GPCR Regulation of TRP Channels

TRPV1 channels have been studied extensively for their modulation by GPCRs. Currents through TRPV1 channels are increased in response to inflammation, which mediates an enhanced depolarization and increased excitability [2]. The sensitization of TRPV1 channels can be mediated by both G*αq*- and G*αs*-coupled receptors. Stimulation of a G*αq*-coupled receptor leads to activation of phospholipase C (PLC), which hydrolyzes membrane bound phosphatitylinositol 1,4, bisphosphate (PIP2) into soluble inositol 1,4,5 trisphosphate (IP3) and membrane bound diacylglycerol (DAG, Figure 1). Subsequently, IP3 binds to IP3 receptors located at the membrane of the endoplasmic reticulum, which triggers the release of Ca2+. DAG in turn activates protein kinase C, which phosphorylates target proteins. Every step of this cascade can interfere with the function of TRPV1 channels [39]. Presence of PIP2 in the membrane is thought to decrease TRPV1 channel function by interfering with agonist binding [26]. If PIP2 is depleted from the membrane in response to PLC activation, TRPV1 activity may increase [30]. The exact role of PIP2 remains debated, as it was also shown to activate TRPV1 channels [30]. Activated PKC was shown to phosphorylate two serine residues at the C-terminus, which is thought to mediate sensitization [40]. A rise in cytosolic Ca2<sup>+</sup> is not considered to contribute to sensitization as it usually leads to rapid channel desensitization in response to prolonged activation [39]. A large number of inflammatory modulators was shown to increase TRPV1 channels via one of these mechanisms (Table 1).

**Figure 1.** TRPV1 channels can be gated by different mechanisms (as indicated). Three major G-proteindependent pathways modulate the function of TRPV1 channels. Activation of G*αq*/11-coupled receptors (**left**) leads to activation of phospholipase C (PLC), which hydrolyzes phosphatitylinositol 1,4, bisphosphate (PIP2) into inositiol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG). DAG activates protein kinase C (PKC), which phosphorylates TRPV1 channels, thereby increasing their function. Activation of a G*αs*-coupled receptor (**center**) stimulates adenylyl cyclase (AC), which produces cyclic adenosine monophosphate (cAMP). Subsequent activation of protein kinase A (PKA) leads to phosphorylation of TRPV1 channels and an increase in current. Stimulation of a G*αi*/*o*-coupled receptor (**right**) decreases AC activity. Therefore, less cAMP is formed, PKA is less active and hence TRPV1 channels are not phosphorylated which decreases their activity.


AC, adenylyl cylcase; DAG, diacylglycerol; PKA, proteinkinase A; PKC, protein kinase C; BAM 8–22, bovine adrenal medulla peptide 8–22.

Activation of a G*αs*-coupled receptor stimulates the activity of adenylyl cyclase, which produces cyclic adenosine monophosphate (cAMP). This nucleotide is needed to activate protein kinase A (PKA), which then phosphorylates its target proteins. PKA-mediated phosphorylation of TRPV1 channels increases their sensitivity towards their agonists and reduces Ca2+-mediated desensitization [54]. Several inflammatory mediators were found to sensitize TRPV1 channels utilizing this pathway (Table 1).

By contrast, activation of a G*αi*-coupled receptor decreases the activity of adenylyl cyclase which reduces the abundance of cAMP and subsequent activation of PKA (Table 1). Indeed, activation of G*αi*-coupled cannabinoid [62,63] and *μ*-opioid (MOP) [60,61] receptors was shown to reduce currents of TRPV1 receptors, which is thought to contribute to the peripheral analgesic action of opioids [60,61] and cannabinoids [62,63].

In addition to the three major GPCR pathways, TRPV1 channels were shown to be sensitized by nerve growth factor (NGF), which requires the early activation of PI3 kinase and the presence of PKC and CamKII (Ca2<sup>+</sup>/calmodulin dependent protein kinase II) [64]. The inflammatory mediator histamine sensitizes TRPV1 channels via G*αq*-coupled H1 receptors. Instead of utilizing the signaling cascade described above, histamine-mediated sensitization requires activation of phospholipase A2 and lipoxigenases [65–67].

In sensory neurons, a variety of G*αi*/*o*-coupled receptors were found to inhibit currents through TRPM3 channels: including GABA*<sup>B</sup>* receptors [68–70], *μ*-opioid receptors [68,69], somatostatin receptors [68,70], CB1- [69], as well as, CB2 receptors [68], and neuropeptide Y receptors [69]. Likewise, low concentrations of noradrenaline reduce TRPM3 activity, hinting towards *α*<sup>2</sup> as mediating receptor. However, the adrenergic receptor involved was not further characterized [68]. Whether *δ*-opioid receptors also mediate a TRPM3 inhibition remains controversial: while deltorphin, a *δ*-selective peptide was able to reduce TRPM3 function [68], the small-molecule *δ*-selective agonist SB205607 was not [69]. Likewise, activation of G*αi*/*o*-coupled metabotropic glutamate receptors (mGluR4/6/7/8) did not reduce TRPM3 function [69]. In a heterologous system, G*αq*/11-coupled M1 receptors were found to inhibit currents through TRPM3 channels [70]. However, in sensory neurons, activation of G*αq*/11-coupled mGluR5 only weakly inhibited TRPM3 [68]. The inhibition of TRPM3 in sensory neurons involves activation pertussis toxin (PTX)-sensitive G*αi*/*o*-coupled receptors [68–70]. The effect did not require signaling downstream of G*αi*/*<sup>o</sup>* activation, but relied on a direct interaction with the *βγ* dimer [68–70].

TRPA1 channels are sensitized by G*αs*- and G*αq*-coupled receptors in sensory neurons. A G*αi*-mediated interaction has not been reported for sensory neurons. Activation of G*αq*-coupled PAR2 receptors increased currents mediated by TRPA1 receptors in dorsal root ganglion neurons. This interaction required the activation of PLC but none of the downstream products. Consequently, depletion of PIP2 from the membrane was shown to be sufficient for this interaction [12]. The inflammatory mediator bradykinin was shown to increase currents through TRPA1 channels in dorsal root ganglion neurons. This effect was mediated by G*αq*-coupled B2 receptors and required the activation of PLC. Interestingly, activation of PKA was further required for the interaction of B2 receptors and TRPA1 channels [71]. An interaction of G*αq*-coupled bradykinin B1 receptors with TRPA1 channels was reported in behavioral experiments. This interaction relied on activation of PLC and PKC [72]. Histamine was shown to cause nocifensive behavior in a TRPA1 dependent manner. It is thought to involve G*αq*-coupled H1 receptors and activation of PLC [73]. Adenosine, another component of the inflammatory soup, was found to sensitize esophageal C-fibers and increase TRPA1 currents via G*αs*-coupled A2*<sup>A</sup>* receptors. Activation of PKA is necessary for this interaction [74]. Electrophilic metabolites of prostaglandins, however, were demonstrated to activate TRPA1 channels directly [75,76].

The inflammatory mediators prostaglandin E2 (PGE2), bradykinin and histamine were tested for their influence on TRPM8 channel function. As opposed to the previously described members of the TRP channel family, the activity of the cool sensor TRPM8 is reduced in the presence of bradykinin and PGE2. However, application of histamine did not interfere with TRPM8 channel function [77]. The action of bradykinin required the mobilization of PKC [77,78], whereas PGE2 involved activation of PKA [77]. Bradykinin is assumed to act via G*αq*-coupled B2 receptors [79] and several modes of action have been suggested: it was found that depletion of PIP2 from the plasma membrane reduced heterologously expressed TRPM8 channel function [80]. However, these experiments were performed in absence of GPCRs and it remains to be established if PIP2 depletion is also sufficient to reduce TRPM8 channel function in a native cell system. PIP2 is hydrolyzed to form IP3 and DAG, which is required to activate PKC. PKC is thought to activate protein phosphatase I (PPI), which is suggested to dephosphorylate TRPM8 channels. The dephosphorylation of TRPM8 channels is proposed to finally inhibit TRPM8 channel function [79]. More recently, both bradykinin and histamine were found to inhibit TRPM8 channels via a direct interaction of G*α<sup>q</sup>* subunits with the channels. The inhibitory effect of both mediators did not require activation of PLC or any of the subsequent steps in the signaling cascade [81]. The receptor via which PGE2 exerts its effect has not been determined, however it was found that activation of a G*αs*-coupled receptor and subsequent PKA stimulation was required. Furthermore, the exact mechanism how PKA modulates TRPM8 channel function remains unknown [79].

#### *2.2. Acid-Sensing Ion Channels*

Acid-sensing ion channels (ASIC) represent one of many ion channel families that detect noxious chemical stimuli. Additional chemical sensors are TRP channels, namely TRPA1 and TRPV1, as well as ATP-gated P2X receptors [82]. As the name suggests, acid sensing ion channels are activated in low pH conditions [83]. Such acidic conditions occur during an inflammatory response, ischemia, or fatiguing exercise [82]. ASICs can be divided into three subtypes ASIC1 to ASIC3. ASIC1 and ASIC2 can even be further subclassified into two splice variants each (ASIC1a, ASIC1b; ASIC2a, ASIC2b) [84]. A fourth analog, sometimes referred to as ASIC4 [85], rather affects expression levels of ASIC1a and ASIC3, instead of producing proton-gated currents [84]. The EC50 for proton-mediated currents via ASIC1 and ASIC3 channels ranges between a pH of 6.2 to 6.8, whereas ASIC2 channels have an EC50 between pH 4.1 and 5 [84]. All forms of ASICs can be detected in somata and peripheral ends of sensory neurons [85]. ASIC1a and ASIC3 channels are preferentially expressed in small diameter DRG neurons which also express TRPV1 and most likely subserve nociceptive function [86]. A functional channel is composed of three subunits and all but one subunit can participate in both homo- and heteromeric channels. Only ASIC2b does not form functional homomeric channels [87]. One subunit consists of two transmembrane domains, having both the N- and C-termini at the intracellular side [88,89]. Most of the protein is located at the extracellular side forming the large extracellular domain (termed ECD). The structure of the extracellular domain was compared to a hand holding a ball, which explains the peculiar terminology for parts of the ECD, such as palm, knuckle, thumb, finger and *β*-ball [90]. The ECD contains a number of regulatory domains: for example, an acidic pocket is formed by acidic amino acid residues at the subunit–subunit interface, which is involved in binding of H<sup>+</sup> ions and subsequent gating [89]. ASIC channels follow a three-state kinetic model, from a closed to an activated to an inactivated state. The recovery from inactivation can only be achieved in high pH conditions [88] and this desensitized state is thought to be regulated by the thumb domain within the ECD [90].

Genetic studies have suggested that ASICs play a role in sensing mechanical signals, but the exact gating mechanism is unknown, and their role remains heavily debated [91].

#### GPCR Regulation of ASICs

A few components of the inflammatory soup have been tested for their modulatory effect on acid-sensing ion channels: histamine was shown to selectively potentiate heterologously expressed ASIC1a channels. This process involved a direct action of histamine and did not require the presence of a GPCR [92]. The nucleotides UTP and ATP were shown to increase acid-induced currents (Figure 2) in rat dorsal root ganglion neurons as well as acid-induced membrane excitability. In this respect, UTP was found to act via G*αq*-coupled P2Y2 receptors and required the activation of PLC, subsequent stimulation of PKC and the presence of the anchoring protein PICK-1 (protein interacting with C-kinase 1) [93]. A similar effect can be observed when serotonin is applied: both ASIC-mediated currents and neuronal excitability are increased [94]. Serotonin was found to act via G*αq*-coupled 5-HT2 receptors [94] and required activation of PKC [94,95]. Two phosphorylation sites, one at the N-terminus and one at the C-terminus of ASIC3, need to be phosphorylated for the full effect. Again, PICK-1 is necessary for PKC-mediated phosphorylation of ASIC channels. This scaffold protein is thought to bind to ASIC2b subunits in heterotrimeric channels and to link PKC to the channel and to enable phosphorylation [95]. Another G*αq*-coupled receptor, PAR2, was found to increase ASIC-mediated currents in rat pulmonary sensory neurons. Interestingly, neither PLC nor PKC were required for the PAR2 mediated current increase but the pathway involved was not studied further [96]. Depending on the activation mechanism of PAR2, one may observe an increase of cytosolic Ca2<sup>+</sup> following the G*αq*-dependent activation of PLC and formation of IP3. On the other hand, PAR2 activation was shown to signal also via G*α*12/13 proteins, which activate Rho kinase and lead to ERK phosphorylation. Additionally, PAR2 activation may lead to *β*-arrestin recruitment. Whether PAR2 activation may also decrease or increase cAMP levels remains controversial [97].

**Figure 2.** Acid sensing ion channels (ASIC) are gated by increasing concentrations of H+. Two G-protein pathways modulate the function of ASICs. Activation of G*αq*/11-coupled receptors (**left**) leads to activation of phospholipase C (PLC) which hydrolyzes phosphatitylinositol 1,4, bisphosphate (PIP2) into inositiol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG). DAG activates protein kinase C (PKC) which phosphorylates ASICs increasing their function. The interaction of PKC with ASICs requires the presence of PICK-1 (protein interacting with C-kinase 1). By contrast, activation of G*αq*/11-coupled V1*<sup>A</sup>* receptors (**center**) were shown to decrease ASIC-mediated currents via an unknown mechanism. Stimulation of G*αi*/*o*-coupled receptors (**right**) was shown to decrease currents through ASICs involving an undetermined mechanism.

By contrast, activation of G*αi*/*o*-coupled receptors was shown to decrease currents through ASIC channels. First, stimulation of cannabinoid CB1 receptors was found to reduce nocifensive behavior triggered by local acidosis which relies on an interaction between CB1 receptors and ASIC channels [98]. Second, activation of *μ*-opioid receptors was shown to decrease ASIC-mediated currents, neuronal excitability in dorsal root ganglion neurons and nocifensive behavior induced by local acidification [99]. In addition, nocifensive behavior provoked by mechanical stimuli [100] or local acidification [101] is reduced by local application of oxytocin. Oxytocin reduces ASIC-mediated currents in dorsal root ganglion neurons via activation of vasopressin V1*<sup>A</sup>* receptors. This effect was found to be dependent on G*α<sup>q</sup>* activation, but further steps of the cascade were not tested [101]. It remains to be determined if the observed differences in G*αq*-mediated effects on ASIC channels, for example, depend on the recruitment of PICK-1.
