*2.3. Mechanosensitive Channels in Pain Sensation*

The variety of mechanical stimuli detected by so-called mechanosensors in the sensory nervous system ranges from light to noxious mechanical stimuli. These specialized neurons express mechanotransducer channels [102]. To sense noxious mechanical stimuli, high-threshold mechanosensors are required, which should express ion channels that open in response to strong mechanical stimuli and lead to a depolarization of these neurons [103]. Acid sensing ion channels (as described above) were suggested to act as mechanotransducer channels in *C. elegans*. However, heterologously expressed mammalian ASICs do not gate in response to mechanical stimuli. Hence, such depolarizing mechanotransducer channels in nociceptive neurons remain to be identified [103].

One family of ion channels that contributes to the sensation of noxious mechanical stimuli is the family of two-pore K<sup>+</sup> channels (K2*P*) [103]. The family of K2*<sup>P</sup>* channels consist of 15 members, which usually provide so-called background or leak currents, which are the major contributors to the resting membrane potential [11]. Three members of the K2*<sup>P</sup>* family were found to be involved in sensing noxious mechanical stimuli: K2*P*2.1 (TREK1), K2*P*4.1 (TRAAK), and K2*P*10.1 (TREK2) [104]. A functional K2*<sup>P</sup>* channel is formed by two subunits consisting of four transmembrane segments each (TMS1–4). Both the N- and C-termini are located in the intracellular space and the linker regions between TMS1 and -2, as well as TMS3 and -4 are located inside the plasma membrane to form one selectivity filter each [105]. Accordingly, a total of eight transmembrane domains and four selectivity filter regions line the ion conduction pore. This structure of the pore is highly homologous to that of voltage-gated K<sup>+</sup> channels [104]. In closed conformation, the ion conduction pathway is blocked by lipid acyl side chains and membrane stretch directly gates K2*<sup>P</sup>* channels [104]. K2*P*2.1 and K2*P*4.1 channels are strongly expressed in small-diameter DRG neurons and only weakly expressed in medium- and large-diameter DRG neurons, whereas K2*P*10.1 channels are exclusively expressed in small-diameter neurons [106]. Interestingly, knock-out of these channels leads to an increased nocifensive response to mechanical stimuli [107]. Since all these channels are selective K<sup>+</sup> channels, opening of K2*<sup>P</sup>* channels leads to an efflux of K<sup>+</sup> ions and subsequent hyperpolarization. It is thought that stretch-activation of K2*<sup>P</sup>* channels counteracts the activation of depolarizing mechanotransducer channels and thereby finetunes the mechanically induced nociceptive signal which is transferred to the brain [102]. Hence, it is clear that these three members of the K2*<sup>P</sup>* family contribute to the perception of noxious mechanical stimuli, but they cannot represent the primary depolarizing mechanotransducer channel [103]. Such a functional entity is rather provided by Piezo channels, in particular Piezo2, which contributes to mechanically activated currents in DRG neurons [108]. However, deletion of Piezo2 impairs touch, but sensitizes mechanical pain in mice [109]. Therefore, additional sensors of mechanical pain remain to be identified.

#### 2.3.1. GPCR Regulation of Mechanosensitive Potassium Channels

The function of mechanosensitive K2*<sup>P</sup>* channels can be adjusted by a number of modulators like arachidonic acid, polyunsaturated fatty acids, glutamate, noradrenaline, acetylcholine, TRH [105] or serotonin [107] (Figure 3). Arachidonic acid and polyunsaturated fatty acids activate these channels directly [105]. The other modulators influence K2*<sup>P</sup>* channel function via activation of GPCR pathways. All three major GPCR pathways affect K2*P*2.1 and K2*P*10.1 channel activity: phosphorylation of two different C-terminally located serine residues either by cAMP activated PKA or PKC leads to an inhibition of both subtypes. Phosphorylation of yet another serine residue by protein kinase G (PKG) on the other hand activates K2*P*2.1 and K2*P*10.1 channels. PKG is activated by an increase of cyclic guanosine monophosphate (cGMP) which in turn is formed by soluble guanylyl cyclase. Soluble guanylyl cyclase is directly activated by nitric oxide and does not involve activation of a GPCR [105]. In dorsal root ganglion neurons, prostaglandin F2*<sup>a</sup>* (PGF2*a*) was shown to decrease K2*<sup>P</sup>* mediated currents [106]. The exact coupling mechanism was not elucidated, however, PGF2*<sup>a</sup>* is the endogenous ligand for G*αq*-coupled FP prostanoid receptors [110] and it is likely that PKC activation is involved

in this process. In addition, prostaglandin E2 (PGE2)-induced nocifensive behavior was reduced in K2*P*10.1 knock-out mice, but the mechanism of action was not investigated [111].

**Figure 3.** K2*<sup>P</sup>* channels are opened in response to mechanical stimuli. Stimulation of a G*αq*/11-coupled receptor (**left**) activates phospholipase C (PLC). Hydrolyzation of phosphatitylinositol 1,4, bisphosphate (PIP2) forms inositiol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG). DAG activates protein kinase C (PKC) which phosphorylates K2*<sup>P</sup>* channels decreasing their function. Activation of G*αs*-coupled receptors (**center**) leads to activation of adenylyl cyclase (AC) which produces cyclic adenosine monophosphate (cAMP). Thereafter, protein kinase A (PKA) is activated which phosphorylates K2*<sup>P</sup>* channels and decreases their currents. Stimulation of G*αi*/*o*-coupled receptors (**right**) decreases AC activity. Therefore, less cAMP is formed, PKA is less active and subsequently K2*<sup>P</sup>* channels are not phosphorylated, which increases their activity.

Activation of a K<sup>+</sup> permeable ion channel leads to an efflux of K<sup>+</sup> ions and a subsequent hyperpolarization. K2*<sup>P</sup>* channels are active at resting conditions and contribute to the formation of the resting membrane potential. Inhibition of these channels leads to a depolarization and a subsequent increase in excitability [107]. Other components of the inflammatory soup were examined for their effects on K2*<sup>P</sup>* channels: for example, serotonin was also found to inhibit K2*P*2.1 and K2*P*10.1 channels via activation of 5-HT4 receptors in a heterologous cell system [112]. These GPCRs are G*αs*-coupled and activate PKA, which is thought to mediate this effect [107]. Application of UTP, which may act through G*αq*-coupled P2Y2, P2Y4 or P2Y6 receptors, leads to an inhibition of K2*<sup>P</sup>* channels in mammary epithelial cells [113]. It remains to be determined if these inflammatory mediators also interact with mechanosensitive K2*<sup>P</sup>* channels in dorsal root ganglion neurons.

On the other hand, activation *μ*-opioid receptors were found to increase K2*P*2.1 currents in hippocampal astrocytes [114], in a heterologous cell system [115], and in substantia gelatinosa neurons of the spinal cord. The latter mechanism is thought to be involved in the antinociceptive actions of opioids [116]. All opioid receptors are coupled to G*αi*/*<sup>o</sup>* G-proteins, which reduce the activity of adenylyl cyclase [110]. Subsequently, less cAMP is formed, which in turn leads to reduced PKA activity and less PKA-mediated phosphorylation of K2*<sup>P</sup>* channels [105]. Since opioid receptors are also expressed in peripheral sensory neurons [117], such an interaction between *μ*-opioid receptors and K2*<sup>P</sup>* channels might also exist in peripheral sensory neurons and contribute to opioid-mediated antinociception.
