*2.4. Calcium-Activated Chloride Channels in Pain Sensation*

Calcium-activated chloride (CaCC) channels occur in a variety of tissues. They are widely expressed in the nervous system but also other tissues like vascular smooth muscle cells. In addition, CaCCs are used as a marker for stroma tumors in the gastrointestinal tract and were initially termed DOG1 [122]. Functionally described CaCCs were linked to transmembrane proteins of unknown function 16A and B (TMEM16) [123–125]. Thereafter, the term anoctamin was introduced to account for its predicted eight (lat. octo) membrane spanning domains and its function as an anion channel [122,126]. The family of TMEM16 proteins consists of ten members termed TMEM16A to TMEM16K (I is left out), which correspond to anoctamin 1 to anoctamin 10 (ANO1 to ANO10) [127]. Only ANO1 and ANO2 were unequivocally identified as mediating calcium-activated anion currents, the other members were either identified as calcium-activated lipid scramblases or as dual-function scramblases/ ion channels [128,129]. The subtypes ANO1, ANO2 and ANO3 were found to be expressed in sensory neurons [130,131]. ANO1 was only detected in TRPV1 positive neurons [132], whereas about 50% of ANO3 positive neurons were also TRPV1 positive [131]. Both ANO1 [133] and ANO3 [131] are involved in nociceptive behavior in mouse models of inflammatory pain. Cyro-EM- [134–136] and X-ray studies [137] revealed that TMEM16 analogs actually consist of ten transmembrane domains. TMEM16A forms homodimers [138,139], where transmembrane domains 3–7 of both subunits form one separate ion conduction pathway creating a two-pore anion channel [134–136]. Calcium, needed for gating, binds to two regions of negatively charged amino acid residues in transmembrane domains 6–8. Subsequently, transmembrane domain 6 is displaced, which ultimately opens the channel [134,135]. In the absence of Ca2<sup>+</sup>, CaCC function is not altered by changes in membrane voltage within physiological limits. Only voltages exceeding 100 mV may gate CaCCs directly if Ca2<sup>+</sup> is absent [140], presence of cytosolic Ca2<sup>+</sup> merely shifts the voltage-dependence to more physiological levels [141]. In sensory neurons, Ca2<sup>+</sup> can either rise in response to activation of a G*αq*-coupled receptor and subsequent release from intracellular stores [142], or due to an influx of extracellular Ca2<sup>+</sup> via Ca2<sup>+</sup>-permeable ion channels like TRPV1 channels [143], or to a lesser extend voltage-gated Ca2<sup>+</sup> channels [142].

Mice lacking TRPV1 receptors, the canonical heat sensor, retain some sensitivity to noxious heat and CaCCs were suggested to fulfill that task [122]. Indeed, heterologously expressed TMEM16A channels produced currents at temperatures that exceeded 44 ◦C [144] and tissue specific knock-out reduced mechanically and thermally induced nocifensive behavior [132,133].

#### GPCR Regulation of Calcium-Activated Chloride Channels

As described above, there are three possible sources for Ca2<sup>+</sup> to activate CaCCs. One of these possibilities is to activate a G*αq*-coupled receptor and the subsequent signaling cascade (Figure 4). In dorsal root ganglion neurons, the inflammatory mediator bradykinin was demonstrated to increase neuronal excitability via gating of CaCCs which leads to a depolarizing Cl− efflux due to comparably high intracellular Cl− concentrations. The induction of Cl− currents through CaCCs by bradykinin required activation of PLC, formation of IP3 and an increase of cytosolic Ca2<sup>+</sup> levels [145]. In addition, it was also shown that activation of proteinase-activated receptor 2 (PAR2) induced currents through CaCCs in dorsal root ganglion neurons. This action is dependent on increasing levels of cytosolic Ca2<sup>+</sup> and a close proximity of IP3 receptors, which are located on the membrane of the endoplasmic reticulum, to ANO1 channels, which are located at the plasma membrane [142]. Furthermore,

expression of both TMEM16A (ANO1) and PAR2 is induced in a model of neuropathic pain and both proteins are co-expressed in the same set of dorsal root ganglion neurons [146]. The inflammatory mediator serotonin was shown to induce currents mediated via CaCCs in dorsal root ganglion neurons. Even though all three types of 5-HT2 receptors are expressed on small-diameter dorsal root ganglion neurons, only activation of 5-HT2*<sup>C</sup>* receptors was able to induce such currents. Furthermore, the according increase in excitability also required activation of TRPV1 channels, which provides an additional Ca2<sup>+</sup> source [44]. Other possible mediators for sensitization include nucleotides: Both, UTP and ATP were found to interact with CaCCs via activation of G*αq*-coupled P2Y1 and P2Y2 receptors. However, this interaction was only investigated in kidney [147] and pancreatic cells [148]. It remains to be established if such an interaction also contributes to nucleotide-mediated sensitization of sensory neurons.

**Figure 4.** Calcium-activated Cl− channels (CaCC) are gated by increasing concentrations of cytosolic Ca2<sup>+</sup> and influenced by membrane voltage. The source of Ca2<sup>+</sup> may be Ca2<sup>+</sup>-permeable ion channels located in proximity to CaCCs (not shown) or Ca2<sup>+</sup> released from intracellular stores. Stimulation of G*αq*/11-coupled receptors (**left**) activates phospholipase C (PLC). The subsequent hydrolysis of phosphatitylinositol 1,4, bisphosphate (PIP2) forms inositiol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG). DAG activates protein kinase C (PKC) which influences CaCC function. IP3 binds to IP3 receptors located in the membrane of the endoplasmic reticulum (ER) which causes Ca2<sup>+</sup> release from the ER. Activation of G*αi*/*o*-coupled receptors (**right**) was shown to decrease CaCC currents via an unknown mechanism.

In animal experiments, it was determined that activation of G*α<sup>i</sup>* coupled cannabinoid CB1 receptors may contribute to peripheral antinociception via an interaction with CaCCs [149]. In addition, central antinociception via activation of G*αi*-coupled *δ*-opioid (DOP) receptors may involve an interaction with CaCCs [150]. However, it remains unclear how G*αi*-coupled receptors interfere with CaCC function.
