:10.1016/j.brainresbull.2014.04.015Find this resource:

Kaneko, H., Putzier, I., Frings, S., & Gensch, T. (2002). Determination of intracellular chloride concentration in dorsal root ganglion neurons by fluorescence lifetime imaging (Vol. 53). San Diego, CA: Academic Press.Find this resource:

Kunzelmann, K., Nilius, B., Owsianik, G., Schreiber, R., Ousingsawat, J., Sirianant, L., … Heemskerk, J. W. (2014). Molecular functions of anoctamin 6 (TMEM16F): A chloride channel, cation channel, or phospholipid scramblase? Pflugers Archive: European Journal of Physiology, 466(3), 407–414. doi:10.1007/s00424-013-1305-1Find this resource:

Labrakakis, C., Tong, C. K., Weissman, T., Torsney, C., & MacDermott, A. B. (2003). Localization and function of ATP and GABAA receptors expressed by nociceptors and other postnatal sensory neurons in rat. The Journal of Physiology, 549(Pt. 1), 131–142.Find this resource:

Lalonde, M. R., Kelly, M. E., & Barnes, S. (2008). Calcium-activated chloride channels in the retina. Channels (Austin), 2(4), 252–260.Find this resource:

LaMotte, R. H., & Campbell, J. N. (1978). Comparison of responses of warm and nociceptive C-fiber afferents in monkey with human judgments of thermal pain. Journal of Neurophysiology, 41(2), 509–528. doi:10.1152/jn.1978.41.2.509Find this resource:

Large, W. A., & Wang, Q. (1996). Characteristics and physiological role of the Ca(2+)-activated Cl- conductance in smooth muscle. The American Journal of Physiology, 271(2, Pt. 1), C435–C454. doi:10.1152/ajpcell.1996.271.2.C435Find this resource:

Lee, B., Cho, H., Jung, J., Yang, Y. D., Yang, D. J., & Oh, U. (2014). Anoctamin 1 contributes to inflammatory and nerve-injury induced hypersensitivity. Molecular Pain, 10(1), 5. doi:10.1186/1744-8069-10-5Find this resource:

Lee, M. G., Zeng, W., & Muallem, S. (1997). Characterization and localization of P2 receptors in rat submandibular gland acinar and duct cells. The Journal of Biological Chemistry, 272(52), 32951–32955.Find this resource:

Lee, Y., Lee, C. H., & Oh, U. (2005). Painful channels in sensory neurons. Molecules and Cells, 20(3), 315–324.Find this resource:

Liu, B., Linley, J. E., Du, X., Zhang, X., Ooi, L., Zhang, H., & Gamper, N. (2010). The acute nociceptive signals induced by bradykinin in rat sensory neurons are mediated by inhibition of M-type K+ channels and activation of Ca2+-activated Cl- channels. The Journal of Clinical Investigation, 120(4), 1240–1252. doi:10.1172/JCI41084Find this resource:

Machaca, K., Qu, Z., Kuruma, A., Hartzell, C., & MCCarty, N. (2002). The endogenous calcium-activated Cl channel in Xenopus oocytes: A physiologically and biophysically rich model system. In C. M. Fuller (Ed.), Calcium-activated chloride channels (Vol. 53, pp. 3–39). Amsterdam, the Netherlands: Academic Press.Find this resource:

Mao, S., Garzon-Muvdi, T., Di Fulvio, M., Chen, Y., Delpire, E., Alvarez, F. J., & Alvarez-Leefmans, F. J. (2012). Molecular and functional expression of cation-chloride cotransporters in dorsal root ganglion neurons during postnatal maturation. Journal of Neurophysiology, 108(3), 834–852.Find this resource:

Marmorstein, A. D., Marmorstein, L. Y., Rayborn, M., Wang, X., Hollyfield, J. G., & Petrukhin, K. (2000). Bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2), localizes to the basolateral plasma membrane of the retinal pigment epithelium. Proceedings of the National Academy of Sciences of the United States of America, 97(23), 12758–12763. doi:10.1073/pnas.220402097Find this resource:

Marquardt, A., Stohr, H., Passmore, L. A., Kramer, F., Rivera, A., & Weber, B. H. (1998). Mutations in a novel gene, VMD2, encoding a protein of unknown properties cause juvenile-onset vitelliform macular dystrophy (Best’s disease). Human Molecular Genetics, 7(9), 1517–1525.Find this resource:

Marty, A., Tan, Y. P., & Trautmann, A. (1984). Three types of calcium-dependent channel in rat lacrimal glands. The Journal of Physiology, 357, 293–325.Find this resource:

Mayer, M. L. (1985). A calcium-activated chloride current generates the after-depolarization of rat sensory neurones in culture. The Journal of Physiology, 364, 217–239.Find this resource:

Melvin, J. E., Arreola, J., Nehrke, K., & Begenisich, T. (2002). Ca2+-activated Cl- currents in salivary and lacrimal glands. Calcium-Activated Chloride Channels, 53, 209–230. doi:10.1016/S1063-5823(02)53035-0Find this resource:

Miledi, R. (1982). A calcium-dependent transient outward current in Xenopus laevis oocytes. Proceedings of the Royal Society of London. Series B, Biological Sciences, 215(1201), 491–497.Find this resource:

Morales-Aza, B. M., Chillingworth, N. L., Payne, J. A., & Donaldson, L. F. (2004). Inflammation alters cation chloride cotransporter expression in sensory neurons. Neurobiology of Disease, 17(1), 62–69. doi:10.1016/j.nbd.2004.05.010Find this resource:

Nishi, S., Minota, S., & Karczmar, A. G. (1974). Primary afferent neurones: The ionic mechanism of GABA-mediated depolarization. Neuropharmacology, 13(3), 215–219.Find this resource:

Park, H., Oh, S. J., Han, K. S., Woo, D. H., Park, H., Mannaioni, G., … Lee, C. J. (2009). Bestrophin-1 encodes for the Ca2+-activated anion channel in hippocampal astrocytes. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 29(41), 13063–13073. doi:10.1523/jneurosci.3193–09.2009Find this resource:

Payne, J. A., Rivera, C., Voipio, J., & Kaila, K. (2003). Cation-chloride co-transporters in neuronal communication, development and trauma. Trends in Neurosciences, 26(4), 199–206. doi:10.1016/S0166-2236(03)00068-7Find this resource:

Pedemonte, N., & Galietta, L. J. (2014). Structure and function of TMEM16 proteins (anoctamins). Physiological Reviews, 94(2), 419–459. doi:10.1152/physrev.00039.2011Find this resource:

Petrukhin, K., Koisti, M. J., Bakall, B., Li, W., Xie, G., Marknell, T., … Wadelius, C. (1998). Identification of the gene responsible for Best macular dystrophy. Nature Genetics, 19(3), 241–247. doi:10.1038/915Find this resource:

Pifferi, S., Dibattista, M., & Menini, A. (2009). TMEM16B induces chloride currents activated by calcium in mammalian cells. Pflugers Archive: European Journal of Physiology, 458(6), 1023–1038. doi:10.1007/s00424-009-0684-9Find this resource:

Pineda-Farias, J. B., Barragan-Iglesias, P., Loeza-Alcocer, E., Torres-Lopez, J. E., Rocha-Gonzalez, H. I., Perez-Severiano, F., … Granados-Soto, V. (2015). Role of anoctamin-1 and bestrophin-1 in spinal nerve ligation-induced neuropathic pain in rats. Molecular Pain, 11, 41. doi:10.1186/s12990-015-0042-1Find this resource:

Price, T. J., Cervero, F., Gold, M. S., Hammond, D. L., & Prescott, S. A. (2009). Chloride regulation in the pain pathway. Brain Research Reviews, 60(1), 149–170.Find this resource:

Price, T. J., Hargreaves, K. M., & Cervero, F. (2006). Protein expression and mRNA cellular distribution of the NKCC1 cotransporter in the dorsal root and trigeminal ganglia of the rat. Brain Research, 1112(1), 146–158.Find this resource:

Qu, Z., & Hartzell, H. C. (2000). Anion permeation in Ca(2+)-activated Cl(-) channels. The Journal of General Physiology, 116(6), 825–844.Find this resource:

Reisert, J., Bauer, P. J., Yau, K. W., & Frings, S. (2003). The Ca-activated Cl channel and its control in rat olfactory receptor neurons. The Journal of General Physiology, 122(3), 349–363. doi:10.1085/jgp.200308888Find this resource:

Reuter, D., Zierold, K., Schroder, W. H., & Frings, S. (1998). A depolarizing chloride current contributes to chemoelectrical transduction in olfactory sensory neurons in situ. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 18(17), 6623–6630.Find this resource:

Schlichter, R., Bader, C. R., Bertrand, D., Dubois-Dauphin, M., & Bernheim, L. (1989). Expression of substance P and of a Ca2+-activated Cl- current in quail sensory trigeminal neurons. Neuroscience, 30(3), 585–594.Find this resource:

Schroeder, B. C., Cheng, T., Jan, Y. N., & Jan, L. Y. (2008). Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell, 134(6), 1019–1029. doi:10.1016/j.cell.2008.09.003Find this resource:

Scott, R. H., McGuirk, S. M., & Dolphin, A. C. (1988). Modulation of divalent cation-activated chloride ion currents. British Journal of Pharmacology, 94(3), 653–662.Find this resource:

Scudieri, P., Sondo, E., Ferrera, L., & Galietta, L. J. (2012). The anoctamin family: TMEM16A and TMEM16B as calcium-activated chloride channels. Experimental Physiology, 97(2), 177–183. doi:10.1113/expphysiol.2011.058198Find this resource:

Sun, H., Tsunenari, T., Yau, K. W., & Nathans, J. (2002). The vitelliform macular dystrophy protein defines a new family of chloride channels. Proceedings of the National Academy of Sciences of the United States of America, 99(6), 4008–4013. doi:10.1073/pnas.052692999Find this resource:

Sun, Y., Birnbaumer, L., & Singh, B. B. (2015). TRPC1 regulates calcium-activated chloride channels in salivary gland cells. Journal of Cellular Physiology, 230(11), 2848–2856. doi:10.1002/jcp.25017Find this resource:

Sung, K. W., Kirby, M., McDonald, M. P., Lovinger, D. M., & Delpire, E. (2000). Abnormal GABAA receptor-mediated currents in dorsal root ganglion neurons isolated from Na-K-2Cl cotransporter null mice. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 20(20), 7531–7538.Find this resource:

Takayama, Y., Uta, D., Furue, H., & Tominaga, M. (2015). Pain-enhancing mechanism through interaction between TRPV1 and anoctamin 1 in sensory neurons. Proceedings of the National Academy of Sciences of the United States of America, 112(16), 5213–5218. doi:10.1073/pnas.1421507112Find this resource:

Tarran, R., Loewen, M. E., Paradiso, A. M., Olsen, J. C., Gray, M. A., Argent, B. E., … Gabriel, S. E. (2002). Regulation of murine airway surface liquid volume by CFTR and Ca2+-activated Cl- conductances. The Journal of General Physiology, 120(3), 407–418.Find this resource:

Taylor, R., & Roper, S. (1994). Ca(2+)-dependent Cl- conductance in taste cells from Necturus. Journal of Neurophysiology, 72(1), 475–478. doi:10.1152/jn.1994.72.1.475Find this resource:

Tian, Y., Schreiber, R., & Kunzelmann, K. (2012). Anoctamins are a family of Ca2+-activated Cl- channels. Journal of Cell Science, 125(Pt. 21), 4991–4998. doi:10.1242/jcs.109553Find this resource:

Tominaga, M., & Julius, D. (2000). Capsaicin receptor in the pain pathway. Japanese Journal of Pharmacology, 83(1), 20–24.Find this resource:

Toyoda, H., Yamada, J., Ueno, S., Okabe, A., Kato, H., Sato, K., … Fukuda, A. (2005). Differential functional expression of cation-Cl- cotransporter mRNAs (KCC1, KCC2, and NKCC1) in rat trigeminal nervous system. Brain Research. Molecular Brain Research, 133(1), 12–18. doi:10.1016/j.molbrainres.2004.09.015Find this resource:

Wang, S., Dai, Y., Fukuoka, T., Yamanaka, H., Kobayashi, K., Obata, K., … Noguchi, K. (2008). Phospholipase C and protein kinase A mediate bradykinin sensitization of TRPA1: A molecular mechanism of inflammatory pain. Brain, 131(Pt. 5), 1241–1251. doi:10.1093/brain/awn060Find this resource:

Waxman, S. G., & Zamponi, G. W. (2014). Regulating excitability of peripheral afferents: Emerging ion channel targets. Nature Neuroscience, 17(2), 153–163. doi:10.1038/nn.3602Find this resource:

Winpenny, J. P., Harris, A., Hollingsworth, M. A., Argent, B. E., & Gray, M. A. (1998). Calcium-activated chloride conductance in a pancreatic adenocarcinoma cell line of ductal origin (HPAF) and in freshly isolated human pancreatic duct cells. Pflugers Archive: European Journal of Physiology, 435(6), 796–803.Find this resource:

Witschi, R., Punnakkal, P., Paul, J., Walczak, J. S., Cervero, F., Fritschy, J. M., … Zeilhofer, H. U. (2011). Presynaptic alpha2-GABAA receptors in primary afferent depolarization and spinal pain control. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 31(22), 8134–8142.Find this resource:

Woo, D. H., Han, K. S., Shim, J. W., Yoon, B. E., Kim, E., Bae, J. Y., … Lee, C. J. (2012). TREK-1 and Best1 channels mediate fast and slow glutamate release in astrocytes upon GPCR activation. Cell, 151(1), 25–40. doi:10.1016/j.cell.2012.09.005Find this resource:

Yamagata, K., Sugimura, M., Yoshida, M., Sekine, S., Kawano, A., Oyamaguchi, A., … Niwa, H. (2016). Estrogens exacerbate nociceptive pain via up-regulation of TRPV1 and ANO1 in trigeminal primary neurons of female rats. Endocrinology, 157(11), 4309–4317. doi:10.1210/en.2016-1218Find this resource:

Yang, H., Kim, A., David, T., Palmer, D., Jin, T., Tien, J., … Jan, L. Y. (2012). TMEM16F forms a Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation. Cell, 151(1), 111–122. doi:10.1016/j.cell.2012.07.036Find this resource:

Yang, Y. D., Cho, H., Koo, J. Y., Tak, M. H., Cho, Y., Shim, W. S., … Oh, U. (2008). TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature, 455(7217), 1210–1215. doi:10.1038/nature07313Find this resource:

Zholos, A., Beck, B., Sydorenko, V., Lemonnier, L., Bordat, P., Prevarskaya, N., & Skryma, R. (2005). Ca(2+)- and volume-sensitive chloride currents are differentially regulated by agonists and store-operated Ca2+ entry. The Journal of General Physiology, 125(2), 197–211.Find this resource:

Zygmunt, A. C., & Gibbons, W. R. (1992). Properties of the calcium-activated chloride current in heart. The Journal of General Physiology, 99(3), 391–414.Find this resource: