Inhibitory G i/O -coupled receptors in somatosensory neurons: Potential therapeutic targets for novel analgesics

Adenylate cyclase

The letter “i” in G_αi stands for “inhibitory” because receptors coupled to G_αi proteins inhibit AC, as opposed to “stimulatory” G_αs proteins. G_s-coupled receptors, such as prostaglandin D receptors, generally increase excitability of DRG neurons; thus contribute to inflammatory hypersensitivity. Concurrent activation of G_i-coupled receptors, in principle, counteracts this effect by decreasing cAMP levels. There are nine mammalian membrane bound AC isoforms. AC5 and AC6 are inhibited by all G_αi isoforms via protein–protein interactions, while AC1 is inhibited by G_αo, and G_βγ may also contribute to inhibition of these AC isoforms. AC2, AC4, and AC7 on the other hand are potentiated by G_βγ subunits in the presence of G_s stimulation (Figure 1). ³³ Further complicating this picture is the finding that sustained stimulation of G_i-coupled receptors paradoxically potentiates cAMP production, especially after cessation of the stimulus. ³⁴ This may underlie hypersensitivity upon repeated application of G_i-coupled agonists such as morphine and adenosine.^35,36 Also, increased cytoplasmic Ca²⁺ stimulates several isoforms of AC (Figure 1), while inhibits others, providing a cross talk from G_q-coupled receptors. ³³

GIRK (Kir3.x) channels

GIRK channels are stimulated by activation of G_i-coupled cell surface receptors, leading to hyperpolarization and thus decreased excitability. GIRKs are members of the inwardly rectifying K⁺ (Kir) family of ion channels ³⁷ ; four subunits, Kir3.1, Kir3.2, Kir3.3, and Kir3.4, form homo- or hetero-tetramers to produce functional GIRK channels. The GIRK1/GIRK4 (Kir3.1/Kir3/4) combination forms the classical cardiac K⁺ channel activated by acetylcholine and contributes to slowing the heart rate, while GIRK2 is generally expressed in the nervous system. Activation of various G_i-coupled receptors including GABA_B and SST receptors have been shown to activate GIRK currents in rat DRG neurons,^38,39 and mRNA has been detected for all four Kir subunits in those cells. ³⁸ Another study reported that GIRK channels were present in rat and human DRG neurons, but they were absent in mouse DRG neurons. In vivo nociceptor-specific transgenic expression GIRK2 in mouse DRG neurons using Na_v1.8 promoter restored peripheral analgesia induced by the μOR agonist DAMGO. ⁴⁰ Unbiased RNA sequencing in mouse DRG neurons detected low levels of GIRK channel expression, with the exception of GIRK1 (KCNJ3), ⁴¹ and single cell RNA sequencing of mouse DRG neurons showed significant enrichment of GIRK2 in the tyrosine-hydroxylase positive subpopulation. ²⁶

GIRK channel activation is mediated by direct interactions between G_βγ and the channel.^42,43 Interestingly, the channels are activated by G_i-coupled receptors, but not by G_q- or G_s-coupled receptors, even in heterologous expression systems. The mechanism of this selectivity has been a subject of intensive research; it cannot be explained by different subunit composition, because all G_β-s (G_β1–4) with the exception of G_β5 activate GIRK channels, ⁴⁴ and no clear differences were identified in subunit composition of G_β and G_γ associating with different G_α-s. The most likely explanation is that the G_αi-G_βγ complex associates with high affinity with GIRK channels in resting cells, and upon receptor activation, a local conformation switch, similar to a clamshell opening, rather than full dissociation of G_βγ from G_αi, activates the channel. This model is based largely on fluorescent resonance energy transfer measurements between the channel, receptor, and the G-proteins.^45,46 The key findings supporting this model are that upon receptor stimulation, fluorescent resonance energy transfer may increase or decrease between Gβ and the channel, ⁴⁶ and between Gβ and G_αi, ⁴⁵ depending on the location of the CFP and YFP tags on the individual proteins. It remains to be seen if other effectors of G_βγ show similar mechanism.

Voltage-gated Ca²⁺ channels

N-type (Ca_v2.2, CACNA1B) and P/Q-type (Ca_v2.1, CACNA1A) voltage-gated Ca²⁺ channels (VGCC) are also classical targets of G_βγ released from G_αi. ⁴⁷ N-type channels are usually found presynaptically, where they play an important role in initiating neurotransmitter release. Inhibition of N-type channels by G_i-coupled receptors reduces transmitter release, an effect expected to take place in the central process in the context of DRG neurons. Indeed, inhibition of N-type Ca²⁺ channels by several G_i-coupled receptors including GABA_B receptors and opioid receptors have been reported in DRG neurons. ⁴⁸

DRG neurons also express low-voltage activated Ca²⁺ channel (T-type), and the GABA_B receptor agonist baclofen has been shown to inhibit both low- and high-voltage activated Ca²⁺ channels in DRG neurons. ⁴⁹ T-type channels (Ca_v3.2) are expressed both in the soma and the in the peripheral nerve termini and play important roles in initiating the receptor potential in response to mechanical stimuli.^50,51

Transient receptor potential melastatin 3

Transient receptor potential melastatin 3 (TRPM3) channels are activated by heat, ⁵² and chemical agonists such as pregnenolone sulphate (PregS) ⁵³ and the synthetic agonist CIM0216. ⁵⁴ These channels are expressed in small nociceptive DRG neurons, and their genetic deletion in mice reduces sensitivity to noxious heat. ⁵² Recent reports from three different laboratories identified TRPM3 as a novel target of G_βγ upon activation of G_i-coupled receptors in DRG neurons using a wide range of overlapping techniques.^55–57 Activation of μ-opioid,^56,57 GABA_B,^55–57 SST,^55,57 or Neuropeptide Y (NPY) receptors ⁵⁷ inhibited Ca²⁺ signals evoked by PregS in DRG neurons. Activation of recombinant GABA_B, M2 muscarinic, and Dopamine 2 receptors also inhibited TRPM3 expressed in HEK cells, and the effect on M2 receptor activation was inhibited by co-expressing the G_βγ binding C-terminal fragment of the β-adrenergic receptor kinase. ⁵⁵ Co-expressing G_β1γ2 in HEK cells inhibited PregS-induced Ca²⁺ signals and currents, but various G_αi/o isoforms including the constitutively active G_α1-Q204L had no effect. ⁵⁶ Similarly, co-expressing G_β1γ2 in Xenopus oocytes inhibited PregS-induced currents, but none of the tested G_αi/o isoforms had a significant effect. ⁵⁵ The effect of G_βγ likely proceeds via direct protein–protein interaction as application of purified G_βγ, but not G_αi2 inhibited TRPM3 currents in excised inside out patches^55,57 and TRPM3 co-immunoprecipitated with G_β.^55,57 Nocifensive responses evoked by hind paw injection of either CIM0216 or PregS were inhibited by co-injection of baclofen,^55,57 DAMGO, ⁵⁶ morphine, ⁵⁷ or NPY. ⁵⁷ On the other hand, Ca²⁺ responses in DRG neurons evoked by agonists of other sensory TRP channels TRPV1, ⁵⁶ TRPA1,^55,56 and TRPM8 ⁵⁵ were not affected by G_i-coupled receptor activation, and accordingly, nocifensive responses to the TRPV1 agonist capsaicin were not inhibited by co-injection of DAMGO, ⁵⁶ and baclofen did not inhibit nocifensive responses to the TRPA1 agonist mustard oil. ⁵⁵ Overall, the three articles described here convincingly demonstrate that TRPM3 is a bona fide novel ion channel target of G_βγ in DRG neurons, see also discussion by Csanady. ⁵⁸

Other targets

Downstream targets of G_βγ also include phosphoinositide 3-kinase-γ (PI3Kγ) and mitogen-activated protein kinases.^59,60 While there is an extensive literature on mitogen-activated protein kinases in DRG neurons, most studies focused on its role in inflammatory hypersensitivity, and little is known if they play any roles in signaling by G_αi/o-coupled receptors. ⁶¹ Similarly, PI3K enzymes have been studied largely in the context of hypersensitivity, NGF-signaling ⁶² and inflammation, ⁶³ and little is known about their role in G_αi/o-coupled receptor signaling.

β-arrestin 1 (arrestin 2) and β-arrestin 2 (arrestin 3) were originally identified to bind to phosphorylated GPCRs and induce their desensitization and internalization ⁸ ; their roles, however, are emerging as independent signaling mediators.^8,64 Arrestins have been extensively studied in the context of opioid receptor signaling, and new biased opioid receptor agonists with minimal arrestin recruitment are being developed with the hope of minimizing the side effects of these drugs. ¹⁰ Relatively little is known about the roles of arrestins in DRG neurons. It has been shown that δ-opioid receptor (δOR) signaling to VGCC was enhanced in β-arrestin1 knockout mice, and the behavioral effects of δOR agonists were enhanced in the absence of β-arrestin1. ⁶⁵ The authors concluded that these effects are due to δOR activation of cofilin through Rho-associated coiled-coil containing protein kinase, LIM domain kinase, and β-arrestin1 to regulate actin polymerization. ⁶⁵ Another article found that the high-internalizing δOR agonist (SNC80) preferentially recruited β-arrestin 1, and genetic deletion of β-arrestin 1 induced a significant increase in the potency of SNC80 to inhibit mechanical pain and decreased acute tolerance. In contrast, the low-internalizing δOR agonists (ARM390) preferentially recruited β-arrestin 2 with unaltered behavioral effects in β-arrestin 2 knockout animals. ⁶⁶

There are several less common targets of G_i signaling; some of them with relevance to DRG neurons are discussed below. Substance P released from nociceptive nerve endings is generally thought to be pro-nociceptive, but acute antinociceptive effects of this peptide have also been described. ⁶⁷ Substance P activates Neurokinin receptors (NK1–3), which are generally thought to couple to G_q and activate PLC, but they may also couple to G_i. Substance P has been shown to inhibit T-type VGCC ⁶⁸ and potentiate M-type K⁺ channels ⁶⁹ in DRG neurons, both of which reduce excitability. These effects were mediated by production of reactive oxygen species, and they were eliminated by overnight pertussis toxin (PTX) treatment showing the involvement of G_i signaling.

While G_i-coupled receptors are generally inhibitory, there are examples where pro-nocicepitive mediators increase excitability with the involvement of G_i-coupled receptors. Three examples are listed below on tetrodotoxin-resistant voltage-gated Na⁺ channels Na_v1.8 and Na_v1.9. The pro-inflammatory prostaglandin PGE2 has been reported to potentiate Na_v1.9 currents in mouse DRG neurons, and PTX inhibited the effect, pointing to the role of G_i signaling. ⁷⁰ The chemokine CCL2 potentiated Na_v1.8 channels in rat DRG neurons; the effect was blocked by PTX and gallein, suggesting the involvement of G_i signaling and G_βγ. ⁷¹ The chemokine CXCL12 increased the activity of Na_v1.8 and Na_v1.9 currents in rat DRG neurons; PTX and the PI3K inhibitor LY294002 eliminated the effect on Na_v1.9, but not on Na_v1.8. ⁷²

Opioid receptors

Morphine and other opioid receptor agonists are mainstream therapy against severe pain. Most clinically relevant effects and many side effects of opioids are mediated by G_i-coupled μOR. The two other opioid receptor subtypes δOR and κ-opioid receptors (κOR) also couple to G_i/o, and have been studied as alternative targets for analgesics. ⁷³ Specific activation of both δOR and κOR has also been reported to induce analgesic effects, but κOR activation has been associated with dysphoria, while δOR activation has been reported to have anxiolytic and antidepressant effects. ⁷⁴ The nociceptin receptor or opioid receptor like 1 shares homology with opioid receptors; it is activated by its endogenous ligand nociceptin, but not by most opioid drugs. ⁷⁵

Opioid receptors are expressed both centrally, in the brain and spinal cord, as well as peripherally in cell bodies and peripheral processes of DRG neurons. DRG neurons express all three opioid receptors and opioid receptor like 1 at different levels and cellular distribution ⁴¹ (Table 1). Both locally administered morphine and opioid receptor agonists such as the μOR agonist DAMGO, which do not cross the blood brain barrier, have been shown to have analgesic effects.^76,77 The idea of peripherally acting opioids targeting DRG neurons, potentially devoid of central side effects, such euphoria and tolerance, have been raised, but so far, there are no clinically useful antinociceptive drugs available.^76,78 Loperamide or Imodium is a peripherally acting μOR agonist, used as an over the counter antidiarrheal medication. ⁷⁹ Loperamide has no antinociceptive effect when taken orally, but it was reported to alleviate painful symptoms of oral or skin ulcers when applied topically. ⁷⁹ The main reason for the lack of the analgesic effect of oral loperamide is that it does not reach the systemic circulation, due to its almost complete degradation by the liver. ⁷⁹ Loperamide has been shown to have analgesic effect when injected subcutaneously^80,81 or applied topically. ⁸²

A recent review on δOR in primary sensory neurons provides a thorough description of the roles of δOR as well as μOR in DRG neurons. ⁸³ Briefly, most research in DRG neurons focused on μOR and δOR, and experiments based on immunocytochemistry suggested that μOR and δOR are expressed in an overlapping set of cells. ⁸⁴ A more recent study by Scherrer et al. ⁸⁵ using a δOR-GFP reporter mouse line showed that μOR and δOR are expressed in different cell populations; δOR were restricted to medium-to-large myelinated NF200 expressing cells and non-peptidergic IB4 positive smaller neurons. μOR on the other hand was mainly expressed in small, peptidergic TRPV1- and substance P-positive neurons. ⁸⁵ These data are also consistent with the distribution of RNA expression of these receptors in a recent single cell RNA sequencing article ²⁶ (see also Table 1). Consistent with μOR and δOR being expressed in different cell populations, selective activation of μOR or δOR also had functionally distinct effects. Intrathecal administration of the μOR selective agonist DAMGO decreased sensitivity to noxious heat, without significant effect on mechanical pain; the δOR-specific SNC80 on the other hand significantly attenuated mechanical pain, without having an effect on heat sensitivity. ⁸⁵

The debate on whether or not μOR and δOR are co-expressed in the same DRG neurons however is not yet settled. Recent studies demonstrated the coexistence of μORs and δORs in small DRG neurons using single-cell PCR, in situ hybridization, immunostaining, and electrophysiology. ⁸⁶ Heteromers of μOR and δOR were shown in DRG neurons using antibodies that recognize those heteromers. ⁸⁷ Finally, facilitation of the degradation of μOR-δOR heteromers by δOR agonists have been shown to be alleviated by disrupting heteromer formation. ⁸⁸

A recent paper showed that nociceptor-specific deletion of μOR had no effect on morphine-induced analgesia, but eliminated both tolerance and opioid-induced hyperalgesia. ⁸⁹ The same study also showed that methylnaltrexone bromide, a peripherally restricted μOR antagonist, was sufficient to abrogate tolerance and hyperalgesia induced by morphine, without diminishing its antinociceptive effect. These data raise doubt about the usefulness of peripherally acting μOR agonists as analgesics. As mentioned earlier, it was suggested that in mice, analgesic effect of the peripherally acting μOR agonist DAMGO required transgenic expression of GIRK2 in DRG neurons. ⁴⁰

Significant recent efforts used innovative approaches to target peripheral opioid receptors for pain relief. ⁹⁰ A recent article reported a peripherally acting μOR agonist, which acts selectively at the site of injury. Spahn et al. ⁹¹ synthesized a fentanyl analog that only activates μOR at low pH, which is characteristic of inflamed and injured tissues, and they showed that the compound reduced inflammatory hyperalgesia to both thermal and mechanical stimuli in rats. Another recent article reported covalently attaching morphine to hyperbranched polyglycerol by a cleavable linker, which prevents blood-brain barrier permeation and selectively releases morphine in injured tissue. This conjugated morphine produced analgesia in inflamed rat paws without major side effects. ⁹²

While RNA levels for κOR are lower than those of other opioid receptors (Table 1) both κOR expression ⁸⁴ and inhibitory effects of κOR agonists on VGCC ⁹³ have been reported in DRG neurons. As mentioned earlier, DRG neurons play important roles not only in pain, but also in itch, which in chronic forms is a significant medical problem. Pruritus, or itch, is one of the side effects of activation of μOR, ⁷³ but activation of κOR has the opposite effect. The κOR agonist nalfurafine, ⁷³ as well as two different peripherally acting κOR agonists, asimadoline and CR845, were shown to be effective against itch. ⁹⁴ CR845 showed promising results in phase II clinical trials against pruritus associated with chronic kidney disease in hemodialysis patients. ⁹⁵

DRG neurons also express ORL-1 nociceptin receptors (Table 1), and a recent study using a mouse line in which the ORL-1 protein was tagged with GFP found that 43% of DRG neurons were GFP-positive. GFP was expressed both in small and large neurons, with a slight dominance (58%) of neurofilament positive myelinated neurons. ⁹⁶ Nociceptin receptors were reported to inhibit N-type VGCC in DRG neurons in a tonic, agonist independent manner. ⁹⁷ Nociceptin receptors were also reported to be expressed in human DRG neurons, and the same study showed that their activation reduced capsaicin-induced Ca²⁺ signals in rat DRG neurons. ⁹⁸

Overall, there are conflicting data on the efficiency of stimulating peripheral opioid receptors in alleviating pain in mice, and there are clear receptor subtype specific effects. Peripheral κOR-s on the other hand are promising targets against itch in humans.

GABA_B receptors

GABA_B receptors are obligate heteromers of GABA_B1 and GABA_B2 subunits; the presence of both subunits is required for functional G-protein signaling for the following two reasons. First, the GABA binding site is on GABA_B1 receptors and the G_i-activating domain is on the GABA_B2 subunit. Second, GABA_B1 subunits have an ER retention signal, which prevents trafficking of the subunit in the absence of GABA_B2 receptors, which masks this signal when they form a dimer with GABA_B1. ⁹⁹

GABA_B receptors are the highest expressing GPCRs in DRG neurons on the RNA level ⁴¹ (Table 1). The only widely available GABA_B agonist baclofen is used clinically as a central muscle relaxant; its effect is attributed to inhibiting neurotransmitter release onto motoneurons in the ventral horn of the spinal cord. ¹⁰⁰ The use of systemic baclofen is limited by its severe side effects at higher doses such as drowsiness, mental confusion, and even coma, ¹⁰¹ which is not surprising, given the abundance of these receptors in the CNS. ⁹⁹ Systemic side effects can be limited by administering baclofen intrathecally, which is often done to reduce spasticity in various conditions. Baclofen is also used to treat pain conditions, as an adjuvant therapy, but its effect is mainly attributed to acting as a central muscle relaxant.

As discussed earlier, GABA_B receptor activation by baclofen was shown to activate GIRK channels, ³⁸ inhibit VGCC, ⁴⁹ and inhibit the heat-activated TRPM3 channels^55–57 in DRG neurons; these effects are mediated by the G_βγ arm of classical heterotrimeric G-protein signaling. All of these mechanisms, in principle, may mediate antinociceptive effects.

GABA_B receptors can also be activated by α-conotoxins. These toxins are generally considered to be inhibitors of nicotinic acetylcholine receptors, but some of them such as Vc1.1 and RgIA also inhibit N-type VGCC via activation of GABA_B receptors ¹⁰² reviewed in Adams et al. ¹⁰³ Accordingly, intramuscular injection of Vc1.1 was shown to induce a long-lasting reversal of mechanical allodynia, which was prevented by the GABA_B receptor antagonist, SCH50911. ¹⁰⁴

Activation of GABA_B receptors in DRG neurons by baclofen was recently shown to inhibit the sensitized state of TRPV1, but not the basal heat or capsaicin activation of TRPV1. The effect was independent of G_βγ signaling; it was mediated by direct protein–protein interaction between GABA_B1 receptors and TRPV1. ³² While GABA_B2 receptors were not detected in the protein complexes of TRPV1 and GABA_B1 receptors in DRG neurons, GABA_B2 receptors were required for the effect of baclofen both in a heterologous expression system and in DRG neurons. Baclofen was effective when injected locally, showing the presence of the receptors in the peripheral processes, and GABA was shown to be released from nociceptive nerve terminals, suggesting an autocrine feedback mechanism. ³² The growing evidence that these receptors have important antinociceptive effects in the periphery, raise the possibility that peripherally acting GABA_B receptor agonists can be developed as novel analgesics with less side effects.

SST receptors

SST receptors are expressed not only in DRG neurons but also centrally, as well as in inflammatory cells, and can affect nociception and inflammation; the topic is reviewed in literature.^105,106 Briefly, it has been shown that SST is released from activated capsaicin-sensitive nerve endings, and it can exert both local and systemic anti-nociceptive and anti-inflammatory effects.^105,107 Intraplantar injection of SST reduced mechanical allodynia in a rat inflammatory pain model. ¹⁰⁸ The SST receptor agonist octreotide inhibited formalin-induced nociceptive behaviors when injected locally, and it also reduced the responses of C-fibers to bradykinin-induced excitation and sensitization to heat. ¹⁰⁹ It was also shown that intraplantar injection of octreotide inhibited capsaicin-induced nocifensive responses in rats, and it also inhibited capsaicin-induced nerve activity in the skin-nerve preparation. ¹¹⁰ Furthermore, intra-articular injection of SST was shown to inhibit knee pain in humans. ¹¹¹ The SST4 receptor agonist J-2156 was shown to inhibit capsaicin-induced Ca²⁺ signals in rat DRG neurons, ¹¹² as well as activate GIRK channels and inhibit VGCC. ³⁹ SST4 receptor deficient mice showed increased mechanical hyperalgesia after carrageenan-induced inflammation, and the antinociceptive effect of the SSTR4 agonist J-2156 was absent in these animals. ¹¹³ Lipopolysaccharide-induced airway inflammation and bronchoconstriction were also markedly enhanced in SSTR4 knockout animals, pointing to the important role of these receptors in inflammatory cells. ¹¹³ SST was also shown recently to inhibit Ca²⁺ signals induced by the TRPM3 agonist PregS in a subset of mouse DRG neurons.^55,56 Targeting SST receptors for pain control is complicated by the fact that activation of these receptors have significant other effects, including inhibition of insulin release and inhibition of exocrine secretion and motor activity of the gastrointestinal tract, which may be overcome by developing subtype specific agonists. ¹⁰⁵

Metabotropic glutamate receptors

Metabotropic glutamate receptors (mGlur-s) can function either as homodimers, or as heterodimers. ¹¹⁴ They are divided into group I receptors (mGluR1 and 5) which signal via G_αq and group II (mGluR2 and 3), and group III (4,6,7, and 8), which signal via G_αi. ¹¹⁵ Group I mGluRs, similar to other PLC-coupled receptors, have been shown to be present on peripheral terminals of DRG neurons and play roles in inflammatory hyperalgesia, ¹¹⁶ reviewed in study by Neugebauer. ¹¹⁷

There are several articles showing antinociceptive effects of the activation of peripheral group II G_i/o-coupled mGluR-s. Subcutaneous injection of a selective group II mGluR agonist (APDC) into the plantar surface of the hind paw inhibited prostaglandin E2 (PGE2)-induced thermal hyperalgesia in mice. ¹¹⁸ The same study also showed that in cultured DRG neurons, APDC blocked PGE2-induced potentiation of capsaicin-induced Ca²⁺ responses, which was abolished when neurons were pretreated with PTX. Another article from the same group showed that subcutaneous injection of group II mGluR agonists into the plantar surface of the mouse hind paw did not alter basal mechanical thresholds, but inhibited PGE2- or carrageenan-induced mechanical allodynia. ¹¹⁹ Group II metabotropic glutamate receptor agonists also inhibited forskolin-induced potentiation of tetrodotoxin-resistant sodium currents in mouse DRG neurons. ¹²⁰ Finally, it was shown that membrane hyperexcitability in mouse and human DRG neurons exposed to PGE2 was prevented by the group II mGluR agonist APDC. ¹²¹

While several studies focused on group II mGluR-s, on the RNA level, group III mGluR-s show substantially higher expression in mouse DRG neurons (Table 1). Recent studies also demonstrated potential antinociceptive roles of this group; mGluR8 was found to be present in peripheral nociceptive terminals, and ipsilateral, but not contralateral hind paw injection of the group III mGluR agonist L-AP-4 inhibited nociceptive behavioral responses to capsaicin in rats. ¹²² Local L-AP-4 injection also attenuated forskolin-induced thermal hyperalgesia. ¹²² It was also shown that mGluR7 was expressed in small peptidergic and large rat DRG neurons. ¹²³ Nerve ligation experiments in the same study also showed that mGluR7 was anterogradely transported from the cell body to the peripheral site, and after peripheral nerve injury, mGluR7 expression was down-regulated. It was also shown that inhibiting peripheral group II/III mGluR-s by intraplantar injection of various antagonists increased capsaicin-induced nociceptive behaviors and nociceptor activity, ¹²⁴ indicating peripheral glutamate release. On the other hand, the mGluR group III agonist L-AP4 did not have a significant effect on TRPM3 activity, as assessed by PregS-induced Ca²⁺ signals, ⁵⁷ while agonists of many other G_αi/o-coupled receptors showed robust inhibition.^55–57

Overall, both excitatory group I and inhibitory groupII/III mGluR-s are expresed at peripheral nerve terminals, but the opposing effects of the two different receptor groups makes the effects of a potential peripheral glutamate release complex. Neverthelesss, in principle, both group I antagonsist and group II/III agonists may induce beneficial antinociceptive effects. ¹¹⁷

Adenosine receptors

ATP is released from many cell types and acts as a paracrine signal; it activates both metabotropic (P2X) and ionotropic (P2Y) receptors. Activation of both P2X and P2Y receptors in DRG neurons is generally excitatory. Secreted ATP becomes dephosphorylated rapidly to adenosine by ectoenzymes. ¹²⁵ Adenosine receptors are distinct from purinergic receptors and couple to different G-proteins. ¹²⁶ Adenosine 1 receptors (A1R, adora1) couple to G_i/o-proteins, and they are the most abundant adenosine receptors in DRG neurons; however, G_s-coupled Adenosine 2A receptors (A2AR) are also expressed in DRG neurons at lower levels^21,41 (see also Table 1). Adenosine release has been detected in response to capsaicin and formalin from nociceptive nerve fibers. ¹²⁷ Due to the presence of receptors with different signal transduction pathways, as well as to the fact that A1R may also couple to G_q, the local effects of adenosine can be quite complex, both pro- and antinociceptive effects have been observed, reviewed in study by Sawynok and Liu. ¹²⁶ The presence of various adenosine receptors on many other cell types including immune and vascular cells makes the overall effects of pharmacological modulation of this pathway quite complex. ¹²⁸

NPY receptors

NPY is a 36 amino acid peptide; it has five receptors Y1R–Y5R, all couple to G_αi/o proteins. DRG neurons express Y1R and Y2R (Table 1). NPY was shown to inhibit VGCC in rat DRG neurons^129,130 and it also inhibited depolarization-induced Ca²⁺ signals and release of substance P from DRG neurons. ¹³⁰ While both nociceptive and antinociceptive effects of NPY have been described, in general, it is believed that this peptide is mainly antinociceptive. ¹³¹ Two independent mouse lines with genetic deletion of Y1R have been generated, and the two studies largely agree that the knockout mice display hyperalgesia to mechanical and thermal stimuli.^132,133 NPY receptors are also expressed in the dorsal horn, and the analgesic effects of NPY may be due to activation of spinal receptors. ¹³⁴ Consistent with the main role of central NPY receptors, it was shown that intrathecal, but not local administration of NPY reduced guarding behavior in a rat model of plantar incision pain. ¹³⁵ As mentioned earlier, application of NPY or peptide YY inhibited PregS-induced activation of TRPM3 in mouse DRG neurons,^55,57 and local injection of peptide YY inhibited nocifensive responses evoked by the TRPM3 agonist PregS. ⁵⁷

Serotonin receptors

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) is part of the inflammatory soup that sensitizes nociceptors. It binds to a variety of receptors including ionotropic 5-HT3 receptors, and a variety of GPCRs coupling to G_q (5HT2), G_s (5HT4,6,7), and G_i/o (5HT1,5). ¹³⁶ Many of these receptors are expressed in DRG and TG neurons, and the overall effect of serotonin is complex, but pro-algesic effects likely dominate. Ionotropic HT3a receptors, for example, are expressed in the central termini and play roles in central sensitization to painful stimuli. ¹³⁷ Injecting serotonin or a 5HT2 receptor agonist in the hind paw of mice evoked hyperalgesia to mechanical stimuli indicating the presence of stimulatory 5HT2 receptors in the peripheral nerve termini. ¹³⁸ Serotonin also induced action potentials and potentiated TRPV1 currents in isolated DRG neurons through 5HT2C receptors. ¹³⁹ Sumatriptam, a drug, which is used to treat migraine headaches, ¹⁴⁰ selectively activates G_i/o-coupled 5HT1B and 5HT1D receptors, which are expressed in DRG neurons (Table 1). It is not clear to what extent direct effects of sumatriptam on TG neurons contribute to its beneficial effects, ¹⁴⁰ but the drug was shown to inhibit TRPV1 activity in TG neurons. ¹⁴¹ Serotonin application was shown to potentiate calcium signals and CGRP release induced by capsaicin in TG neurons, but sumatriptam had an inhibitory effect, showing opposing effects of activating different 5HT receptors expressed in those neurons. ¹⁴² Sumatriptam was also shown to induce hyperalgesic priming in rats, which may explain the clinical finding that the drug may contribute to migraine chronification. ³⁶

Designer receptors exclusively activated by designer drugs

Designer receptors exclusively activated by designer drugs (DREADDs) are mutated GPCRs that do not respond to endogenous ligands, but can be activated by synthetic compounds. Most of them are based on muscarinic acetylcholine receptors; they are activated by the inert clozapine derivative clozapine-N-oxide (CNO). ¹⁴³ DREADDs based on other receptors are also available, and are being developed. ¹⁴⁴ By expressing various forms of these receptors in specific cell types, the effect of activating G_i-, G_q-, or G_s-coupled receptors can be studied by applying their chemical activator. Together with optogenetic approaches, ¹⁴⁵ DREADDs, in principle, are promising selective tools to study the effects of activation or inhibition of specific neuronal populations in various pain conditions. DREADDs can be expressed in vivo either by crossing mice expressing cre-dependent DREADDs with cell-type specific cre-mice ¹⁴⁶ or by injecting DREADD-expressing viral particles.

Expressing inhibitory DREADDs in DRG neurons is a compelling strategy to achieve pain relief. Currently, there are two published articles using this strategy.

Iyer et al. ¹⁴⁷ showed that viral expression of the hM4-based G_i-coupled DREADD in small-diameter nociceptors enabled chemogenetic increase of mechanical and thermal nociception thresholds. In the same article, the authors found that transdermal illumination in mice expressing an inhibitory channelrhodopsin inhibited pain.

Another article however raised doubts about the peripheral G_i-coupled DREADD-based approach to inhibit pain. Saloman et al. ¹⁴⁸ expressed the G_i-coupled hM4Di receptor in nociceptive DRG neurons expressing the heat- and capsaicin-sensitive TRPV1 ion channel, by crossing TRPV1-cre mice with floxed hM4Di expressing mice. As expected, injection of CNO produced a significant increase in the heat threshold in these animals. Consistent with TRPV1 positive cells being largely insensitive to mechanical stimuli, mechanical sensitivity was not affected by CNO. Surprisingly, however, expression of these receptors induced significant changes in the absence of CNO, including changes in voltage-gated Na⁺ and Ca²⁺ currents, as well as an increase in the expression of Na_v1.7 channels. Expression of the G_i-coupled DREADD also reduced the effectiveness of stimulating endogenous μOR by DAMGO on PGE2-induced inflammatory thermal hyperalgesia. The authors concluded that while DREADDs are useful tools, they need additional refinement, especially for potential clinical use. Recognizing the imperfections in currently available DREADDs, novel receptors and compounds are being developed.^149,150

Additional caution on using these designer receptors have been raised by a recent paper showing that CNO is converted to clozapine in vivo, and the latter is responsible for activating them. ¹⁵¹ Clozapine is an atypical antipsychotic medication; its mechanism of action is not fully understood, but it inhibits certain dopamine and serotonin receptors. The doses required in vivo activation of DREADDs were below that required to exert effects in animals not expressing DREADDs, suggesting that this compound can be more useful than CNO for in vivo use. ¹⁵¹

Optogenetic approaches

Optogenetic approaches classically use light-activated ion channels to study the effects of activating or inhibiting specific neurons and have been used in pain research, see Copits et al. ¹⁴⁵ for review. In addition to light-activated ion channels, various GPCRs have also been engineered to become light sensitive. Among G_i-coupled receptors, a photoactivatable μOR was created by splicing together the transmembrane and extracellular parts of the light-activated GPCR rhodopsin, and the intracellular loops and C-terminus of μ-opioid receptor. ¹⁵² This opto-μOR was virally expressed in isolated DRG neurons, where they were shown to increase the phosphorylation of extracellular signaling-regulated kinase. Opto-μOR and other light inducible G_i-coupled receptor constructs are promising tools to study the effects of G_i-coupled receptor activation in DRG neurons.