Decision letter: The signaling lipid sphingosine 1-phosphate regulates mechanical pain

Abstract

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Somatosensory neurons mediate responses to diverse mechanical stimuli, from innocuous touch to noxious pain. While recent studies have identified distinct populations of A mechanonociceptors (AMs) that are required for mechanical pain, the molecular underpinnings of mechanonociception remain unknown. Here, we show that the bioactive lipid sphingosine 1-phosphate (S1P) and S1P Receptor 3 (S1PR3) are critical regulators of acute mechanonociception. Genetic or pharmacological ablation of S1PR3, or blockade of S1P production, significantly impaired the behavioral response to noxious mechanical stimuli, with no effect on responses to innocuous touch or thermal stimuli. These effects are mediated by fast-conducting A mechanonociceptors, which displayed a significant decrease in mechanosensitivity in S1PR3 mutant mice. We show that S1PR3 signaling tunes mechanonociceptor excitability via modulation of KCNQ2/3 channels. Our findings define a new role for S1PR3 in regulating neuronal excitability and establish the importance of S1P/S1PR3 signaling in the setting of mechanical pain thresholds. https://doi.org/10.7554/eLife.33285.001 Introduction Pain is a complex sensation. It serves to protect organisms from harmful stimuli, but can also become chronic and debilitating following tissue injury and disease. Distinct cells and molecules detect noxious thermal and mechanical stimuli. Thermal pain is detected by thermosensitive TRP channels in subsets of nociceptors (Caterina et al., 2000; Vriens et al., 2011), and gentle touch is detected by Piezo2 channels in low-threshold mechanoreceptors (LTMRs) (Ranade et al., 2014; Woo et al., 2014). Aδ high-threshold mechanoreceptors (HTMRs) have been shown to play a key role in responses to painful mechanical stimuli (Arcourt et al., 2017; Ghitani et al., 2017). Recent studies have shown that there are at least two populations of HTMRs that mediate responses to noxious mechanical stimuli. The Npy2r+ subpopulation of HTMRs mediates fast paw withdrawal responses to pinprick stimulation and terminates as free nerve endings in the epidermis (Arcourt et al., 2017). The Calca+ subpopulation of circumferential-HTMRs responds to noxious force and hair pulling, and terminates as circumferential endings wrapped around guard hair follicles (Ghitani et al., 2017). Additionally, somatostatin-expressing interneurons of laminae I-III in the dorsal horn of the spinal cord receive input from nociceptors and are required for behavioral responses to painful mechanical stimuli (Duan et al., 2014). Despite these advances in defining the cells and circuits of mechanical pain, little is known about the molecular signaling pathways in mechanonociceptors. Here, we show that sphingosine 1-phosphate (S1P) is required for mechanical pain sensation. S1P is a bioactive lipid that signals via 5 G-protein coupled S1P Receptors (S1PRs 1–5). S1P signaling, mainly via S1PR1, plays a well-known role in immune cell migration and maturation (Spiegel and Milstien, 2003; Matloubian et al., 2004; Schwab et al., 2005). Additionally, recent studies have shown that S1PRs are expressed throughout the nervous system (Janes et al., 2014; Mair et al., 2011; Camprubí-Robles et al., 2013) and S1P signaling is associated with a variety of neuroinflammatory disorders, including multiple sclerosis (Brinkmann et al., 2010) and Alzheimer’s disease (Couttas et al., 2014). S1P has been implicated in spontaneous pain (Camprubí-Robles et al., 2013) and thermal pain hypersensitivity (Mair et al., 2011; Finley et al., 2013; Weth et al., 2015), but due to conflicting accounts of S1P receptor expression in the CNS (Janes et al., 2014; Weth-Malsch et al., 2016) and PNS (Mair et al., 2011; Camprubí-Robles et al., 2013; Usoskin et al., 2015) as well as inconsistent reports on the effects of S1P on neuronal excitability (Camprubí-Robles et al., 2013; Zhang et al., 2006; Li et al., 2015) and pain behaviors (Mair et al., 2011; Camprubí-Robles et al., 2013; Finley et al., 2013; Weth et al., 2015), the role of S1P in somatosensation remains controversial. We found that mice lacking the S1P receptor S1PR3 display striking and selective deficits in behavioral responses to noxious mechanical stimuli. Likewise, peripheral blockade of S1PR3 signaling or S1P production impairs mechanical sensitivity. We show that S1P constitutively enhances the excitability of A mechanonociceptors (AMs) via closure of KCNQ2/3 potassium channels to tune mechanical pain sensitivity. The effects of S1P are completely dependent on S1PR3. While previous studies have shown that elevated S1P triggers acute pain and injury-evoked thermal sensitization (Mair et al., 2011; Camprubí-Robles et al., 2013), we now demonstrate that baseline levels of S1P are necessary and sufficient for setting normal mechanical pain thresholds. By contrast, elevated S1P selectively triggers thermal sensitization via activation of TRPV1+ heat nociceptors, with no effect on mechanical hypersensitivity. Our findings uncover an essential role for constitutive S1P signaling in mechanical pain. Results To identify candidate genes underlying mechanosensation, we previously performed transcriptome analysis of the sensory ganglia innervating the ultra-sensitive tactile organ (the star) of the star-nosed mole (Gerhold et al., 2013). Immunostaining revealed the tactile organ is preferentially innervated by myelinated Aδ fibers (Gerhold et al., 2013), which are primarily mechanosensitive. While our original analysis focused on ion channels enriched in the neurons of the star organ, our dataset also revealed enrichment of several components of the S1P pathway, including S1pr3. Likewise, single-cell RNA seq of mouse dorsal root ganglion (DRG) neurons revealed S1pr3 expression in a subset of myelinated mechanoreceptors (Usoskin et al., 2015) in addition to a subpopulation of peptidergic C nociceptors. S1P promotes excitability in small-diameter, capsaicin-sensitive nociceptors (Mair et al., 2011; Camprubí-Robles et al., 2013; Zhang et al., 2006; Li et al., 2015). In addition, S1PR3 has been shown to mediate spontaneous pain triggered by elevated S1P and thermal sensitization following sterile tissue injury (Camprubí-Robles et al., 2013). However, no studies have examined the role of S1PR3 in mechanosensation or in regulating somatosensory behaviors under normal conditions. Given the enrichment of S1pr3 in mechanosensory neurons of the star-nosed mole and mouse, we hypothesized that S1P signaling via S1PR3 may also play a role in mechanosensation. Thus, we set out to define the role of S1P signaling and S1PR3 in somatosensory mechanoreceptors. S1PR3 mediates acute mechanical pain We first examined a variety of somatosensory behaviors in mice lacking S1PR3 (Kono et al., 2004) (S1pr3tm1Rlp/Mmnc; referred to herein as S1PR3 KO). We initially investigated baseline responses to mechanical stimuli. S1PR3 KO mice displayed a dramatic loss of mechanical sensitivity (Figure 1A; see Figure 1—source data 1), as von Frey paw withdrawal thresholds were significantly elevated in S1PR3 KO mice relative to WT and S1PR3 HET littermates (mean thresholds: 1.737 g vs. 0.736 and 0.610 g, respectively). Moreover, S1PR3 KO mice demonstrated decreased responses to a range of noxious tactile stimuli (2–6 g; Figure 1B) and to noxious pinprick stimulation (Figure 1C), but normal responsiveness to innocuous tactile stimuli (0.6–1.4 g; Figure 1B). S1PR3 KO mice exhibited normal tape removal attempts (Ranade et al., 2014) (Figure 1D), righting reflexes (Figure 1—figure supplement 1A), radiant heat withdrawal latencies (Figure 1E), and itch-evoked scratching (Figure 1—figure supplement 1B). These results demonstrate a selective role for S1PR3 in acute mechanical pain. Figure 1 with 1 supplement see all Download asset Open asset S1PR3 mediates acute mechanical pain. (A) von Frey 50% withdrawal threshold measurements for S1pr3+/+ (WT, N = 8), S1pr3+/- (HET, N = 7) and S1pr3-/- (KO, N = 12) mice. p<0.0001 (one-way ANOVA). Tukey-Kramer post hoc comparisons for KO and HET to WT indicated on graph. (B) von Frey force-response graph for WT (N = 8) versus KO (N = 12) animals; pgenotype <0.0001 (two-way ANOVA). Tukey HSD comparisons between genotypes are indicated for given forces. (C) % withdrawal to pinprick stimulation of hindpaw for HET versus KO animals; p<0.0001 (unpaired t-test; N = 5–7 mice per group). (D) Number of attempted removal bouts in tape assay for WT (N = 2), HET (N = 2), and KO (N = 5) mice; p=0.172 (one-way ANOVA). (E) Baseline radiant heat measurements for WT (N = 8), HET (N = 3), and KO (N = 5) mice; p=0.444 (one-way ANOVA). (F) von Frey 50% withdrawal threshold measurements for mice pre- and post-injection of 500 µM TY 52156 (N = 10), 10 µM W146 (N = 6), or 1% DMSO-PBS vehicle (N = 17); p=0.016, 0.650 (two-tailed paired t-test comparing vehicle- vs. drug-injected paw). (G) von Frey force-response graph for mice injected with either 1% DMSO-PBS (N = 4) or 500 µM TY 52156 (N = 4); ptreatment <0.0001 (two-way ANOVA). Tukey HSD comparisons were made between treatment groups and significant differences at a given force are indicated on graph. Error bars represent mean ± SD. https://doi.org/10.7554/eLife.33285.002 Figure 1—source data 1 S1PR3 mediates acute mechanical pain. Related to Figure 1. https://doi.org/10.7554/eLife.33285.004 Download elife-33285-fig1-data1-v2.xlsx As a complement to our analysis of somatosensation in S1PR3 KO animals, we employed a pharmacological approach, using the S1PR3-selective antagonist TY 52156 (TY) (Nussbaum et al., 2015). Similar to the phenotype of knockout animals, intradermal injection of 500 µM TY into the mouse hindpaw (the site of testing) triggered a rapid and significant elevation in von Frey paw withdrawal thresholds (Figure 1F) and decreased responsiveness to noxious (2–6 g), but not innocuous (0.6–1.4 g), tactile stimuli (Figure 1G), without affecting noxious heat sensitivity (Figure 1—figure supplement 1C). By contrast, blockade of S1PR1 with the selective antagonist W146 (Finley et al., 2013) had no effect on baseline mechanical or thermal thresholds (Figure 1F; Figure 1—figure supplement 1C). Overall, these data show that S1PR3 signaling sets mechanical pain sensitivity. Endogenous S1P mediates acute mechanical pain We next asked whether peripheral S1P was required for the S1PR3-dependent effects on mechanosensation. We decreased S1P levels via injection of the sphingosine kinase inhibitor SKI II to block local production of S1P (Chiba et al., 2010) or elevated S1P levels via intradermal injection of S1P and measured behaviors 30 min after injection. Decreasing local S1P levels with SKI II significantly reduced mechanical sensitivity (Figure 2A; see Figure 2—source data 1), comparable to the hyposensitivity phenotype observed in S1PR3 KO mice (Figure 1A). Again, similar to what was observed in S1PR3 KO animals (Figure 1E), peripheral blockade of S1P production had no effect on baseline thermal sensitivity (Figure 1—figure supplement 1C). Surprisingly, injecting exogenous S1P (10 µM; maximum solubility in saline vehicle) had no effect on mechanical sensitivity (Figure 2A–B). However, as previously reported (Mair et al., 2011; Camprubí-Robles et al., 2013), S1P injection triggered S1PR3-dependent thermal hypersensitivity and spontaneous pain (Figure 2C–D), demonstrating that the lack of effect on mechanical hypersensitivity is not due to problems with S1P delivery or degradation. Figure 2 Download asset Open asset Endogenous S1P mediates acute mechanical pain. (A) von Frey 50% withdrawal measurements for mice pre- and post-injection of 50 µM SKI II (N = 8) or 10 µM S1P (N = 7); p=0.003, 0.604 (two-tailed paired t-tests). (B) von Frey force-response graph for animals injected with 10 µM S1P or 0.1% MeOH-PBS; pgenotype >0.05 (two-way ANOVA; N = 8 mice per group). No Tukey HSD comparisons at any force between genotypes were significant. (C) Intradermal cheek injection of 10 µM S1P, 2 µM, 0.2 µM, and 20 µL 0.3% methanol PBS (vehicle), with quantification of number of forepaw wipes over the 5 min post-injection interval; p<0.0001 (one-way ANOVA; N = 3 mice per condition). Dunnett’s multiple comparisons p-values are represented on graph for comparisons made between treated and vehicle groups. (D) Radiant heat normalized paw withdrawal latencies 20–30 min post injection of 15 µL 10 µM S1P, 0.2 µM S1P. or 0.3% methanol-PBS vehicle (i.d.) into the hind paw of S1PR3 WT or KO mice; p=0.0129 (one-way ANOVA; N = 3–10 mice per condition). Dunnett’s multiple comparisons p-values are represented on graph for comparisons made between treated and vehicle groups. (E) von Frey 50% withdrawal measurements for mice pre- (baseline) and post-injection of 50 µM SKI II (N = 14) and 0 (N = 4), 10 (N = 3), 75 (N = 4), or 200 nM S1P (N = 3; one-way ANOVA; p=0.0001). Tukey Kramer comparisons are indicated on graph. Error bars represent mean ± SD. https://doi.org/10.7554/eLife.33285.005 Figure 2—source data 1 Endogenous S1P mediates acute mechanical pain. Related to Figure 2. https://doi.org/10.7554/eLife.33285.006 Download elife-33285-fig2-data1-v2.xlsx These data support a model whereby S1P constitutively activates S1PR3 to set normal mechanical pain thresholds. To further test this model, we asked if the mechanical hyposensitivity elicited after endogenous S1P depletion (via SKI II) could be rescued by local injection of exogenous S1P. Indeed, we found that injection of exogenous S1P reversed SKI II-induced mechanical hyposensitivity in a dose-dependent manner, and observed a maximal effect with 200 nM S1P (Figure 2E). Although quantification of native S1P levels in skin is inaccurate owing to avid lyase activity (Shaner et al., 2009), our data establish that baseline S1P levels are sufficient to maximally exert their effect on S1PR3-dependent mechanical pain, such that increased S1P does not evoke mechanical hypersensitivity, but diminished S1P leads to mechanical hyposensitivity. These data show that constitutive activation of S1PR3 by S1P is required for normal mechanosensitivity. S1PR3 is expressed in A mechanonociceptors and thermal nociceptors Our behavioral data showing distinct roles for S1PR3 in mechanonociception and thermal hypersensitivity suggest that S1PR3 is expressed in distinct subsets of somatosensory neurons. While a previous study suggested that all somatosensory neurons express S1PR3 (Camprubí-Robles et al., 2013), single cell RNA seq data suggests S1pr3 is not expressed by all DRG neurons (Usoskin et al., 2015), and no studies have performed quantitative analysis of S1PR3 staining or co-staining to define subpopulations of S1PR3+ neurons. We thus set out to characterize the somatosensory neuron subtypes expressing S1pr3 using in situ hybridization (ISH) of wild-type somatosensory ganglia and immunohistochemistry (IHC) in an S1pr3mCherry/+ reporter mouse (Sanna et al., 2016). We first used in situ hybridization (ISH) with a specific S1pr3 probe to examine expression patterns of S1pr3 (Figure 3A–B; see Supplementary file 1). In our experiments, 43% of cells from wild-type DRG expressed S1pr3. Co-ISH revealed that one population of S1pr3+ neurons represents Aδ mechanonociceptors (AMs). These cells expressed Scn1a (39.9% of all S1pr3+), a gene that encodes the Nav1.1 sodium channel, which mediates mechanical pain in Aδ fibers (Osteen et al., 2016). S1pr3+ cells also co-expressed Npy2r (20.4% of all S1pr3+), a marker of a subset of mechanonociceptive A fibers (Arcourt et al., 2017). S1pr3 was expressed in 70.6% of Scn1a+ cells and 72% of Npy2r+ cells, comprising a majority of both of these populations. Interestingly, a subset of cells co-expressed S1pr3 and the mechanically sensitive channel Piezo2, which is expressed by Aβ, Aδ, and C fibers (Ranade et al., 2014). The remaining S1pr3+ cells were Trpv1+ and/or Trpa1+ C nociceptors (67.1% of all S1pr3+), which are reported to overlap minimally with the Scn1a+ and Npy2r+ populations (Arcourt et al., 2017; Osteen et al., 2016). Figure 3 with 1 supplement see all Download asset Open asset S1pr3 is expressed in A mechanonociceptors and C thermal nociceptors. (A) (Top) Representative co-ISH of S1pr3 (green; left) with Scn1a, Npy2r, Piezo2, and Trpv1 (magenta; center) in sectioned DRG. Right column: overlay with co-localized regions colored white (10x air objective; scale = 100 µm). (B) Bar chart showing the % of total cells expressing the indicated marker (grey) and the % of total cells co-expressing both marker and S1pr3 (green). See Table S1 for quantification. (C) Representative IHC images of sectioned DRG from S1pr3mCherry/+ animals stained with anti-DsRed (green, S1PR3) and anti-Peripherin (left, magenta) or anti-NF200 (right, magenta). Arrows indicate co-stained cells. Images were acquired using a 10x air objective (scale = 100 µm). (D) Whole-mount skin IHC confocal images with anti-DsRed antibody (S1PR3, green) and anti-NefH antibody (NF200, magenta) in an S1pr3mCherry/+ animal (20x water objective; scale = 50 µm). Arrows indicate co-positive free nerves (left image). Arrowheads indicate NF200- free nerves (left) or S1PR3- circumferential fibers (right image). (E) Sectioned skin IHC with anti-DsRed (S1PR3) and anti-NefH (NF200, left, top right) or anti-DsRed (S1PR3) and anti-beta-tubulin III (BTIII, bottom right) antibody (magenta) in S1pr3mCherry/+ skin (20x air objective; scale = 50 µm). Arrows indicate co-positive free nerve endings (left), S1PR3-negative lanceolate/circumferential hair follicle endings (top right, arrow = circumferential, arrowhead = lanceolate), or S1PR3-negative putative Merkel afferent (bottom right). (F) (Left) Quantification of sectioned DRG IHC experiments showing % of S1PR3+ cells that co-stained with indicated markers (n > 250 cells per marker). (Right) Quantification of sectioned skin IHC experiments showing % of fibers positive for indicated marker that co-stained with S1PR3 (anti-DsRed; n = 10 images per marker from two animals). https://doi.org/10.7554/eLife.33285.007 We next used an S1pr3mCherry/+ reporter mouse, which produces a functional S1PR3-mCherry fusion protein (Sanna et al., 2016), as an independent strategy to explore S1PR3 expression and localization. This strategy was used because we found that anti-S1PR3 antibodies showed broad immunoreactivity in DRG from mice lacking S1PR3, and so we instead used anti-DsRed antibodies to probe expression of the S1PR3 fusion protein (Figure 3—figure supplement 1E). We found that 42.4% of S1PR3+ cells co-stained with anti-Peripherin, demonstrating that S1PR3 is expressed in a subset of small-diameter neurons. We also observed that 69.5% of S1PR3+ cells co-stained with anti-NF200, which marks medium and large-diameter myelinated neurons. Furthermore, we observed that S1PR3+ cells were primarily of small to medium diameter (11.3–35.1 µm), whereas all cells in the DRG ranged from 11.3 to 53.9 µm. Overall, these data support the expression of S1PR3 in subsets of small-diameter thermal nociceptors and medium-diameter mechanonociceptors (Figure 3F). Additionally, no significant differences were observed between WT and S1PR3 KO DRG in number of Trpa1+, Trpv1+, Peripherin+, NF200+, or IB4+ cells (Figure 3—figure supplement 1B–C,F,G). The mean diameters of Trpv1+ neurons (Figure 3—figure supplement 1D, left), NF200+ neurons (Figure 3—figure supplement 1G), or all neurons (Figure 3—figure supplement 1D, right) in WT versus KO DRG were not significantly different, suggesting no loss of major sensory neuronal subtypes in the S1PR3 KO. We then visualized S1PR3 expression in nerve fibers that innervate the skin using anti-DsRed antibodies in whole-mount immunohistochemistry (IHC; Figure 3D). The reporter animals showed no specific antibody staining in epidermal or dermal cells (Figure 3—figure supplement 1I), and single-cell RNA seq of a diverse array of mouse epidermal and dermal cells corroborates this lack of expression (Joost et al., 2016). We observed overlap of S1PR3-expressing free nerve endings with NF200+ myelinated free nerves and NF200- putative C-fiber endings (Figure 3F), but did not observe expression of S1PR3 in NF200+ circumferential or lanceolate hair follicle receptors, or in putative Merkel afferents (Figure 3D–E). β-tubulin III, PGP9.5 (pan-neuronal markers), and NF200 staining in S1PR3 KO skin displayed patterns of epidermal and dermal innervation similar to WT skin, suggesting the phenotypes observed in the S1PR3 KO mice are not due to developmental loss of sensory neuronal innervation (pPGP9.5= 0.443 (n = 93, 38 fibers); pNefH = 0.405 (n = 61, 28 fibers); pBTIII = 0.353 (n = 104, 89 fibers); two-tailed t-tests based on average number of fibers per field of view). These results support expression of S1PR3 in subsets of myelinated A mechanonociceptors and unmyelinated C nociceptors that terminate as free nerve endings. S1P activates thermal nociceptors but not putative AMs Live imaging of cultured DRG neurons from adult reporter animals showed expression of S1PR3-mCherry fusion protein in 48.3% of neurons, mirroring our ISH and IHC results (Figure 4A). To examine the effects of S1P on A mechanonociceptors and C nociceptors, we performed ratiometric calcium imaging and electrophysiology on DRG cultures from reporter mice. Interestingly, only 56.1 ± 22.4% of mCherry-expressing neurons were activated by 1 µM S1P (Representative trace in Figure 4B; representative images in Figure 4C), which our dose-response showed to be the saturating concentration for calcium influx (Figure 4D; EC50 = 155 nM). All S1P-responsive neurons were also capsaicin-sensitive (n > 2000 neurons). And while sensory neurons from S1PR3 KO animals did not respond to S1P, as expected (Camprubí-Robles et al., 2013), they exhibited capsaicin responses that were not significantly different from WT neurons (Figure 3—figure supplement 1H). The mean diameter of S1P-responsive mCherry+ neurons was 22.4 ± 1.0 µm, whereas the mean diameter of non-responsive mCherry+ neurons was 28.7 ± 3.2 µm (p=0.0002, two-tailed t-test). We also performed whole cell current clamp experiments and, consistent with other studies (Mair et al., 2011; Zhang et al., 2006; Li et al., 2015), found that S1P evoked action potential firing in capsaicin-sensitive small diameter cells (Figure 4E). This shows that only the small-diameter, S1PR3+ putative nociceptors are excited by S1P. We next asked whether the S1PR3+ medium-large diameter neurons represent the mechanonociceptors observed by ISH (Figure 3A). To this end, we asked whether the spider toxin Hm1a, a selective activator of AM nociceptors (Osteen et al., 2016), triggers calcium influx in S1PR3-expressing trigeminal neurons. Indeed, we found that 44.2 ± 15.1% of Hm1a-responsive neurons expressed mCherry (Figure 4F), consistent with our staining showing expression of S1pr3 in AM nociceptors and the role of Hm1a-responsive neurons in mediating mechanical pain in vivo (Osteen et al., 2016). Figure 4 Download asset Open asset S1P activates thermal nociceptors but not mechanonociceptors. (A) (Left) Representative image of mCherry signal in live, cultured adult DRG neurons from one S1pr3mCherry/+ animal. (Right) Quantification of % of total cells expressing S1pr3 from DRG ISH and mCherry from dissociated DRG cultures (N = 2 animals each experiment). (B) Representative traces depicting F340/F380 signal from Fura2-AM calcium imaging showing two neurons, one which responded to 1 µM S1P, 1 µM Capsaicin, and high K + Ringer’s (red) and one which only responded to high K+ (black). (C) (Left) Fura-2 AM calcium imaging before (left) and after (center) addition of 1 µM S1P in S1pr3 mCherry/+ cultured mouse DRG neurons. Bar indicates fluorescence ratio. Right-hand image indicates mCherry fluorescence. (Right) % of mCherry neurons that are responsive to 1 µM S1P in ratiometric calcium imaging (n > 1000 cells from 16 imaging wells from three animals). (D) Dose-response curve of mean neuronal calcium responders to varying concentrations of S1P. Concentrations used: 1, 10, 50, 100, 200, 1000, and 10,000 nanomolar (N = 2 animals). Error bars represent mean ± SD. Black dotted line indicates sigmoidal fit for all S1P responders from which EC50 was derived. All S1P responders were also capsaicin-responsive. (E) Current-clamp trace of a single wild-type neuron firing action potentials in response to bath addition of 1 µM S1P and 1 µM capsaicin, with Ringer’s wash in-between. Four of tenneurons responded to S1P and one of one S1P-responsive also responded to capsaicin. Bar = 2 s. (F) (Left) Fura-2 AM calcium imaging after addition of 500 nM Hm1a in S1pr3mCherry/+ P0 TG neurons, which were used instead of adult DRG neurons because they respond to Hm1a without prior PGE2 sensitization. Right-hand image indicates mCherry fluorescence. (Right) % of Hm1a-responsive P0 TG neurons that are mCherry+ (N = 1 animal, 1230 total neurons). https://doi.org/10.7554/eLife.33285.009 S1PR3 modulates KCNQ2/3 channels to regulate AM excitability We next interrogated the molecular mechanism by which S1P signaling in AM nociceptors may regulate mechanical pain. We performed whole-cell current clamp on the medium-diameter S1pr3mCherry/+ dissociated DRG neurons (membrane capacitance = 61.05 ± 1.92 pF), which did not display S1P-evoked calcium influx (Figure 4B–C). In these cells, 1 µM S1P application did not change membrane potential (Figure 5—figure supplement 1A; see Figure 5—source data 1) or elicit firing in the absence of current injection (Figure 5—figure supplement 1A; Figure 5A). However, S1P dramatically lowered the threshold to fire action potentials (rheobase) in an S1PR3-dependent manner (Figure 5A, Figure 5—figure supplement 1B). Figure 5 with 1 supplement see all Download asset Open asset S1PR3 modulates KCNQ2/3 channels to regulate AM excitability. All experiments were performed in S1pr3mCherry/+ or -/- DRG neurons. (A) (Left) Example traces of a single mCherry +neuron in whole cell current clamp before and after S1P application. (Right) % change in rheobase after S1P application for S1pr3mCherry/+ (left, n = 7) and KO (right, n = 12) neurons (pWT,KO = 0.012, 0.287; two-tailed paired t-tests). (B) % ∆ in input resistance after S1P or vehicle application (p=0.017; two-tailed paired t-test; n = 4 cells per group). (C) The S1P-sensitive current is carried by potassium. The current-voltage relationship was determined by subtraction of the post-S1P current from the pre-S1P current and reverses at −60.125 mV; n = 6 cells. Data were fitted with a Boltzmann equation. Pre- and post-S1P currents were measured at the indicated voltage (−100 mV to +80 mV, 20 mV increments) following a +100 mV step (100 ms). Current was quantified using the peak absolute value of the slowly-deactivating current 0–10 ms after stepping to indicated voltage. Unless indicated otherwise, all error bars represent mean ± SEM. (D) (Graphic, top) Averaged current traces of a single mCherry+ neuron in whole cell voltage clamp recording comparing tail currents (∆I tail) pre- and post-S1P using indicated voltage step protocol. (graphic, bottom) Averaged current traces of a single mCherry+ neuron in whole cell voltage clamp recording with XE991 treatment. Holding phase (−40 mV, 150 ms) was truncated in traces. (Left graph) % ∆ in outward tail current (average ±SD after indicated treatments (1 µM S1P, 3 µM XE 991, or both) for S1pr3mCherry/+ medium-diameter neurons; (p=0.58; one-way ANOVA; n = 6, 8, 14 cells) using protocol depicted at right. (Right graph) % ∆ in inward tail current after indicated treatments (LINO = 100 µM linopirdine) for S1pr3mCherry/+ medium-diameter neurons; (p=0.47; two-tailed paired t-test; n = 12 cells). https://doi.org/10.7554/eLife.33285.010 Figure 5—source data 1 S1PR3 modulates KCNQ2/3 channels to regulate AM excitability. Related to Figure 5. https://doi.org/10.7554/eLife.33285.012 Download elife-33285-fig5-data1-v2.xlsx We then set out to determine the mechanism by which S1PR3 activity increases neuronal excitability using whole-cell voltage clamp recording. Previous studies showed that S1P excites capsaicin-sensitive nociceptors by increasing voltage-gated sodium currents and reducing steady-state potassium currents (Zhang et al., 2006; Li et al., 2015). We found that S1P had no such effects on S1PR3+ medium-diameter cells (Figure 5—figure supplement 1C–E). By contrast, S1P triggered a robust increase in input resistance (Figure 5B), consistent with the closure of potassium channels. I-V analysis revealed that the current inhibited by S1P application was carried by potassium (Figure 5C). Additionally, S1P significantly reduced slow, voltage-dependent tail current amplitudes (Figure 5—figure supplement 1F; Figure 5D (top)) in an S1PR3-dependent manner (Figure 5—figure supplement 1F, center). As tail currents in Aδ neurons are primarily mediated by KCNQ2/3 potassium channels (Schütze et al., 2016; Passmore et al., 2012), we postulated that S1P may alter tail currents through modulation of these channels. Furthermore, the above properties of the S1P-sensitive cur