GPR171 Activation Modulates Nociceptor Functions, Alleviating Pathologic Pain

biomedicines

Article GPR171 Activation Modulates Nociceptor Functions, Alleviating Pathologic Pain

Pyung Sun Cho 1,2, Han Kyu Lee 3 , Young In Choi 3 , Seung In Choi 1,2, Ji Yeon Lim 1,2, Minseok Kim 1,2 , Hyun Kim 4 , Sung Jun Jung 3,* and Sun Wook Hwang 1,2,*

1 Department of Biomedical Sciences, College of Medicine, Korea University, Seoul 02841, Korea; [email protected] (P.S.C.); [email protected] (S.I.C.); [email protected] (J.Y.L.); [email protected] (M.K.) 2 Department of Physiology, College of Medicine, Korea University, Seoul 02841, Korea 3 Department of Physiology, College of Medicine, Hanyang University, Seoul 04763, Korea; [email protected] (H.K.L.); [email protected] (Y.I.C.) 4 Department of Anatomy, College of Medicine, Korea University, Seoul 02841, Korea; [email protected] * Correspondence: [email protected] (S.J.J.); [email protected] (S.W.H.)

Abstract: Modulation of the function of somatosensory neurons is an important analgesic strategy, requiring the proposal of novel molecular targets. Many G-protein-coupled receptors (GPRs) have been deorphanized, but the receptor locations, outcomes due to their activations, and their signal transductions remain to be elucidated, regarding the somatosensory nociceptor function. Here we report that GPR171, expressed in a nociceptor subpopulation, attenuated pain signals via Gi/o- coupled modulation of the activities of nociceptive ion channels when activated by its newly found

ligands. Administration of its natural peptide ligand and a synthetic chemical ligand alleviated nociceptor-mediated acute pain aggravations and also relieved pathologic pain at nanomolar and micromolar ranges. This study suggests that functional alteration of the nociceptor neurons by Citation: Cho, P.S.; Lee, H.K.; Choi, Y.I.; Choi, S.I.; Lim, J.Y.; Kim, M.; Kim, GPR171 signaling results in pain alleviation and indicates that GPR171 is a promising molecular H.; Jung, S.J.; Hwang, S.W. GPR171 target for peripheral pain modulation. Activation Modulates Nociceptor Functions, Alleviating Pathologic Keywords: GPR171; pain; analgesia; nociceptor; TRP channel Pain. Biomedicines 2021, 9, 256. https://doi.org/10.3390/ biomedicines9030256 1. Introduction Academic Editor: Anand Rotte Nociceptor neurons in the dorsal root ganglia (DRG) are in charge of the initiation of pain-related signals in the body [1]. Upon activation by ligands, G-protein-coupled Received: 1 February 2021 receptors (GPRs) modulate the excitability of nociceptors, negatively or positively tuning Accepted: 21 February 2021 the strength of the initiated signals. While some GPRs, such as prostaglandin EP2 and EP4 Published: 5 March 2021 receptors and bradykinin B2 receptors strongly facilitate nociceptor excitation, other GPRs, including µ-opioid receptors, GABA receptors, and chemokine-like receptor 1 receptors, Publisher’s Note: MDPI stays neutral B reduce excitation, highly contributing to relieving pain [2–6]. Better understanding of the with regard to jurisdictional claims in published maps and institutional affil- identities of pain-relieving GPRs and the molecular mechanisms underlying the reduction iations. in excitability may help to contribute to the development of novel painkilling strategies. GPR171 was initially cloned two decades ago but was recently deorphanized [7,8]. Here we examined its expression in the nociceptor population and hypothesized that GPR171 activation by its specific ligands may modulate nociceptor functions, consequently regulating pain phenotypes. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 2. Materials and Methods This article is an open access article 2.1. Nociceptive Behavioral Tests distributed under the terms and conditions of the Creative Commons Six-week-old male C57BL/6J mice were used. Time engaged in hind paw licking Attribution (CC BY) license (https:// and flicking behavior was quantitated for ~10 min as previously described [9–11] (Supple- creativecommons.org/licenses/by/ mentary Figure S8A). For eliciting TRPV1-activation-induced behaviors, 100 ng capsaicin- 4.0/). containing 10 µL saline was intraplantarily injected; for eliciting TRPA1-activation-induced

Biomedicines 2021, 9, 256. https://doi.org/10.3390/biomedicines9030256 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 256 2 of 17

behaviors, 100 ng AITC in 10 µL saline or 26.4 µg cinnamaldehyde in 20 µL saline was injected. For eliciting depolarization-induced behaviors mediated by voltage-gated channel activation, 140 mM KCl in 10 µL water was injected. For conventional TRPV4-mediated flinch assays, we used intraplantar injection of 10 µL deionized water for hypotonic stimulation 30 min after prostaglandin E2 (PGE2) priming (intraplantar pretreatment with 10 µL saline containing 100 ng PGE2) [11]. TRPV4-mediated nociceptive behaviors were observed by counting the number of hind paw flinching events for 10 min immediately after the hypotonic stimulus. For observing formalin-induced acute and tonic pain, 5% or 0.5% formalin in saline (20 µL) was intraplantarily injected and the cumulative time that a mouse spent licking and flinching the injected paw was measured every 5 min for 50 min. For CFA-induced inflammation, 10 µL CFA was injected into a hind paw (Supplemen- tary Figure S8D). For determining changes in mechanical or thermal behaviors by 24 and 48 h CFA inflammation, mice were acclimated to the test environment for 30 min before performing the following assays. Hargreaves assay (using a Plantar Analgesia meter; IITC, Woodland Hills, CA, USA) for acute heat avoidance or thermal hyperalgesia, von Frey assay by up-and-down paradigm (using von Frey filaments; Stoelting, Dale Wood, IL, USA) for mechanical allodynia, and Randall–Selitto assay (using Analgesy-meter, UGO Basile, Italy) for flexion reflex to noxious pressures, were carried out as described previously [12–16] (Supplementary Figure S8C). For neuropathic pain development, the CCI model was established as described previously [17] (Supplementary Figure S8D). Briefly, the mice were anesthetized with inhalation of 3% isoflurane in a mixture of N2O/O2 gas. The full circumference of the sciatic nerve of the left hind limb was exposed and loosely tied three times with two silk sutures (7-0; Ailee, Busan, Korea). Sham surgery was conducted by exposing the sciatic nerve in the same manner but without tying the nerve. Hargreaves and von Frey assays were performed with the injured mice 14 days after the surgery. For a postoperative pain model, an incision pain model was modified according to a previous study [18] (Supplementary Figure S8D). Briefly, mice were anesthetized with 2% isoflurane and hind paws was sterilized with 10% povidone–iodine solution. With a no. 10 scalpel blade, a 5 mm longitudinal incision was made in the glabrous skin and fascia of a hind paw plantar. The underlying muscle was elevated with a sterile curved forcep, with the muscle origin and insertion intact. The incised skin was apposed with two nylon sutures (8-0; Ailee, Korea). Sham mice were not incised or sutured but anesthetized for the same duration.

2.2. Open Field Behavioral Tests The open field was a square opaque white box (40 × 40 × 40 cm) that was divided into a central field (center, 20 × 20 cm) and an outer field (periphery). After drug injection, individual mice were exposed for 30 min to the open field box under dim light, and the total distance traveled, the time spent in the central field, and rearing numbers were monitored with TSE ActiMot/MoTil system (TSE systems, Bad Homburg, Germany) as described previously [19] (Supplementary Figure S8E).

2.3. Sticky-Tape Removal Tests A circular piece of tape, 9 mm in diameter, was placed on the bottom of a hind paw of the animals. The latency of biting or licking to remove the tape was measured as described previously [20] (Supplementary Figure S8B).

2.4. Measurements of Food Intake, Water Intake, and Body Weight Experiments were conducted as described previously [21]. Brieﬂy, mice fasted for 12 h were treated with drugs and then returned to their individual cages. Cumulative food and water consumptions were recorded at 1, 2, 4, and 8 h after drug treatment. For intraperitoneal vehicle, 1% DMSO in sterile saline was used. For long-term measurement, drugs were administered on every third day for forty-eight days and food and water con- Biomedicines 2021, 9, 256 3 of 17

sumptions were recorded at 8 h after every drug treatment. Body weights were measured on every fifth day for fifty days from the first treatment.

2.5. Immunostaining Analysis Lumbar DRG, spinal cord, and glabrous skin biopsies were prepared from mice at 6 weeks. Sections (14 µm for DRG and 30 µm for spinal cord and glabrous skin) prepared using a Leica CM3050s cryotome (Leica Microsystems, Wetzlar, Germany) were perme- abilized with 0.2% Triton-X100 for 1 h. For cultured DRG neurons, cultures were ﬁxed in 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton-X100 for 10 min and blockade with 3% bovine serum albumin in phosphate-buffered saline. Samples were then stained with anti-GPR171 (rabbit, 1:200; Abcam) anti-TRPV1 (mouse, 1:100; Ab- cam), anti-TRPA1 (sheep, 1:200; Antibodies-online, Aachen, Germany), anti-TRPV4 (sheep, 1:500; Invitrogen, Rockford, IL. USA), anti-CGRP (goat, 1:2000; Abcam), anti-NF200 (mouse, 1:100; Sigma-Aldrich), anti-NeuN (mouse, 1:100; Chemicon, Temecula, CA, USA), and/or anti-PGP9.5 (mouse, 1:50; Abcam) antibodies. After 24 h at 4 ◦C, sections were stained with secondary antibodies (Alexa 594-labeled or Alexa 488-labeled secondary antibodies; Life Technologies) for 1 h at room temperatures and placed under a coverslip, followed by image acquisition with an iRiS Digital imaging system (Logos Biosystems, Anyang, Korea) or Zeiss LSM 800 confocal microscope (Carl Zeiss, Oberkochen, Germany).

2.6. Reverse Transcription–Polymerase Chain Reaction (RT-PCR) and Quantitative RT-PCR GPR171 mRNA expression was validated using RT-PCR or qRT-PCR as described previously [22]. We reverse-transcribed 1 µg RNA with the amﬁRivert Platinum cDNA synthesis Master Mix (GenDEPOT, Baker, TX, USA). cDNA was subjected to qRT-PCR with the SYBR Green PCR Master Mix (Applied Biosystems, Woolston, UK) or RT-PCR with 5 × PCR Master Mix (Elpisbio, Daejeon, Korea). β-actin mRNA was the internal control. Primer sequences are listed below. For mouse β-Actin: F-GGCTCTTTTCCAGCCTTCCTT, R-CCACCGATCCACACA: GAGTACT; NM_007393 For mouse GPR171 version 1: F-CTGGCGGTGTCTAATTTGTG, R-TTTCTTCCAG AGGCTTGCTC; NM_173398 For mouse GPR171 version 2: F-TGCCGAACATGGTGATTCCC, R-TATGGGACTGAC AGCGTG; NM_173398 For mouse GPR171 version 3: F-GCTTTGTTCCCTACCACGCT, R-TGAG GCGAAAGT CTCGGTG; NM_173398

2.7. Cell Cultures and Transfections Cultures of DRG neurons and HEK293 cells were prepared and transfected as de- ◦ scribed previously [11,23,24]. All cells were grown at 37 C and 5% CO2 and neurons were used at 48–72 h after culture. For in vivo transfection of small-interfering RNA (siRNA) or scrambled RNA (scRNA), 3 µg of RNA and 5% d-glucose in 7 µg lipofectamine 2000 (Invitrogen) were injected into the spinal cord or peri-sciatic region 24 or 48 h before experiments. Mouse GPR171-siRNA (target sequence: sense-CUGUACUAUCAUCUGUCAA, antisense-UUGACAGAUGAUAGUACAG) and a scrambled RNA were purchased from Bioneer Corporation (Daejeon, Korea). cDNA for human GPR171 was generously gifted by Dr. Jae Young Seong of Korea University.

2.8. Fluorescence Intracellular Ca2+ Imaging Experiments The Ca2+ imaging experiments were conducted as described previously [25]. Brieﬂy, cells were loaded with 5 µM Fura-2AM dye and 0.02% pluronic F127 for 30 min. The cells were resuspended in 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (titrated to pH 7.4 with NaOH). Images of dye-loaded cells were obtained with a cooled charge-coupled device (CCD) camera (Retiga-SRV, Q-imaging Corp., Burnaby, BC, Canada) at a perfusion rate of approximately 4 mL/min of test solutions and Biomedicines 2021, 9, 256 4 of 17

at room temperatures. The ratio of ﬂuorescence intensity at 340 nm/380 nm wavelengths in each experiment was analyzed using MetaFluor (Molecular Devices, Sunnyvale, CA, USA).

2.9. Spinal Cord Slice Preparation Mice were decapitated under anesthesia with isoflurane (VetOne, Boise, ID, USA); the lumbar spinal cord was rapidly removed and placed in ice-cold modified artificial cerebrospinal fluid (ACSF) containing 80 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 0.5 mM CaCl2, 3.5 mM MgCl2, 25 mM NaHCO3, 75 mM sucrose, 1.3 mM sodium ascorbate, and 3 mM sodium pyruvate, with pH at 7.4 and osmolality at 310–320 mOsm/kg, bubbled with 95% O2 and 5% CO2. Spinal cord slices (250–350 µm) were cut horizontally by a VT1200s vibratome (Leica, Germany). The slice was then incubated for approximately 1 h ◦ at 33 C in oxygenated (95% O2 and 5% CO2) cutting solution containing 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, 25 mM D-glucose, 1.3 mM sodium ascorbate, and 3 mM sodium pyruvate, with pH at 7.2 and osmolality at 310–320 mOsm/kg.

2.10. Whole-Cell Patch Clamp Recordings The slices after incubation as described were placed in a recording chamber and perfused with oxygenated recording solution at a rate of 5 mL/min at room temperatures. Whole-cell recording experiments were then performed on lamina 2 dorsal horn neurons. Borosilicate glass pipettes (Sutter Instrument, Novato, CA, USA) with resistance of 3–6 MΩ were filled with internal solution containing 130 mM potassium gluconate, 5 mM KCl, 4 mM Na2ATP, 0.5 mM NaGTP, 20 mM HEPES, and 0.5 mM EGTA with pH 7.28 adjusted with KOH and osmolality at 310–320 mOsm/kg. Data were acquired by pClamp 10.0 software (Molecular Devices) with MultiClamp 700B patch-clamp amplifier and Digidata 1550B (Molecular Devices). Data were low-pass filtered at 2 kHz and digitized at 5 kHz.

2.11. Ex Vivo Nociceptor Recordings Ex vivo single-fiber recordings were carried out as previously described [26]. The two-chambered organ bath system (perfusion and recording chambers) was used. The perfusion chamber was continuously superfused with a synthetic interstitial fluid (SIF) com- posed of 3.5 mmol/L KCl, 107.8 mmol/L NaCl, 0.69 mmol/L MgSO4·7H2O, 1.53 mmol/Ll CaCl2·2H2O, 1.67 mmol/L NaH2PO4·2H2O, 26.2 mmol/L NaHCO3, 9.64 mmol/L C6H11NaO7, 7.6 mmol/L sucrose, and 5.55 mmol/L glucose, which was saturated with a mixture of 95% ◦ O2 and 5% CO2 and maintained at 31 ± 1 C. The hairy skin of the hind paw innervated by the saphenous nerve was dissected from mice sacrificed with CO2 inhalation. The preparation was put in the perfusion chamber with the epidermal side down. The nerve connected to the skin flap was drawn to the recording chamber filled with paraffin oil. The nerve filaments teased from the nerve were used for single-fiber recordings using gold electrodes. Spikes from single C-fiber nociceptors were recorded with a differential amplifier (DP 311; Warner Instruments, Hamden, CT, USA), and the data were transferred to a personal computer via a data acquisition system (DAP5200a; Microstar Laboratory, Redmond, WA, USA). Data were analyzed using the window discrimination feature of the software (Dapsys 8; Bethel University, http://dapsys.net/ accessed on 5 March 2021) [27].

2.12. Compounds All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless other- wise described. BigLEN and MS15203 were purchased from Tocris Bioscience (Ellisville, MO, USA). Cinnamaldehyde was purchased from MP Biomedicals (Solon, OH, USA). Stock solutions were prepared using water or dimethyl sulfoxide and diluted with test solutions before use. Biomedicines 2021, 9, 256 5 of 17

2.13. Data Analysis Statistical signiﬁcance of data was assessed using the two-tailed unpaired Student’s t-test, one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test, or two-way ANOVA followed by Tukey’s post hoc test (*** p < 0.001, ** p < 0.01, * p < 0.05). Data are shown as means ± S.E.M.

2.14. Data Availability All relevant data are available from the authors upon request.

3. Results 3.1. GPR171 Is Expressed in Nociceptors We first examined GPR171 immunostaining in DRG, which initiate nociceptive signals. We found GPR171 expression in a subpopulation of DRG neurons (502 of 1498; 33.5%). We further examined whether other subpopulation markers were co-expressed with GPR171 (Figure1A,B). Peptidergic and non-peptidergic nociceptor markers including calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4)-binding components were co-localized with GPR171 expression as well as the marker for myelinated DRG neurons, neurofila- ment 200 (NF200). Over fifty percent of the GPR171-positive subpopulation co-expressed CGRP and at least a third of IB4-positive non-peptidergic nociceptors were also GPR171- positive (Figure1B). Neuronal diameter analysis was also performed on the DRG neurons. Small-diameter neurons, which are mostly comprised of C- and or Aδ-fiber nociceptors, constituted the majority of GPR171-expressing neurons, particularly around neurons with 15–20 µm in diameter (Figure1C). Approximately half of these small-diameter neurons expressed GPR171, and these proportions were two times greater than the subpopulation with diameters 35 µm and larger. These data suggest that considerable numbers of nociceptor DRG neurons express GPR171. Immunostaining of the glabrous skin layers, spinal cord, and cultured DRG neurons of mice also revealed GPR171 expression (Supplementary Figure S1). DRG nerve termini in the skin epidermis partly showed its co-expression with CGRP but not with IB4 (Supplementary Figure S1A) [28,29]. Thus, these data indicate a potential role of GPR171 in nociceptor DRG neurons. We then speculated whether GPR171 functions in nociception and examined the effect of its specific activation using the conventional formalin test. Intraplantar and intrathecal administration of endogenous or synthetic GPR171 agonists (bigLEN and MS15203, respectively) both significantly mitigated phase 2 nociceptive behaviors induced by 5% formalin injection (Figure1D,E). Interestingly, when we performed the Transient receptor potential ankyrin subtype 1 (TRPA1) activation-specific formalin test using a lower dose of formalin (0.5%) [30,31], both phase 1 and 2 nociceptive behaviors were reduced by GPR171 activation (Figure1F,G). The data suggest that GPR171 expressed in peripheral nociceptors may play a role in modulating their excitability, implicating that modality-specific components such as Transient receptor potential (TRP) ion channels may be involved in this mechanism.

3.2. Peripheral GPR171 Activation Attenuates TRP-Specific Acute Pain Based on the data shown above, we hypothesize possible co-expressions of nociceptive TRP ion channels (TRPV1, TRPA1, and TRPV4). When immunostained, over a half of TRP-positive neurons co-expressed GPR171 and approximately a quarter of the DRG neuronal population expressing GPR171 also expressed at least one of those three TRP channels (Figure2A,B). Therefore, we then focused on these TRP-specific pain modalities. Licking and flicking induced by intraplantar injection of cinnamaldehyde, which is a TRPA1 activator, and induced by the TRPV1 activator capsaicin as well as flinches in response to TRPV4 activation using hypotonic insult were all blunted when the GPR171 activator, MS15203, was peripherally pre-treated (Figure2C,E). In contrast, nociceptive behaviors by the activation of voltage-sensing components were not affected (Figure2F). Thus, rather than general excitability of the nociceptors, TRP-channel-specific mechanisms may be Biomedicines 2021, 9, 256 6 of 17

Biomedicines 2021, 9, x FOR PEER REVIEW 6 of 17 modulated by GPR171 action in these neurons, which may also explain the attenuation of phase 1 nociception because, at least partly, TRPA1 activation is known to be involved.

FigureFigure 1. PR171 1. PR171 expression expression in a insubset a subset of dorsal of dorsal root root ganglia ganglia (DRG (DRG)) neurons. neurons. (A (A) Double) Double immunostaining immunostaining of of GPR171 with otherwith DRG other markers DRG in markers the lumbar in the DRG lumbar (scale DRG bar, (scale 50 μm bar,). 50 Arrowheadsµm). Arrowheads indicate indicate the marke the marker-expressingr-expressing somas somas for example. for (B) Coexample.-expression (B) Co-expression ratios of GPR171 ratios ofand GPR171 other andDRG other markers. DRG markers. The ratios The were ratios calculated were calculated as mean as mean percent percent values values by by counting thecounting number the of number GPR171 of GPR171-positive-positive neurons neurons among among that of that marker of marker-positive-positive neurons neurons from from three three different different mice andand vice versa.vice Data versa. are Datapresented are presented as means as means± S.E.M.± S.E.M. (C) Size (C) distribution Size distribution of collected of collected DRG DRG neurons neurons and and GPR171 GPR171-positive-positive DRG neuronsDRG according neurons according to soma to diameters. soma diameters. (D) Time (D) Timecourse course of the of theduration duration of of5% 5% formalin formalin-induced-induced nociceptivenociceptive behaviors behaviors in mice.in Animals mice. Animals were wereintraplantarily intraplantarily (i.pl. (i.pl.)) pretreated pretreated with with vehicle vehicle ( (red),red), oror GPR171-speciﬁc GPR171-specific agonists, agonists, MS15203 MS15203 (2.5 µ(g,2.5 μg, greengreen)) or bigLEN or bigLEN (2.5 (2.5 μg,µg, blue blue),), 30 30 min priorprior to to the the formalin formalin injection. injection. The The formalin formalin responses responses were quantiﬁed were quantified into the two into the two phases ofof the the right right histogram. histogram. (E )( ExperimentsE) Experiments were were conducted conducted as in ( Das) in but (D animals) but animals were intrathecally were intrathecally (i.t.) pretreated (i.t.) pretreated with withvehicle vehicle or or drugs. drugs. (F )(F Experiments) Experiments were we conductedre conducted as in (asD) in but (D animals) but animals were treated were withtreated 0.5% with formalin. 0.5% (G)formalin. Experiments (G) Exper- imentswere were conducted conducted as in as (F) in but (F animals) but animals were intrathecally were intrathecally pretreated pretreated with vehicle with or drugs.vehicle Five or drugs. animals Five were animals used for were each used for eachdata data point point of (D –ofG ).(D Effects–G). Effects on the on durations the durations of the nociceptive of the nociceptive responses responses of drugs in of (D drugs–G) at in every (D–G 5 min) at every (left panels) 5 min (left panelswere) were compared compared with with those those with vehiclewith vehicle injection injection by two-way by two ANOVA-way ANOVA and Tukey’s and test Tukey’s (* for MS15203test (* for treatment MS15203 and treatment # and #for for bigLEN bigLEN treatment), treatment and), and the phasethe phase comparisons comparisons (right ( panels)right panels were) conducted were conducted by one-way by one ANOVA-way ANOVA and Bonferroni’s and Bonfer- roni’stest. test. Statistical Statistical differences differences are indicatedare indicated by * for by the * for effects the effects of MS15203 of MS15203 and # for and those # for of bigLEN,those of comparedbigLEN, compared to those of theto those ### ## # of thevehicles. vehicles. *** ***p < p 0.001, < 0.001, ** p **< 0.01,p < 0.01, * p < * 0.05; p < 0.05;### p < 0.001, p < 0.001,## p < 0.01, p < 0.01,# p < 0.05. p < 0.05.

We then speculated whether GPR171 functions in nociception and examined the effect of its specific activation using the conventional formalin test. Intraplantar and intrathecal administration of endogenous or synthetic GPR171 agonists (bigLEN and MS15203, respectively) both significantly mitigated phase 2 nociceptive behaviors induced by 5% formalin injection (Figure 1D,E). Interestingly, when we performed the Tran- sient receptor potential ankyrin subtype 1 (TRPA1) activation-specific formalin test using a lower dose of formalin (0.5%) [30,31], both phase 1 and 2 nociceptive behaviors were Biomedicines 2021, 9, x FOR PEER REVIEW 7 of 17

reduced by GPR171 activation (Figure 1F,G). The data suggest that GPR171 expressed in peripheral nociceptors may play a role in modulating their excitability, implicating that modality-specific components such as Transient receptor potential (TRP) ion channels may be involved in this mechanism.

3.2. Peripheral GPR171 Activation Attenuates TRP-Specific Acute Pain Based on the data shown above, we hypothesize possible co-expressions of nociceptive TRP ion channels (TRPV1, TRPA1, and TRPV4). When immunostained, over a half of TRP-positive neurons co-expressed GPR171 and approximately a quarter of the DRG neuronal population expressing GPR171 also expressed at least one of those three TRP channels (Figure 2A,B). Therefore, we then focused on these TRP-specific pain modalities. Lick- ing and flicking induced by intraplantar injection of cinnamaldehyde, which is a TRPA1 activator, and induced by the TRPV1 activator capsaicin as well as flinches in response to TRPV4 activation using hypotonic insult were all blunted when the GPR171 activator, MS15203, was peripherally pre-treated (Figure 2C,E). In contrast, nociceptive behaviors by the activation of voltage-sensing components were not affected (Figure 2F). Thus, rather than general excitability of the nociceptors, TRP-channel-specific mechanisms may Biomedicines 2021, 9, 256 be modulated by GPR171 action in these neurons, which may also explain the 7attenuation of 17 of phase 1 nociception because, at least partly, TRPA1 activation is known to be involved.

FigureFigure 2. 2. GPR171GPR171 expressions expressions in Transient Transient receptorreceptor potential potential (TRP)-expresser (TRP)-expresser neurons neurons and and the the effecteffect on on TRP TRP-mediated-mediated nociceptions nociceptions of GPR171of GPR171 activation. activation. (A) Double (A) Double immunostaining immunostaining of GPR171 of with TRP channels in the lumbar DRGs (scale bar, 50 µm). (B) Co-expression ratios of GPR171 and TRP channels of the neurons from three different mice. Data are presented as means ± S.E.M. (C) Time course of the duration of 10 mM cinnamaldehyde (CA)-induced nociceptive behaviors in mice. Animals were intraplantarily pretreated with vehicle (red), or GPR171-specific agonist, MS15203 (2.5 µg, green), 30 min prior to the CA injection. From (C) to (E), black circles represent the mean data from the animals pretreated with MS150203, 30 min prior to the vehicle injection instead of TRP activators. Total durations of nociceptive responses for 5 min are quantified in the right histogram. (D) Time course of the duration of 0.1% capsaicin (CAP)-induced nociceptive behaviors and the effect on it of MS15203 were monitored in the same way as in (C). (E) Time course of the number of hypotonicity-induced flinches in prostaglandin E2 (PGE2)-primed mice. Animals were intraplantarily pretreated with vehicle (red) or MS15203 (2.5 µg, green), 30 min prior to the hypotonic stimulation. Total number of flinches for 10 min are quantified in the right histogram. (F) Time course of the duration of 140 mM KCl-induced nociceptive behaviors in mice. Animals were intraplantarily pretreated with vehicle (red) or MS15203 (2.5 µg, green), 30 min prior to the KCl injection. Total durations of nociceptive responses for 10 min are quantified in the right histogram. In (C–F), five animals were used for each data. Statistical differences are indicated by * for the effects of MS15203 compared to the responses upon chemical stimulations. *** p < 0.001. Biomedicines 2021, 9, 256 8 of 17

3.3. GPR171 Modulates DRG Neuronal Functions We further analyzed the mechanism of GPR171 action using in vitro and ex vivo preparations. In cultured DRG neurons, the TRPV1-mediated increase in intracellular Ca2+ upon capsaicin perfusion was blunted by the GPR171-specific agonist, MS15203 or bigLEN, incubation (Figure3A and Supplementary Figure S3A). This result is reproducible in whole-cell voltage clamp experiments (Figure3J). The inhibitory effect disappeared when pretreated with pertussis toxin but not with gallein, which confirmed that Gi/o signaling is coupled and that Gα than Gβγ is critical in this GPR action (Figure3B,C; Supplementary Figure S3A–C) [7]. The inhibition was also observed with TRPA1-, TRPV2-, TRPV4-, TRPM3-, and TRPC4/5-mediated increases in intracellular Ca2+ (Figure3D,E; Supplementary Figure S3D–F). Again, Ca2+ responses of DRG neurons by activation of voltage-sensing components that were readily modulable by µ-opioid receptor activation were resistant (Figure3F) , which is confirmed by whole-cell voltage clamp assays (Supplementary Figure S4A–H) and suggests that TRP-channel-specific events were particularly modulable by GPR171 action. Ex vivo C-fiber recording in skin–nerve preparation also showed the same outcomes from peripheral perfusion of bigLEN for inhibition of capsaicin-induced firing (Supplementary Figure S4M,N). Therefore, TRP-mediated excitatory events can be regulated by GPR171 activation in the peripheral termini of nociceptors. We recorded the excitatory post-synaptic current (EPSC) of dorsal horn neurons to observe the effect of GPR171 in the spinal cord. The frequency of EPSC but not its amplitude evoked by capsaicin perfusion was significantly reduced with treatment with either bigLEN or MS15203, indicating that TRPV1-mediated presynaptic events in the DRG central termini were selectively attenuated by GPR171 activation (Figure3G–I; Supplementary Figure S4I–L). Altogether, these data suggest that TRP activation in nociceptor DRG neurons may be a crucial target of GPR171 action in peripheral nociceptive signaling.

3.4. Peripheral GPR171 Activation Attenuates Pathologic Pain To determine whether the anti-nociceptive mechanisms of GPR171 work in pathologic pain states in vivo, we first tested peripheral injection of GPR171-specific agonists in complete Freund’s adjuvant (CFA)-induced hind paw inflammatory pain. Two injection routes were selected to examine whether the targeting of peripheral and central termini of nociceptors works. Allodynic mechanical hypersensitivity and hyperalgesic heat hypersensitivity were both improved by intrathecal agonist injection (Figure4A,B) or by its ipsilateral intraplantar injection (Figure4C,D). These analgesic effects increased in a dose- dependent manner, which indicates that the analgesia is receptor-dependent and that doses of hundreds nanomolar to micromolar concentrations are relatively effective (Figure4E,F and Supplementary Figure S5). We also tested the effect of the specific agonists on chronic constriction injury (CCI)-induced pain. The analgesic effects were largely similar but more prominent in mechanical allodynia compared with those observed in CFA-induced pain (Figure5A–D). These effects also showed dose dependence (Figure5E,F; Supplementary Figure S6). In an incisional pain model of mice, GRP171 activation was also effective at allodynic and hyperalgesic pain alleviation (Figure6). Biomedicines 2021, 9, x FOR PEER REVIEW 9 of 17 Biomedicines 2021, 9, 256 9 of 17

FigureFigure 3. GPR171 3. GPR171 modulates modulates TRP TRP channel channel functions functions in in DRG DRG neurons. neurons. ((AA)) MS15203 MS15203 attenuated attenuated intracellular intracellular Ca2+ Caincreases2+ increases by 0.3 byμM 0.3 capsaicinµM capsaicin-induced-induced TRPV1 TRPV1 activation activation in in Fura Fura-2-2 Ca Ca2+2+ imagingimaging experiments using using cultured cultured murine murine DRG DRG neurons neurons (n = 30(n). =Averaged 30). Averaged Ca2+ Capeaks2+ peaks are quantified are quantified and and normalized normalized in in the the right histogramhistogram in in (A ()A to) to (F ).(F (B).) ( PreincubationB) Preincubation of of pertussispertussis toxin toxin(PTX (PTX)) for 18 for h 18 prevented h prevented the the effect effect of of GPR171 GPR171 agonists agonists shownshown in in (A) (A ()n (=n 10).= 10 ()C. ()C Co-application) Co-application of 100 of µ100M µM galleingallein did not did prevent not prevent the theeffect effect of ofGPR171 GPR171 agonists agonists shown shown in ((AA)() n(n= = 10). 10) (. D(D) MS15203) MS15203 attenuated attenuated intracellular intracellular Ca2+ Ca2+ increasesincreases by 300 by μM 300 cinnamaldehydeµM cinnamaldehyde-induced-induced TRPA1 TRPA1 activation activation in in Fura Fura-2-2 Ca Ca2+2+ imagingimaging using culturedcultured DRG DRG neurons neurons (n = 23). ((En)= MS15203 23). (E) MS15203attenuated attenuated intracellular intracellular Ca2+ increases Ca2+ increases by 0.1 byμM 0.1 GSK1016790AµM GSK1016790A (abbreviated (abbreviated to GSK to GSK)-induced)-induced TRPV4 activationTRPV4 in activationFura-2 Ca in2+ Fura-2imaging Ca 2+usingimaging cultured using DRG cultured neurons DRG neurons (n = 12) (.n (=F) 12). The (F µ) The-opioidµ-opioid receptor receptor agonist agonist [D [D-Ala-Ala2, 2NMe, - Phe4, GlyNMe-Phe-ol5]-enkephalin4, Gly-ol5]-enkephalin (DAMGO (DAMGO),), but not but MS15203, not MS15203, attenuated attenuated intracellular intracellular Ca Ca2+2+ increasesincreases by by 60 60 mM mM KCl-induced KCl-induced voltagevoltage-gated-gated channel channel activation activation in Fur ina Fura-2-2 Ca2+ Ca imaging2+ imaging using using cultured cultured DRG DRG neurons neurons (n ( n= =36 36).). Experiments Experiments shown shown in in (A)– (F) were(A– performedF) were performed in triplicate. in triplicate. KCl stimulation KCl stimulation was was used used for for monitoring monitoring cell cell survival at at the the end end of theof the fluorescence fluorescence monitoring.monitoring. (G–I) ( GRepresentative–I) Representative traces traces of ofcapsaicin capsaicin-induced-induced miniatureminiature excitatory excitatory postsynaptic postsynaptic currents currents (mEPSCs) (mEPSCs in lamina) in lamina 2 dorsal2 dorsal horn horn neurons neurons ( (GG)) and and its cumulativecumulative probability probability plot plot of theof the inter-event inter-event intervals intervals (H) and (H) the and peak the amplitudes peak amplitudes (I) (I) of recordingsof recordings with with or or without without 10 10 μMµM MS15203 MS15203 treatment ( n(n= = 3). 3). ( J()J) Representative Representative traces traces of capsaicin-inducedof capsaicin-induced inward inward currentscurrents in whole in whole-cell-cell voltage voltage clamp clamp recordings recordings of ofcultured cultured DRG DRG neurons neurons (left)(left) and and its its quantification quantification of peakof peak amplitudes amplitudes (right,(right, n = 5) nwith= 5) withor without or without 10 μM 10 µ MMS15203 MS15203 treatment treatment ( (inset:inset: aa current–voltagecurrent–voltage curve cur ofve capsaicin-induced of capsaicin-induced current current without without MS15203MS15203 treatment treatment subtractedsubtracted by by that that with with MS15203 MS15203 treatment). treatment The break). The indicates break indicates a 5 min omission a 5 min on omission the trace. on Data the are trace. Data arepresented presented as means as means± S.E.M. ± S.E.M.

3.4. Peripheral GPR171 Activation Attenuates Pathologic Pain To determine whether the anti-nociceptive mechanisms of GPR171 work in pathologic pain states in vivo, we first tested peripheral injection of GPR171-specific agonists in complete Freund’s adjuvant (CFA)-induced hind paw inflammatory pain. Two injection routes were selected to examine whether the targeting of peripheral and central termini of nociceptors works. Allodynic mechanical hypersensitivity and hyperalgesic heat hypersensitivity were both improved by intrathecal agonist injection (Figure 4A,B) or by its ipsilateral intraplantar injection (Figure 4C,D). These analgesic effects increased in a dose- dependent manner, which indicates that the analgesia is receptor-dependent and that doses of hundreds nanomolar to micromolar concentrations are relatively effective (Fig- ure 4E,F and Supplementary Figure S5). We also tested the effect of the specific agonists on chronic constriction injury (CCI)-induced pain. The analgesic effects were largely similar but more prominent in mechanical allodynia compared with those observed in CFA- induced pain (Figure 5A–D). These effects also showed dose dependence (Figure 5E,F; Supplementary Figure S6). In an incisional pain model of mice, GRP171 activation was also effective at allodynic and hyperalgesic pain alleviation (Figure 6). BiomedicinesBiomedicines 2021, 9, x 2021FOR, PEER9, 256 REVIEW 10 of 17 10 of 17

Figure 4. GPR171 activationFigure 4. alleviatesGPR171 activation inflammatory alleviates pain. inflammatory (A) Time course pain. of von (A) FreyTime paw-withdrawal course of von Frey thresholds paw-with- (PWTs) in complete Freund’sdrawal adjuvant thresholds (CFA)-inflamed (PWTs) in mice. complete Animals Freund’s were intrathecally adjuvant (CFA treated)-inflamed with vehicle mice. (red),Animals MS15203 were (2.5 µg, green), or bigLEN (2.5intrathecallyµg, blue) treated immediately with vehicle after 24 (red and), 48MS15203 h threshold (2.5 μg, monitoring. green), orThe bigLEN right ( line2.5 μg, plot blue shows) immedi- the mean thresholds. BLs indicateately baselineafter 24 and thresholds 48 h threshold or latencies monitoring. under normal The right condition line plot before shows inflammation. the mean thresholds. Red arrows BLs indicate the time for treatments.indicate (B) Timebaseline course thresholds of Hargreaves or latencies paw-withdrawal under normal latencies condition (PWLs) before in inflammation. CFA-inflamed Red mice. arrows Animals indicate the time for treatments. (B) Time course of Hargreaves paw-withdrawal latencies (PWLs) were treated with drugs in the same manner as in (A). The right line plot shows the mean latencies. (C) Time course of in CFA-inflamed mice. Animals were treated with drugs in the same manner as in (A). The right von Frey thresholds in CFA-inflamed mice. Animals were intraplantarily treated with vehicle (red), MS15203 (2.5 µg, line plot shows the mean latencies. (C) Time course of von Frey thresholds in CFA-inflamed mice. green), or bigLENAnimals (2.5 µg, blue)were inintraplantarily the ipsilateral treated hind pawswith vehicle immediately (red), MS15203 after 24 and (2.5 48μg, h green threshold), or bigLEN monitoring. (2.5 The right line plot showsμg, the blue mean) in thresholds.the ipsilateral (D hind) Time paws course immediately of Hargreaves after latencies24 and 48 in h CFA-inflamed threshold monitoring. mice. Animals The were treated with drugs inright the line same plot manner shows as the in mean (C). The thresholds. right line (D plot) Time shows course the meanof Hargreaves latencies. latencies Five animals in CFA were-in- used for each data point. Statisticalflamed mice. significance Animals is representedwere treated using with differentdrugs in the symbols same (*manner for MS15203 as in (C treatment). The right and line # forplot bigLEN treatment). (E) Maximumshows possiblethe mean analgesic latencies. effects Five animals (percent were M.P.E.) used were for produced each data from point. treatments Statistical with significance different is doses of GPR171 activatorsrepresented (details are listedusingin different Supplementary symbols Figure(* for MS15203 S5). Percent treatment M.P.E.s and were # for calculated bigLEN treatment from comparisons). (E) of the subtractions ofMaximum withdrawal possible thresholds analgesic (or latencies) effects ( ofpercent the ipsilateral M.P.E.) were side beforeproduced and from after treatments inflammation with with differ- vehicle ent doses of GPR171 activators (details are listed in Supplementary Figure S5). Percent M.P.E.s treatment, to those with drug treatment. Symbols indicate different drug treatments in CFA-inflamed mice (circle, single were calculated from comparisons of the subtractions of withdrawal thresholds (or latencies) of intrathecal treatment of MS15203 24 h after CFA inflammation; square, double intrathecal treatments of MS15203 24 and 48 h the ipsilateral side before and after inflammation with vehicle treatment, to those with drug treat- after CFA inflammation;ment. diamond,Symbols indicate single intrathecal different drug treatment treatments of bigLEN in CFA 24-inflamed h after CFA mice inflammation; (circle, single inverted intrathecal triangle, double intrathecal treatmentstreatment of of MS15203 bigLEN 24 h and after 48 CFA h after inflammation; CFA inflammation). square, double (F) Percent intrathecal M.P.E.s treatments were produced of from treatments with differentMS15203 doses 24 and of GPR17148 h after activators CFA inflammation; (details are diamond, listed in Supplementarysingle intrathecal Figure treatment S5). Symbolsof bigLEN indicate 24 different drug treatmentsh after CFA (circle, inflammatio single intrathecaln; inverted treatment triangle, of double MS15203 intrathecal 24 h after treatments CFA inflammation; of bigLEN 24 square, and 48 double h intrathecal treatmentsafter of MS15203CFA inflammation 24 and 48) h. after(F) Percent CFA inflammation; M.P.E.s were diamond, produced single from intrathecaltreatments treatmentwith different of bigLEN doses 24 h after CFA inflammation;of GPR171 inverted activators triangle, (details double are intrathecal listed in Supplementary treatments of bigLEN Figure S5 24) and. Symbols 48 h after indicate CFA different inflammation). Statistical differencesdrug are treatments indicated ( bycircle, * for single the effects intrathecal of MS15203 treatment and of # MS15203 for those 24 of h bigLEN, after CFA compared inflammation; to those of the square, double intrathecal treatments of MS15203 24 and 48 h after CFA inflammation; diamond, vehicles. *** p < 0.001, ** p < 0.01, * p < 0.05; ### p < 0.001, ## p < 0.01, # p < 0.05. Biomedicines 2021, 9, x FOR PEER REVIEW 11 of 17

single intrathecal treatment of bigLEN 24 h after CFA inflammation; inverted triangle, double in- Biomedicines 2021, 9, 256 trathecal treatments of bigLEN 24 and 48 h after CFA inflammation). Statistical differences are 11 of 17 indicated by * for the effects of MS15203 and # for those of bigLEN, compared to those of the vehicles. *** p < 0.001, ** p < 0.01, * p < 0.05; ### p < 0.001, ## p < 0.01, # p < 0.05.

Figure 5. GPR171 activationFigure 5. alleviatesGPR171 activation neuropathic alleviates pain. neuropathic (A) Time course pain. of (A von) Time Frey course paw-withdrawal of von Frey paw thresholds-with- in mice with chronic constrictiondrawal injury thresholds (CCI). in Animals mice with were chronic intrathecally constriction treated injury with (CCI vehicle). Animals (red), were MS15203 intrathecally (2.5 µ g, green), or treated with vehicle (red), MS15203 (2.5 μg, green), or bigLEN (2.5 μg, blue) immediately after 14 bigLEN (2.5 µg, blue) immediately after 14 days of threshold monitoring. The right line plot shows the mean thresholds. days of threshold monitoring. The right line plot shows the mean thresholds. (B) Time course of B ( ) Time course of HargreavesHargreaves paw-withdrawalpaw-withdrawal latenc latenciesies in in mice mice with with CCI. CCI. Animals Animals were were treated treated with with drugs drugs in the in the same manner as in (A). Thesame right manner line plot as in shows (A). The the meanright line latencies. plot shows (C) Time the mean course latencies. of von Frey(C) Time thresholds course inof micevon Frey with chronic constriction injury (CCI).thresholds Animals in mice were with intraplantarily chronic constriction treated injury with (CCI vehicle). Animals (red), were MS15203 intraplantarily (2.5 µg, green), treatedor bigLEN (2.5 µg, blue) in thewith ipsilateral vehicle hind (red) paws, MS15203 immediately (2.5 μg, green after), 14or daysbigLEN of threshold(2.5 μg, blue monitoring.) in the ipsilateral The right hind line paws plot shows the mean thresholds.immediately (D) Time course after 14 of days Hargreaves of threshold latencies monitoring. in mice The with right CCI. line Animals plot shows were the treated mean thresholds. with drugs in the same manner as in ((CD).) TheTime right course line of plot Hargreaves shows the latencies mean latencies.in mice with Five CCI. animals Animals were were used treated for each with data drugs point. in the Statistical same manner as in (C). The right line plot shows the mean latencies. Five animals were used for signiﬁcance is represented using different symbols (* for MS15203 treatment and # for bigLEN treatment). (E) Percent each data point. Statistical significance is represented using different symbols (* for MS15203 treat- M.P.E.s were produced from treatments with different doses of GPR171 activators (details are listed in Supplementary Figure S6). Symbols indicate different drug treatments in mice with CCI (circle, 1 h after intrathecal treatment of MS15203; square, 2 h after intrathecal treatment of MS15203; diamond, 1 h after intrathecal treatment of bigLEN; inverted triangle, 2 h after intrathecal treatment of bigLEN). (F) Percent M.P.E.s were produced from treatments with different doses of GPR171 activators (details are listed in Supplementary Figure S6). Symbols indicate different drug treatments in mice with CCI (circle, 1 h after intraplantar treatment of MS15203; square, 2 h after intraplantar treatment of MS15203; diamond, 1 h after intraplantar treatment of bigLEN; inverted triangle, 2 h after intraplantar treatment of bigLEN). *** p < 0.001, ** p < 0.01; ### p < 0.001, ## p < 0.01. Biomedicines 2021, 9, x FOR PEER REVIEW 12 of 17

ment and # for bigLEN treatment). (E) Percent M.P.E.s were produced from treatments with different doses of GPR171 activators (details are listed in Supplementary Figure S6). Symbols indicate different drug treatments in mice with CCI (circle, 1 h after intrathecal treatment of MS15203; square, 2 h after intrathecal treatment of MS15203; diamond, 1 h after intrathecal treatment of bigLEN; inverted triangle, 2 h after intrathecal treatment of bigLEN). (F) Percent M.P.E.s were produced from treatments with different doses of GPR171 activators (details are listed in Supple- mentary Figure S6). Symbols indicate different drug treatments in mice with CCI (circle, 1 h after Biomedicines 2021, 9, 256 intraplantar treatment of MS15203; square, 2 h after intraplantar treatment of MS15203; diamond,12 of 17 1 h after intraplantar treatment of bigLEN; inverted triangle, 2 h after intraplantar treatment of bigLEN). *** p < 0.001, ** p < 0.01; ### p < 0.001, ## p < 0.01.

FigureFigure 6. 6. GPR171GPR171 activation activation alleviates alleviates incision incision-induced-induced pain. pain. (A (A) )Time Time course course of of von von Frey Frey thresh- thresholds oldsin mice in mice of an of incisionan incision pain pain model. model. Animals Animals were were intrathecally intrathecally treated treated with with vehicle vehicle (red), (red), MS15203 MS15203 (2.5 μg, green), or bigLEN (2.5 μg, blue) immediately after the first post-incision thresh- (2.5 µg, green), or bigLEN (2.5 µg, blue) immediately after the first post-incision threshold monitoring. old monitoring. Threshold monitoring began immediately after the completion of incision opera- Threshold monitoring began immediately after the completion of incision operation. The right tion. The right line plot shows the mean thresholds. (B) Time course of Hargreaves latencies in miceline before plot shows and after the meanincision. thresholds. Animals were (B) Time treated course with ofdrugs Hargreaves in the same latencies manner in as mice in ( beforeA). The and rightafter line incision. plot shows Animals the mean were latencies. treated with (C) Time drugs course in the of same von mannerFrey thresholds as in (A in). Themice right before line plot andshows after the incision. mean latencies.Animals were (C) Time intraplantarily course of vontreated Frey with thresholds vehicle in(red mice), MS15203 before and(2.5 afterμg, incision. greenAnimals), or werebigLEN intraplantarily (2.5 μg, blue) treated in the ipsilateral with vehicle hind (red), paws MS15203 immediately (2.5 µ g,after green), 24 and or 48 bigLEN h thresh- (2.5 µg, oldblue) monitoring. in the ipsilateral The right hind line pawsplot shows immediately the mean after thresholds. 24 and 48(D) h Time threshold course monitoring. of Hargreaves The right latenciesline plot in shows mice before the mean and after thresholds. incision. (AnimalsD) Time were course treated of Hargreaves with drugs in latencies the same in manner mice before as in (C). The right line plot shows the mean latencies. In (A–D), five animals were used for each data and after incision. Animals were treated with drugs in the same manner as in (C). The right line point. Statistical significance is represented using different symbols (* for MS15203 treatment and #plot for bigLEN shows thetreatment mean) latencies.. *** p < 0.001, In (**A –pD <), 0.01; five ### animals p < 0.001, were ## p used< 0.01, for # p each< 0.05. data point. Statistical significance is represented using different symbols (* for MS15203 treatment and # for bigLEN treatment). *** p < 0.001, ** p < 0.01; ### p < 0.001, ## p < 0.01, # p < 0.05.

To determine whether the agonist-induced analgesic effect is GPR171 specific, we conducted RNA interference to downregulate GPR171 expression in the DRG by pre-sciatic or intrathecal injection of its small-interfering RNA and observed the in vivo parameters from the CFA model. Despite bigLEN or MS15203 treatment, pain deteriorated in a similar fashion to those treated with vehicle (Figure7), indicating that these agonists may specifically act by GPR171 activation. We further determined whether any contribution of psychiatric or motor mechanisms may occur by this GPR activation in the central nervous system, particularly when intrathecally injected, but no such sign was detected in the open field test (Figure8). We also wondered whether our local intraplantar and intrathecal administrations of GPR modulators may affect eating behaviors, which was previous Biomedicines 2021, 9, x FOR PEER REVIEW 13 of 17

To determine whether the agonist-induced analgesic effect is GPR171 specific, we conducted RNA interference to downregulate GPR171 expression in the DRG by pre-sciatic or intrathecal injection of its small-interfering RNA and observed the in vivo parameters from the CFA model. Despite bigLEN or MS15203 treatment, pain deteriorated in a similar fashion to those treated with vehicle (Figure 7), indicating that these agonists may specifically act by GPR171 activation. We further determined whether any contribution of psychiatric or motor mechanisms may occur by this GPR activation in the central nervous system, par- Biomedicines 2021, 9, 256 13 of 17 ticularly when intrathecally injected, but no such sign was detected in the open field test (Figure 8). We also wondered whether our local intraplantar and intrathecal administrations of GPR modulators may affect eating behaviors, which was previous shown by hypotha- shown by hypothalamiclamic modulation modulation [21]. We [21 reproduced]. We reproduced the mild the increase mild increase in food inintake food when intake intraperitone- when intraperitoneallyally injected, injected, but our butlocal our injections local injections did not significantly did not signiﬁcantly affect food affect and foodwater intake and and water intakeweight and gain weight (Supplementary gain (Supplementary Figure S2I– FigureM). This S2I–M). tolerance This was tolerance repeated was in mice with repeated in miceGRP171 with knoc GRP171kdown knockdown (Supplementary (Supplementary Figure S7 Figure). We S7).further We checked further checked whether innocuous whether innocuoussomatosensory somatosensory functions functions are affected are affected and showed and showed that those that are those largely are largely ignorable (Supple- ignorable (Supplementarymentary Figure Figure S2A–H S2A–H).). Overall,Overall, our results ourresults suggest suggest that the that inhibitory the inhibitory actions of GPR171 actions of GPR171activati activationon on the on nociceptor the nociceptor activity activity can be can extrapolated be extrapolated to the to in the vivoin vivo levellevel with pathologic with pathologicpain, pain, and and thus, thus, GPR171 GPR171 may may be bea novel a novel peripheral peripheral target target for for pain pain modulation. modulation.

Figure 7. AnimalsFigure with 7. AnimalsGPR171 knockdown with GPR171 are knockdown tolerant to areits pharmacological tolerant to its pharmacological modulation. (A modulation.) A cartoon describing (A)A the intrathecal (i.t.cartoon) treatment describing of siRNA the intrathecaldownregulating (i.t.)treatment GPR171 expression of siRNAdownregulating for experiments GPR171from (B expression–D). Scrambled for RNAs (SC) were injectedexperiments in the same from way. (B– (DB).) Time Scrambled course RNAsof von (SC)Frey werethresholds injected in incomplete the same Freund’s way. (B adjuvant) Time course (CFA) of-inflamed mice. Animalsvon were Frey intrathecally thresholds treated in complete with drugs Freund’s listed adjuvant in the inset (CFA)-inflamed immediately mice. after Animals 24 and 48 were h threshold intrathecally monitoring. The left dot plottreated shows with individual drugs listedthreshold in the values. inset ( immediatelyC) Time course after of Hargreaves 24 and 48 h latencies threshold in monitoring.CFA-inflamed The mice. Ani- mals were treated with drugs in the same manner as in (B). The left dot plot shows individual latency values. (D) Expres- left dot plot shows individual threshold values. (C) Time course of Hargreaves latencies in CFA- sion levels of GPR171 observed by quantitative RT-PCR with lumbar DRGs of the animals intrathecally treated with siRNA inflamed mice. Animals were treated with drugs in the same manner as in (B). The left dot plot compared to those with scrambled RNA. (E) A cartoon describing the peri-sciatic nerve treatment of siRNA downregulating GPR171 expshowsression individual for experiments latency from values. (F– (HD)). Scrambled Expression RNAs levels were of GPR171 injected observed in the same byquantitative way. (F) Time RT- course of PCR with lumbar DRGs of the animals intrathecally treated with siRNA compared to those with scrambled RNA. (E) A cartoon describing the peri-sciatic nerve treatment of siRNA downregulating GPR171 expression for experiments from (F–H). Scrambled RNAs were injected in the same way. (F) Time course of von Frey thresholds in complete Freund’s adjuvant (CFA)-inflamed mice. Animals were intraplantarily treated with drugs listed in the inset immediately after 24 and 48 h threshold monitoring. The left dot plot shows individual threshold values. (G) Time course of Hargreaves latencies in CFA-inflamed mice. Animals were treated with drugs in the same manner as in (F). The left dot plot shows individual latency values. (H) Expression levels of GPR171 observed by quantitative RT-PCR with lumbar DRGs of the animals peri-sciatically treated with siRNA compared to those with scrambled RNA. *** p < 0.001, ** p < 0.01; ### p < 0.001. Biomedicines 2021, 9, x FOR PEER REVIEW 14 of 17

von Frey thresholds in complete Freund’s adjuvant (CFA)-inflamed mice. Animals were intraplantarily treated with drugs listed in the inset immediately after 24 and 48 h threshold monitoring. The left dot plot shows individual threshold values. (G) Time course of Hargreaves latencies in CFA-inflamed mice. Animals were treated with drugs in the same manner as in (F). The left dot plot shows individual latency values. (H) Expression levels of GPR171 observed by quantitative RT- PCR withBiomedicines lumbar DRGs2021 ,of9, 256the animals peri-sciatically treated with siRNA compared to those with scrambled RNA. *** p 14 of 17 < 0.001, ** p < 0.01; ### p < 0.001.

Figure 8. TheFigure effect 8. of The GPR171 effect agonist of GPR171 injection agonist on injection open field on behaviors.open field behaviors. (A) Time spent (A) Time in the spent central in the field of an open field box by micecentral was field monitored of an open for field 30 min. box Meanby mice seconds was monitored± S.E.M. arefor shown30 min. for Mean five seconds mice with ±S.E.M. or without are intrathecal shown for five mice with or without intrathecal drug treatment. (B) Percent time spent in the cen- drug treatment. (B) Percent time spent in the central field by mice for 30 min as in (A) are shown in the histogram. Mean tral field by mice for 30 min as in (A) are shown in the histogram. Mean seconds ±S.E.M. are seconds ± S.E.M. are shown for five mice with or without intrathecal drug treatment. (C) Distance traveled with or without shown for five mice with or without intrathecal drug treatment. (C) Distance traveled with or intrathecal drugwithout treatment intrathecal in five drug mice treatment was averaged in five duringmice was every averaged 5 min during period. every (D) Representative5 min period. (D tracks) Rep- for 30 min of a mouse underresentative open fieldtracks test forare 30 min shown of a (left, mouse vehicle under treated; open field middle, test are MS15203 shown treated; (left, vehicle right, treated; bigLEN mid- treated). The central field isdle, indicated MS15203 as atreated; green square.right, bigLEN (E) Numbers treated of). rearingThe central of mice field inside is indicated open field as a box green were square. monitored (E) for 30 min. Mean numbersNumbers± S.E.M. of arerearing shown of mice for five inside mice open with field orwithout box were intrathecal monitored drug for 30 treatment. min. Mean (F )numbers Time spent by mice for rearing inside±S.E.M. an open are field shown box wasfor five monitored mice with for 30or without min. Mean intrathecal seconds ±drugS.E.M. treatment. are shown (F) Time for five spent mice by with or without intrathecal drugmice treatment. for rearing (G inside) Time an spent open by field five micebox was for rearingmonitored inside for an 30 open min. field Mean box seconds with or ±S.E.M. without are intrathecal drug treatment wasshown averaged for five every mice 5 min with period. or without No statistical intrathecal difference drug treatment. was detected (G) Time at any spent data by point five mice in one-way for ANOVA rearing inside an open field box with or without intrathecal drug treatment was averaged every 5 and Tukey’s analysis. min period. No statistical difference was detected at any data point in one-way ANOVA and Tukey’s analysis. 4. Discussion Our study proposes a new GPR-coupled regulatory mechanism of pain. Use of the neuropeptide ligand, bigLEN, and synthetic ligand, MS15203, has confirmed GPR171 action, as shown in previous studies [7,21]. In peripheral nociceptors, the similar Gi/o- mediated effect of GPR171 activation operates with these two ligands. Either intraplantar injection or intrathecal application of these ligands avoided brain-related behaviors observed in the studies above, suggesting that pain-specific phenotypes can be produced when peripherally modulated. We did not detect any GPR171 in tissues near the DRG termini, which indicates that GPR171 action in nociceptors rather than non-neuronal actions may be predominant for neuronal modulation. However, the secretion of endogenous ligands for GPR171 Biomedicines 2021, 9, 256 15 of 17

including bigLEN and other unknown ones remains as an open question because of the lack of available antibodies. Since neuron-specific expression of Pcsk1n protein, which is the precursor protein for bigLEN cleavage, has been shown, including in embryonic DRG, but not neighboring non-neuronal tissues, paracrine and/or autocrine actions on the nociceptor-secreted bigLEN in some inflamed conditions can be hypothesized [32,33]. In the spinal cord superficial laminae, some neuronal components, but no glial com- ponent, expressed GPR171 as shown by immunohistochemical assays. Nonetheless, firing of dorsal horn neurons was only presynaptically modulated by perfusion of GPR171 ligands. Further studies will clarify whether and/or how these GPR171-positive dorsal horn neurons contribute to the modulation of the ascending pain signals. This may also help to fully understand why phase 2 nociception in the formalin test was more consistently affected than that of phase 1 because many other components as well as presynaptic TRPs are known to contribute to phase 2-inducing synaptic sensitization. DRG is an important painkilling target because DRG-specific modulation may avoid central adverse effects. Neuropeptide-activated and Gi/o-coupled GPRs such as somatostatin and neuropeptide Y receptors, subtypes of which are expressed in a subset of DRG neurons, are currently being revisited in exploring peripheral analgesic efficacy [34–36]. Of note, the studies proposed TRP channel inhibition as the downstream effector mechanism. Although Gi/o-coupled signaling may affect the general excitability of DRG neurons, which mainly involves the altered functions of voltage-gated channels or cyclic nucleotide- gated channels, only TRP channel-mediated effects were detected in our study. Thus, the analgesic outcomes observed here may share a common molecular paradigm with previous studies in which peripheral GPR modulation prompts TRP inhibition. Nonetheless, with the current lack of approaches using voltage-dependent subunits, the possibilities need to be carefully monitored to determine whether specific voltage-dependent effectors are influenced in some particular pathological states and locations in the future. TRP ion channels expressed in nociceptors play a central role in monitoring harmful events in tissues and transforming those into pain signals. In addition to the direct and specific inhibition of each TRP, triggering an inhibitory upstream signal that may commonly attenuate different TRP actions can be an efficient approach for controlling many aspects of pain events. In this context, our results indicate a novel GPR-mediated mechanism that inhibits multiple TRP actions. Our study demonstrated TRPA1, TRPV1, TRPV2, TRPV4, TRPM3, and TRPC4/C5 inhibitions by GPR171 activation. Many of these TRPs have been shown to be engaged in mechanical and thermal nociceptions to some extent, which seems to explain our analgesic phenotypes from the treatment of GRP171 agonists [37]. Such effector-specific outcomes may not be limited to these TRP-mediated ones in this GPR action and other possible important pathologies should be explored in the future. Very recently, GRP171 is proposed to be present in the periaqueductal gray (PAG) and to regulate opioid signaling there in µ-opioid receptor-dependent fashions [38]. Its activation alone in PAG was not significantly effective, but reinforced µ-receptor-mediated signals, finally enhanced the opioid-induced analgesia. Thus, the outcomes observed here regarding the peripheral mechanisms and those potentially involving the descending inhibitory pathways appear to commonly contribute to pain relief. Further attempts on these analgesic concepts operating in different circuits in more practical ways such as quantitative comparisons in pharmacology and target accessibility may extend the applicable options for promoting the utilities of GPR171 modulators. In conclusion, our results suggest a novel GPR171-mediated pain-relieving mechanism. Such findings may lead to future opportunities to devise novel analgesic strategies.

Supplementary Materials: The following are available online at https://www.mdpi.com/2227-905 9/9/3/256/s1. Figure S1. GPR171 expression in tissues. Figure S2. Effects of treatments of GPR171 activators on acute somatosensations, food intake, water intake, and weight gain. Figure S3. BigLEN modulates DRG neuronal TRP channel functions. Figure S4. BigLEN modulates dorsal horn and DRG neuronal current response upon capsaicin treatment but not voltage-dependent currents in DRG neurons. Figure S5. Administration of GPR171 agonists at various doses alleviates inﬂammatory Biomedicines 2021, 9, 256 16 of 17

pain. Figure S6. Administration of GPR171 agonists at various doses alleviates neuropathic pain. Figure S7. Effects of treatments of GPR171 activators on food intake, water intake, and weight gain in mice with knockdown. Figure S8. Summarized illustrations for animal experiments conducted in this study. Author Contributions: Conceptualization and supervision, S.W.H. and S.J.J.; methodology, P.S.C., J.Y.L., S.I.C., M.K. and H.K.; formal analysis and investigation, P.S.C., H.K.L., Y.I.C. and S.I.C.; resources, H.K.; data curation, P.S.C., H.K.L. and Y.I.C.; writing—original draft preparation, S.W.H. and P.S.C.; writing—review and editing, S.W.H., S.J.J. and P.S.C.; funding acquisition, S.W.H. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by grants from the National Research Foundation of Korea (2017R1A2B2001817, 2017M3C7A1025600, and 2019R1A4A1029000). Institutional Review Board Statement: The study was conducted under the approval of the Insti- tutional Animal Care and Use Committee of Korea University College of Medicine (approval No.: KOREA-2019-0168). Informed Consent Statement: Not applicable. Conﬂicts of Interest: The authors declare that there is no conﬂict of interest regarding the publication of this article.

References 1. von Hehn, C.A.; Baron, R.; Woolf, C.J. Deconstructing the neuropathic pain phenotype to reveal neural mechanisms. Neuron 2012, 73, 638–652. [CrossRef][PubMed] 2. Choi, G.; Hwang, S.W. Modulation of the Activities of Neuronal Ion Channels by Fatty Acid-Derived Pro-Resolvents. Front. Physiol. 2016, 7, 523. [CrossRef] 3. Choi, S.I.; Hwang, S.W. Depolarizing Effectors of Bradykinin Signaling in Nociceptor Excitation in Pain Perception. Biomol. Ther. 2018, 26, 255–267. [CrossRef][PubMed] 4. Moriyama, T.; Higashi, T.; Togashi, K.; Iida, T.; Segi, E.; Sugimoto, Y.; Tominaga, T.; Narumiya, S.; Tominaga, M. Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins. Mol. Pain 2005, 1, 3. [CrossRef][PubMed] 5. Xu, Z.Z.; Zhang, L.; Liu, T.; Park, J.Y.; Berta, T.; Yang, R.; Serhan, C.N.; Ji, R.R. Resolvins RvE1 and RvD1 attenuate inflammatory pain via central and peripheral actions. Nat. Med. 2010, 16, 592–597. [CrossRef][PubMed] 6. Yudin, Y.; Rohacs, T. Inhibitory Gi/O-coupled receptors in somatosensory neurons: Potential therapeutic targets for novel analgesics. Mol. Pain 2018, 14, 1744806918763646. [CrossRef][PubMed] 7. Gomes, I.; Aryal, D.K.; Wardman, J.H.; Gupta, A.; Gagnidze, K.; Rodriguiz, R.M.; Kumar, S.; Wetsel, W.C.; Pintar, J.E.; Fricker, L.D.; et al. GPR171 is a hypothalamic G protein-coupled receptor for BigLEN, a neuropeptide involved in feeding. Proc. Natl. Acad. Sci. USA 2013, 110, 16211–16216. [CrossRef][PubMed] 8. Jacobs, K.A.; Collins-Racie, L.A.; Colbert, M.; Duckett, M.; Golden-Fleet, M.; Kelleher, K.; Kriz, R.; LaVallie, E.R.; Merberg, D.; Spaulding, V.; et al. A genetic selection for isolating cDNAs encoding secreted proteins. Gene 1997, 198, 289–296. [CrossRef] 9. Bandell, M.; Story, G.M.; Hwang, S.W.; Viswanath, V.; Eid, S.R.; Petrus, M.J.; Earley, T.J.; Patapoutian, A. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 2004, 41, 849–857. [CrossRef] 10. Bang, S.; Yoo, S.; Yang, T.J.; Cho, H.; Hwang, S.W. Isopentenyl pyrophosphate is a novel antinociceptive substance that inhibits TRPV3 and TRPA1 ion channels. Pain 2011, 152, 1156–1164. [CrossRef][PubMed] 11. Choi, G.; Yang, T.J.; Yoo, S.; Choi, S.I.; Lim, J.Y.; Cho, P.S.; Hwang, S.W. TRPV4-Mediated Anti-nociceptive Effect of Suberanilohy- droxamic Acid on Mechanical Pain. Mol. Neurobiol. 2019, 56, 444–453. [CrossRef] 12. Bang, S.; Yoo, S.; Yang, T.J.; Cho, H.; Hwang, S.W. 17(R)-resolvin D1 specifically inhibits transient receptor potential ion channel vanilloid 3 leading to peripheral antinociception. Br. J. Pharmacol. 2012, 165, 683–692. [CrossRef][PubMed] 13. Chaplan, S.R.; Bach, F.W.; Pogrel, J.W.; Chung, J.M.; Yaksh, T.L. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods 1994, 53, 55–63. [CrossRef] 14. Kim, Y.H.; Park, C.K.; Back, S.K.; Lee, C.J.; Hwang, S.J.; Bae, Y.C.; Na, H.S.; Kim, J.S.; Jung, S.J.; Oh, S.B. Membrane-delimited coupling of TRPV1 and mGluR5 on presynaptic terminals of nociceptive neurons. J. Neurosci. 2009, 29, 10000–10009. [CrossRef] 15. Moqrich, A.; Hwang, S.W.; Earley, T.J.; Petrus, M.J.; Murray, A.N.; Spencer, K.S.; Andahazy, M.; Story, G.M.; Patapoutian, A. Impaired thermosensation in mice lacking TRPV3, a heat and camphor sensor in the skin. Science 2005, 307, 1468–1472. [CrossRef] 16. Trevisani, M.; Siemens, J.; Materazzi, S.; Bautista, D.M.; Nassini, R.; Campi, B.; Imamachi, N.; Andrè, E.; Patacchini, R.; Cottrell, G.S.; et al. 4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1. Proc. Natl. Acad. Sci. USA 2007, 104, 13519–13524. [CrossRef] 17. Bennett, G.J.; Xie, Y.K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988, 33, 87–107. [CrossRef] 18. Pogatzki, E.M.; Raja, S.N. A mouse model of incisional pain. Anesthesiology 2003, 99, 1023–1027. [CrossRef] Biomedicines 2021, 9, 256 17 of 17

19. Kim, J.I.; Lee, H.R.; Sim, S.E.; Baek, J.; Yu, N.K.; Choi, J.H.; Ko, H.G.; Lee, Y.S.; Park, S.W.; Kwak, C.; et al. PI3Kgamma is required for NMDA receptor-dependent long-term depression and behavioral flexibility. Nat. Neurosci. 2011, 14, 1447–1454. [CrossRef] [PubMed] 20. Duan, B.; Cheng, L.; Bourane, S.; Britz, O.; Padilla, C.; Garcia-Campmany, L.; Krashes, M.; Knowlton, W.; Velasquez, T.; Ren, X.; et al. Identification of spinal circuits transmitting and gating mechanical pain. Cell 2014, 159, 1417–1432. [CrossRef][PubMed] 21. Wardman, J.H.; Gomes, I.; Bobeck, E.N.; Stockert, J.A.; Kapoor, A.; Bisignano, P.; Gupta, A.; Mezei, M.; Kumar, S.; Filizola, M.; et al. Identification of a small-molecule ligand that activates the neuropeptide receptor GPR171 and increases food intake. Sci. Signal. 2016, 9, ra55. [CrossRef][PubMed] 22. Chung, S.; Lee, E.J.; Yun, S.; Choe, H.K.; Park, S.B.; Son, H.J.; Kim, K.S.; Dluzen, D.E.; Lee, I.; Hwang, O.; et al. Impact of circadian nuclear receptor REV-ERBalpha on midbrain dopamine production and mood regulation. Cell 2014, 157, 858–868. [CrossRef] [PubMed] 23. Bang, S.; Yoo, S.; Yang, T.J.; Cho, H.; Hwang, S.W. Nociceptive and pro-inflammatory effects of dimethylallyl pyrophosphate via TRPV4 activation. Br. J. Pharmacol. 2012, 166, 1433–1443. [CrossRef] 24. Chiu, I.M.; Heesters, B.A.; Ghasemlou, N.; Von Hehn, C.A.; Zhao, F.; Tran, J.; Wainger, B.; Strominger, A.; Muralidharan, S.; Horswill, A.R.; et al. Bacteria activate sensory neurons that modulate pain and inflammation. Nature 2013, 501, 52–57. [CrossRef] 25. Chatzigeorgiou, M.; Bang, S.; Hwang, S.W.; Schafer, W.R. tmc-1 encodes a sodium-sensitive channel required for salt chemosensa- tion in C. elegans. Nature 2013, 494, 95–99. [CrossRef] 26. Zimmermann, K.; Hein, A.; Hager, U.; Kaczmarek, J.S.; Turnquist, B.P.; Clapham, D.E.; Reeh, P.W. Phenotyping sensory nerve endings in vitro in the mouse. Nat. Protoc. 2009, 4, 174–196. [CrossRef][PubMed] 27. Jang, J.H.; Liang, D.; Kido, K.; Sun, Y.; Clark, D.J.; Brennan, T.J. Increased local concentration of complement C5a contributes to incisional pain in mice. J. Neuroinflamm. 2011, 8, 80. [CrossRef] 28. Lindfors, P.H.; Voikar, V.; Rossi, J.; Airaksinen, M.S. Deficient nonpeptidergic epidermis innervation and reduced inflammatory pain in glial cell line-derived neurotrophic factor family receptor alpha2 knock-out mice. J. Neurosci. 2006, 26, 1953–1960. [CrossRef][PubMed] 29. Zylka, M.J.; Rice, F.L.; Anderson, D.J. Topographically distinct epidermal nociceptive circuits revealed by axonal tracers targeted to Mrgprd. Neuron 2005, 45, 17–25. [CrossRef] 30. Macpherson, L.J.; Xiao, B.; Kwan, K.Y.; Petrus, M.J.; Dubin, A.E.; Hwang, S.; Cravatt, B.; Corey, D.P.; Patapoutian, A. An ion channel essential for sensing chemical damage. J. Neurosci. 2007, 27, 11412–11415. [CrossRef] 31. McNamara, C.R.; Mandel-Brehm, J.; Bautista, D.M.; Siemens, J.; Deranian, K.L.; Zhao, M.; Hayward, N.J.; Chong, J.A.; Julius, D.; Moran, M.M.; et al. TRPA1 mediates formalin-induced pain. Proc. Natl. Acad. Sci. USA 2007, 104, 13525–13530. [CrossRef] 32. Feng, Y.; Reznik, S.E.; Fricker, L.D. ProSAAS and prohormone convertase 1 are broadly expressed during mouse development. Brain Res. Gene Expr. Patterns 2002, 1, 135–140. [CrossRef] 33. Morgan, D.J.; Mzhavia, N.; Peng, B.; Pan, H.; Devi, L.A.; Pintar, J.E. Embryonic gene expression and pro-protein processing of proSAAS during rodent development. J. Neurochem. 2005, 93, 1454–1462. [CrossRef][PubMed] 34. Badheka, D.; Yudin, Y.; Borbiro, I.; Hartle, C.M.; Yazici, A.; Mirshahi, T.; Rohacs, T. Inhibition of Transient Receptor Potential Melastatin 3 ion channels by G-protein betagamma subunits. eLife 2017, 6, e26147. [CrossRef][PubMed] 35. Gorham, L.; Just, S.; Doods, H. Somatostatin 4 receptor activation modulates TRPV1 [correction of TPRV1] currents in dorsal root ganglion neurons. Neurosci. Lett. 2014, 573, 35–39. [CrossRef][PubMed] 36. Quallo, T.; Alkhatib, O.; Gentry, C.; Andersson, D.A.; Bevan, S. G protein betagamma subunits inhibit TRPM3 ion channels in sensory neurons. eLife 2017, 6, e26138. [CrossRef][PubMed] 37. Wang, H.; Woolf, C.J. Pain TRPs. Neuron 2005, 46, 9–12. [CrossRef] 38. McDermott, M.V.; Afrose, L.; Gomes, I.; Devi, L.A.; Bobeck, E.N. Opioid-induced signaling and antinociception are modulated by the recently deorphanized receptor, GPR171. J. Pharmacol. Exp. Ther. 2019, 371, 56–62. [CrossRef]