2.8.2. GPCR Regulation of K*V*7 Channels

A plethora of neurotransmitters and neuropeptides modulate K*V*7 channel function via GPCR signaling (Figure 7), specifically of the G*αq*/11 class [224]. One of the early reports was of the nociception-relevant neuropeptide Substance P (SP) that inhibited K*V*7 currents in bullfrog DRG neurons [225]. Subsequently it was revealed that neurokinin A (NKA inhibited currents through K*V*7 channels in bullfrog DRG neuronsvia NK1 receptors which were coupled to PTX-insensitive G proteins [226], even though this receptor may impinge on the functions of K*V*7 channels through G protein-independent mechanisms as well [227]. The activation of G*αq*/11-coupled receptors leads phospholipase C*β* (PLC*β*) to hydrolyze the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) [228]. The function of K*V*7 channels is governed by the presence of sufficient membrane PIP2 pools and depletion of membrane PIP2 levels leads K*V*7 channels to close [229]. The proalgesic mediator bradykinin mediates inhibition of K*V*7 currents via its actions on the B2 receptor, a G*αq*/11-coupled receptor [230]. The active nociception mediated by bradykinin, consequent to enhanced neuronal excitability can be attenuated by prior application of the K*V*7 channel opener retigabine [145]. In addition to membrane PIP2 depletion, the inhibition of K*V*7 channels via IP3-mediated increase in intracellular Ca2<sup>+</sup> levels and subsequent binding to calmodulin is well known [231,232]. In sympathetic

neurons, the governing factor for substrate (PIP2)- versus product (Ca<sup>2</sup>+)-mediated inhibition consequent to application of bradykinin is contingent upon Ca2<sup>+</sup> availability and rate of PIP2 synthesis [233]. B2 receptors are closely opposed to the endoplasmic reticulum (ER) where IP3 can diffuse and consequently mobilize Ca2<sup>+</sup> reserves [216]. In DRG neurons, bradykinin primarily employs the Ca2<sup>+</sup> axis to inhibit K*V*7 currents as evidenced by the fact that inhibition of Ca2<sup>+</sup> release from IP3-sensitive stores with pharmacological tools as well as chelation of intracellular Ca<sup>2</sup><sup>+</sup> prevents bradykinin-mediated inhibition of K*V*7 channels, akin to direct activation of PLC [145]. One of the targets of the inflammatory soup is the protease-activated receptor 2 (PAR2), a G*αq*/11-coupled receptor expressed in nociceptors [49,234]. Activation of these receptors has an inhibitory impact on K*V*7 currents leading to nociception which requires concurrent increase in cytosolic Ca2<sup>+</sup> levels in addition to depletion of PIP2 levels [235]. Another example is the modulation of excitability in nociceptors by nucleotides. The P2Y1 and P2Y2 receptors are G*αq*/11-coupled receptors [236]. Activation of these receptors by the nucleotides adenosine diphosphate (ADP), and 2-thio-uridine triphosphate (2-thio-UTP), respectively, leads to the inhibition of currents through K*V*7 channels [47]. Moreover, the observed effects were prevented by the application of U73122, a PLC inhibitor, inhibition of Ca2<sup>+</sup>ATPases by thapsigargin, and chelation of intracellular Ca2<sup>+</sup> levels by BAPTA-AM [47].

**Figure 7.** K*V*7 channels are activated by depolarizing voltages (as indicated). G*αq*/11-coupled receptors (**left**) activate phospholipase C (PLC), which cleaves phosphatitylinositol 4,5, bisphosphate (PIP2) into inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG). Depletion of PIP2 from the plasma membrane is sufficient to decrease currents through K*V*7 channels. In addition, Ca2<sup>+</sup> is released from the endoplasmic reticulum subsequent to formation of IP3. Ca2<sup>+</sup> decreases K*V*7 currents via an interaction with calmodulin. Activation of a G*αi*/*o*-coupled receptor (**right**) was shown to decrease K*V*7 currents in a PLC-dependent manner.

The GPCRs encoded by Mas-related genes (Mrgs) are a more recently identified subset of GPCRs that are widely expressed in sensory neurons and implicated in the modulation of nociceptive information [237,238]. Specifically, the MrgD isoform is expressed in DRG neurons especially in non-peptidergic, small-diameter IB4-postive C-fiber somata [239,240]. Activation of endogenous MrgD with the agonist alanine results in the inhibition of K*V*7 currents in DRG neurons, mainly employing a pertussis toxin-sensitive pathway implicating the involvement of G*αi*/*o*. Such an inhibition translates into enhanced neuronal firing in phasic DRG neurons, which classically shoot single single APs [241]. Recombinant cell-lines coexpressing MrgD receptors and K*V*7.2/7.3 heteromers exhibit an inhibition of K*V*7 currents upon stimulation with alanine, an effect that could be reversed partially by pharmacologically blocking G*αi*/*<sup>o</sup>* and reversed completely by PLC inhibition [241].
