Author response: Anti-nociceptive action of peripheral mu-opioid receptors by G-beta-gamma protein-mediated inhibition of TRPM3 channels
2017-08-07

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Opioids, agonists of μ-opioid receptors (μORs), are the strongest pain killers clinically available. Their action includes a strong central component, which also causes important adverse effects. However, μORs are also found on the peripheral endings of nociceptors and their activation there produces meaningful analgesia. The cellular mechanisms downstream of peripheral μORs are not well understood. Here, we show in neurons of murine dorsal root ganglia that pro-nociceptive TRPM3 channels, present in the peripheral parts of nociceptors, are strongly inhibited by μOR activation, much more than other TRP channels in the same compartment, like TRPV1 and TRPA1. Inhibition of TRPM3 channels occurs via a short signaling cascade involving Gβγ proteins, which form a complex with TRPM3. Accordingly, activation of peripheral μORs in vivo strongly attenuates TRPM3-dependent pain. Our data establish TRPM3 inhibition as important consequence of peripheral μOR activation indicating that pharmacologically antagonizing TRPM3 may be a useful analgesic strategy. https://doi.org/10.7554/eLife.26280.001 eLife digest There are very few treatments available for people suffering from strong or long-lasting pain. Currently, substances called opioids – which include the well-known drug morphine – are the strongest painkillers. However, these drugs also cause harmful side effects, which makes them less useful. Like all drugs, opioids mediate their effects by interacting with molecules in the body. In the case of opioids, these interacting molecules belong to a group of receptor proteins called G-protein coupled receptors (or GPCRs for short). These opioid receptors are widely distributed in the nerve cells and brain regions that detect and transmit pain signals. It was poorly understood how activation of opioid receptors reduces the activity of pain-sensing nerve cells, however several lines of evidence had suggested that a protein called TRPM3 might be involved. TRPM3 is a channel protein that allows sodium and calcium ions to enter into nerve cells by forming pores in cell membranes, and mice that lack this protein are less sensitive to certain kinds of pain. Dembla, Behrendt et al. now show that activating opioid receptors on nerve cells from mice, with morphine and a similar substance, rapidly reduces the flow of calcium ions through TRPM3 channels. Further experiments confirmed that activating opioid receptors in a mouse’s paw also reduced the pain caused when TRPM3 proteins are activated. GPCRs interact with a group of small proteins called G-proteins that, when activated by the receptor, split into two subunits. Based on studies with human kidney cells, Dembla, Behrendt et al. found the so-called G-beta-gamma subunit then carries the signal from the opioid receptor to TRPM3. Two independent studies by Quallo et al. and Badheka, Yudin et al. also report similar findings. These new findings show that drugs already used in the treatment of pain can indirectly alter how TRPM3 works in a dramatic way. These results might help scientists to find drugs that work in a more direct way to dial down the activity of TRPM3 and to combat pain with fewer side effects. Though first it will be important to confirm these new findings in human nerve cells. https://doi.org/10.7554/eLife.26280.002 Introduction Throughout the peripheral and central parts of the nociceptive system, μ-opioid receptors (μORs) are widely expressed and strongly control neuronal excitation (Stein, 2016). Agonists of μORs are the most potent analgesic drugs clinically available (Pasternak and Pan, 2013) and are therefore often prescribed for the treatment of severe pain. These opioid substances are especially effective against acute pain states, such as post-operative pain, but they are also used, more controversially, for the treatment of longer lasting or chronic pain (Rowbotham et al., 2003; Chou et al., 2015). Much of the controversy around opioids arises because these substances cause important unwanted effects, such as addiction, tolerance (Volkow and McLellan, 2016), opioid-induced hyperalgesia (Roeckel et al., 2016) and, when overdosed, respiratory depression (Pattinson, 2008). Because of this unfavorable profile of unwanted effects, clinically used opioids are often implicated in fatal overdosing due to drug addiction or dosing accidents (Compton et al., 2016; Ray et al., 2016). While many actions of opioids are triggered by activation of μORs in the central nervous system, opioid receptors are also located on the peripheral nerve endings of nociceptor neurons (Stein et al., 1990a, 1990b; Stein, 2013). Physiologically, in the skin, where many peripheral nociceptor nerve endings reside, opioid receptors are targeted by endogenous opioid substances, such as β-endorphin, released in the periphery from immune cells (Stein et al., 1990b) or skin keratinocytes (Ibrahim et al., 2005; Fell et al., 2014). Activation of peripheral opioid receptors can provide clinically meaningful analgesia (Farley, 2011; Stein and Machelska, 2011). On the contrary, inhibiting peripheral μORs by antagonist application increases pain (Jagla et al., 2014). Targeting peripheral μORs thus has been proposed as a strategy to provide analgesia with reduced adverse effects and an improved safety profile (Stein et al., 2003). An alternative strategy, in which not the μORs themselves but downstream effectors of μOR signaling pathways are targeted, may also prove to be beneficial. However, such strategies have received less attention, partly because the downstream targets of peripheral μOR signaling are not well documented. At central synapses, several intracellular mechanisms leading to reduced neuronal excitation during μOR activation have been worked out in considerable detail. Activation of μORs causes inhibition of L-type voltage-gated Ca2+ channels (Bourinet et al., 1996; Heinke et al., 2011) and activation of several types of K+ channels (Law et al., 2000; Marker et al., 2005). Importantly, also at several central synapses, μORs have been shown to interact with TRP channels, notably TRPV1 (recently reviewed by Bao et al., 2015). The interaction of TRPV1 and μOR occurs at multiple anatomical locations in the central nervous system and its physiological consequences and mechanisms are highly diverse (Maione et al., 2009; Stein, 2016). Proposed mechanisms range from influencing the cAMP-PKA pathway and the participation of several other kinases (ERK, MAPK, JNK) to β-arrestin2 and the PI3-kinase pathway (Law et al., 2000; Rowan et al., 2014; Bao et al., 2015). In peripheral nerves and their endings, several, partially different, intracellular signaling events have been proposed to account for the effectiveness of peripherally restricted opioids, including inhibition of voltage-gated Na+ channels (Gold and Levine, 1996), inhibition of HCN channels (Ingram and Williams, 1994), activation of several classes of K+ channels (Cunha et al., 2010; Nockemann et al., 2013; Baillie et al., 2015), and, importantly, again the inhibition of TRPV1 channels (Vetter et al., 2006; Endres-Becker et al., 2007; Spahn et al., 2013) However, despite the plethora of proposed targets, the magnitude and the interplay of these individual effects has not been determined. Furthermore, many of these ion channels are not inhibited by μOR activation per se, but rather their upregulation or sensitization by the cAMP/PKA pathway is blocked by μOR signaling. It is therefore unclear, how much each of these targets contributes to the overall effect of opioids on peripheral nerve endings, and how this contribution may vary during shifts from resting to inflamed states (and back). Further, it is unclear, and perhaps even unlikely, that all important downstream targets of μORs have already been identified. Recently, TRPM3 channels (Oberwinkler and Philipp, 2014) were described in primary nociceptive neurons (Nealen et al., 2003; Lechner et al., 2009; Vriens et al., 2011; Straub et al., 2013a, 2013b; Held et al., 2015; Usoskin et al., 2015) and it was shown that these divalent-permeable cation channels (Grimm et al., 2003; Oberwinkler et al., 2005; Wagner et al., 2010) play an important role in noxious heat sensation since these channels are intrinsically thermosensitive (Vriens et al., 2011). Activation of TRPM3 leads to release of pro-inflammatory CGRP from peripheral nociceptor nerve terminals (Held et al., 2015), while TRPM3-deficient animals show severe defects in the development of inflammatory hyperalgesia (Vriens et al., 2011). In this context, it is interesting that inhibitors of TRPM3 channels have been identified that exhibit strong anti-nociceptive properties (Straub et al., 2013b; Chen et al., 2014; Suzuki et al., 2016; Krügel et al., 2017). Hence, TRPM3 channels in peripheral nociceptors have pro-nociceptive and pro-inflammatory properties, making them an interesting target to study the mechanism of peripheral nociception and inflammation. Here, we demonstrate that TRPM3 channels in primary nociceptive neurons are rapidly and strongly inhibited by agonists of μORs such as morphine, through an intracellular signaling cascade relaying μORs to TRPM3 channels via Gβγ proteins. We show that local activation of peripheral μORs causes strong analgesia of TRPM3-dependent pain. Our results indicate that it may be worthwhile to further investigate TRPM3 channels as potential targets for the treatment of pain with reduced adverse effects in the central nervous system. Results Activation of μORs inhibits TRPM3 channels in DRG neurons TRPM3 channels possess pro-nociceptive properties in DRG neurons (Vriens et al., 2011; Straub et al., 2013a, 2013b; Chen et al., 2014; Held et al., 2015). We therefore investigated clinically used analgesic drugs for their effects on TRPM3 channels. To address this question, we first activated TRPM3 channels in isolated mouse DRG neurons by applying the known TRPM3 agonist pregnenolone sulfate (PS) (Wagner et al., 2008; Vriens et al., 2011). Under our recording conditions, the ensuing Ca2+ signals were almost completely dependent on the presence of TRPM3 proteins, as demonstrated in control experiments with neurons prepared from TRPM3-deficient (TRPM3-knockout) mice (Figure 1—figure supplement 1). Testing whether morphine, a traditional agonist of opioid receptors, or its synthetic, μOR-specific analog DAMGO (Handa et al., 1981), influences TRPM3 channel activity, we found that both μOR agonists strongly reduced the Ca2+ signals induced by PS (Figure 1a–c). The reduction of these Ca2+ signals was rapid and readily reversible within a time frame of 1–2 min (Figure 1d). We ascertained that DAMGO acted via activating opioid receptors by co-applying the opioid receptor antagonist naloxone, which unspecifically blocks several opioid receptor subtypes (Figure 1e–g). Naloxone alone did not affect TRPM3-dependent Ca2+ signals in DRG neurons (Figure 1e,g), but, when co-applied with DAMGO, it prevented the action of DAMGO completely (Figure 1f,g). In addition, we tested two further, structurally different, μOR agonists, herkinorin and loperamide. Both substances strongly inhibited PS-induced Ca2+ signals in isolated mouse DRG neurons (Figure 1—figure supplement 2). These data indicate that activation of μORs, independent of the chemical nature of the agonist, leads to inhibition of TRPM3 channels in DRG neurons. Figure 1 with 6 supplements see all Download asset Open asset Activated μORs inhibit TRPM3-induced Ca2+ signals in somatosensory neurons and in an overexpression system. (a) DRG neurons were stimulated three times with PS (25 μM) and morphine (1 μM, red trace, n = 188 neurons, five recordings) or vehicle as control (black trace, n = 130 neurons, four recordings). (b) Instead of morphine, DAMGO (0.3 μM, blue trace, 47 neurons, three recordings) was applied (control, black trace, 59 neurons, three recordings). Images of cells isolated from DRGs as used in these experiments are presented in Figure 1—figure supplement 5, example traces of individual cells in Figure 1—figure supplement 6a. (c) Quantification of the inhibition measured in (a) and (b); each dot-like symbol next to the bars represents the inhibition measured and calculated for a single, individual cell. Control experiments showing that the recorded Ca2+ signals are TRPM3-dependent are presented in Figure 1—figure supplement 1. Similar data with two chemically distinct μOR agonists are shown in Figure 1—figure supplement 2. (d) Inhibition by 0.3 μM DAMGO and recovery in DRG neurons stimulated with 25 μM PS, 0.3 μM DAMGO as indicated (n = 111 neurons, seven recordings). (e) The opioid receptor antagonist naloxone (5 μM) did not have an effect on its own (n = 412 neurons, 14 recordings). (f) Naloxone blocked the inhibitory effect of DAMGO on TRPM3 (n = 364 neurons, 15 recordings). Example traces of individual cells from these experiments are shown in Figure 1—figure supplement 6b. (g) Statistical analysis of the recordings shown in panels (d–f). The inhibition caused by DAMGO or naloxone was evaluated. The symbols on the right side of the bars represent inhibition measured in individual cells. (h) In HEK cells overexpressing TRPM3 and μORs, morphine (1 μM) inhibited Ca2+ signals evoked by 25 μM PS (orange trace, n = 115 cells, four recordings), but not in control cells expressing only TRPM3 (green trace, n = 97 cells, same four recordings). (i) DAMGO (0.3 μM) inhibits TRPM3-dependent Ca2+ signals induced by 50 μM nifedipine (orange trace: n = 58 cells, green trace: n = 75 cells, both from three recordings). (j) DAMGO (0.3 μM) inhibits TRPM3-dependent Ca2+ signals induced by heat (upper panel). Orange trace (n = 59 cells from four recordings) is the average of cells overexpressing TRPM3 and μORs. The blue trace (n = 89 cells, three recordings) represents control measurements from HEK cells expressing only μORs; there, TRPM3-independent heat-evoked Ca2+ signals are not inhibited by μOR activation. The lower panel shows the time course of the applied temperature. (k) Exemplary patch-clamp recording of a HEK cell overexpressing TRPM3 (activated by 25 μM PS) and μORs (activated by 0.3 μM DAMGO). Traces were obtained at +80 and −80 mV from voltage ramps. (l) I/V-curves measured at time points indicated in (k). (m) Statistical analysis of the baseline-subtracted current densities (n = 15 cells). A graphical representation of the recorded values for each cell is given in Figure 1—figure supplement 3. A dose response curve for the DAMGO-induced inhibition of TRPM3 currents is shown in Figure 1—figure supplement 4. https://doi.org/10.7554/eLife.26280.003 We successfully reconstituted the signaling pathway from μORs to TRPM3 channels in a heterologous overexpression system by co-expressing μORs and TRPM3 in HEK cells (Figure 1h). Importantly, omitting μOR cDNA from the transfection abolished the effect of morphine (Figure 1h) and DAMGO (as seen, for example, in Figure 6f), providing strong evidence that these agonists act through μORs and do not interfere with TRPM3 channel activity directly. Like many members of the TRP superfamily, TRPM3 channels are activated by several different agonists (Oberwinkler and Philipp, 2014). Taking advantage of the overexpression system, which avoids the complication of other nifedipine- or heat-sensitive conductances (Fajardo et al., 2008; Julius, 2013), we demonstrated that TRPM3 channels activated by the application of nifedipine (Wagner et al., 2008; Drews et al., 2014) or by heat applied in the absence of chemical agonists (Vriens et al., 2011) were also inhibited by μOR activation (Figure 1i,j). Whole-cell patch-clamp electrophysiological recordings from HEK cells overexpressing TRPM3 and μORs demonstrated that currents through TRPM3 channels were inhibited by activating μORs rapidly in less than 5 s (Figure 1k–m, Figure 1—figure supplement 3). After measuring a dose-response curve by varying the concentration of DAMGO, we found that the IC50 values at +80 mV and −80 mV did not differ, providing an indication that inhibition of TRPM3 channels by μOR activation was voltage-independent (Figure 1—figure supplement 4). Similarly, performing patch-clamp experiments on isolated DRG neurons, we observed that the currents evoked by TRPM3 agonists were rapidly (less than 5 s) inhibited by μOR activation (Figure 2a–c). Upon washout of DAMGO, the inhibition was at least partially reversible. Activation of opioid receptors causes many cellular responses in DRG neurons (Law et al., 2000), including the activation of K+ channels (Yoshimura and North, 1983; Ocaña et al., 2004). Importantly, stimulation of K+ channels might reduce Ca2+ signals in DRG neurons through hyperpolarization-induced closure of voltage-gated Ca2+ channels. Because K+ channels cannot directly be studied in Ca2+ imaging experiments, we continued to use the whole-cell patch-clamp technique on isolated DRG neurons. Aiming for small-diameter nociceptors, we selected cells that responded either to TRPV1- or to TRPM3-activating (or to both types of) agonists. In these cells, we could not detect outward currents upon application of the μOR agonist DAMGO (Figure 2d–f). This indicates that the reduction of PS-induced currents by μOR activation is not due to the activation of outward currents. Consequently, the DAMGO-induced reduction of PS-evoked Ca2+ signals observed in Ca2+ imaging experiments (e.g. in Figure 1a–g) is unlikely to be caused by K+ channel-dependent hyperpolarization. Together, these data argue that TRPM3 channels in DRG neurons are inhibited by activating μORs. Figure 2 with 2 supplements see all Download asset Open asset Inhibition of TRPM3 by μORs in DRG neurons is variable. (a) Example of an electrophysiological whole-cell patch-clamp recording from an isolated DRG neuron responding to TRPM3 agonists (PS and nifedipine at 50 μM each), DAMGO (0.3 μM) and capsaicin (0.1 μM). The recording was performed in monovalent-free solution in order to reduce currents through endogenous voltage-gated channels. (b) Statistical analysis of 11 cells responding with an inward current to the combined application of PS and nifedipine. Baseline-subtracted current densities were evaluated at similar time points as indicated by arrows and numbers in (a). (c) To illustrate the variability, the inhibition observed in individual cells is shown. (d–f) Application of DAMGO alone (in standard extracellular solution containing K+) did not evoke significant currents in DRG neurons responsive to TRPM3 or TRPV1 agonists. (d) Exemplary recording of a neuron at −60 mV holding potential sensitive to 1 μM capsaicin and TRPM3 agonists (PS and nifedipine at 50 μM each), (e) statistical analysis of 13 different DRG neurons, (f) DAMGO-induced increase in current density in individual cells, evaluated as difference in current density for each cell between time points 1 and 2 marked with arrows in (d). In (c) and (f), the box indicates mean and 25 and 75 percentiles, the whiskers represent standard deviations. (g–i) Similarly, in Ca2+ imaging experiments, Ca2+ signals evoked by 25 μM PS in DRG neurons were variably sensitive to inhibition by DAMGO (3 μM). PS-sensitive DRG neurons (17% of total neurons, i.e. 614 out of 3576 neurons, obtained from 5 mice in 35 recordings) were classified as sensitive (g) or insensitive (h) to 2 μM capsaicin, and each subgroup was further divided into DAMGO-sensitive and DAMGO-insensitive cell populations and averaged separately. (i) Overview of the number of cells in each subgroup (which also corresponds to the number of neurons averaged for the traces in (g) and (h). Overall, 91.5% (562 of 614) of the PS-sensitive neurons were inhibited by DAMGO. The size distribution of the cells analyzed in (g–i) is shown in Figure supplement 1. A graphical representation of the response of the individual cells analyzed in this is given in Figure supplement 2. inhibition of TRPM3 channels occurs in DRG neurons Our patch-clamp data of PS-sensitive DRG neurons indicated that the inhibition of TRPM3-dependent currents in DRG neurons was (Figure We therefore measured a number of DRG neurons in Ca2+ imaging experiments (Figure and found that not in all PS-sensitive neurons activation of μORs caused a reduction of Ca2+ signals. we these neurons, the to (562 in a total of 614 PS-sensitive cells analyzed (Figure We tested whether TRPM3 inhibition by μOR activation with the presence of TRPV1 channels in these cells, the of this that in mice, TRPV1 channels are restricted to nociceptors et al., in which μORs are also expressed (Vetter et al., 2006; et al., In with we found that most neurons of cells, which a reduction of TRPM3-dependent Ca2+ signals due to application of DAMGO, also Ca2+ the presence of TRPV1 channels. only a of the cells effect of DAMGO on TRPM3-dependent Ca2+ signals of DAMGO-insensitive cells, i.e. of the DAMGO-insensitive cells, to of all PS-sensitive a response to capsaicin (Figure These data are by the size distribution of DAMGO-sensitive and of PS-sensitive cells (Figure supplement 1). PS-sensitive cells of both the distribution of small-diameter and neurons (Vriens et al., 2011; and 2016), with only very few cells of 614 cells, to On the other the group of neurons that responded to PS to capsaicin to a number of cells of cells, to Figure supplement Together, these data show that activation of μORs is of rapidly and strongly currents through TRPM3 channels in a of DRG neurons, but not to TRPM3 channels are inhibited by receptors coupled to proteins DRG neurons a of different receptors (Gold and 2010; et al., 2015), many of which have been in the of these neurons. μORs to members of the i.e. proteins (Law et al., In GPCRs that to proteins have been in the activity of peripheral We therefore tested other GPCRs that to proteins and found that all of them inhibited TRPM3-dependent Ca2+ signals in DRG neurons, the number of neurons responding to the agonists was (Figure 3). we found that TRPM3 was inhibited by activating receptors, receptors, receptors, and receptors (Figure 3). we activated five receptors which to proteins et al., we observed the inhibition of all agonists, which was only in a small number of neurons. In addition, the inhibition caused by did not upon washout (Figure These data show that the activity of TRPM3 channels can be inhibited by a of Figure with 1 supplement see all Download asset Open asset A of different receptor types inhibits TRPM3 channels in isolated DRG neurons. sensitive to pregnenolone sulfate 25 μM) were selected and as sensitive or insensitive with to the agonist In (c) and neurons were in to capsaicin μM) similar to the analysis in Figure Traces represent within these numbers in the number of neurons used for the (a) μM was used to receptors cells from (b) 1 μM was used to receptors cells from (c) 1 μM was used to receptors cells from three (d) 2 μM was used to cells from two (e) μM was used to receptors cells from two (f) receptors were stimulated with μM four cells from Traces of individual cells from the shown in panels (a) – (f) are shown in Figure supplement 1. (g) of inhibition of the PS-evoked Ca2+ signal of all PS-sensitive neurons. The dot-like symbols on the right side of the represent the inhibition measured for each individual cell. (h) of the experiments showing the of PS-sensitive neurons responding with a reduction of PS-induced Ca2+ signals than the of see Materials and methods to the stimulation with the indicated The of the of cells is indicated on the (i) as panel but the average inhibition of only cells that show an inhibition than the The data for the μOR agonist DAMGO in panels (g–i) are from Figure these data indicate that the inhibition of TRPM3-dependent signals is especially when μORs or receptors Activation of μORs not strongly inhibit other TRP channels expressed in DRG neurons from other TRP channels are in and inflammatory in peripheral somatosensory neurons 2013). We tested therefore whether other TRP channels expressed in DRG neurons were inhibited by activation of μORs, by the same as on TRPM3 channels. Ca2+ signals induced by the TRPV1 agonist capsaicin the same reduction in activity whether DAMGO or the control was applied to the DRG neurons (Figure Furthermore, when we used a different and co-applied DAMGO with longer applied capsaicin, there was a much inhibition to the inhibition observed with TRPM3 (Figure supplement 1). we did not difference when we between PS-sensitive and neurons (Figure supplement green red of the PS-sensitive neurons are to possess μORs Figure the Ca2+ signals did not DAMGO washout (Figure supplement again in marked to the of TRPM3 channels. We then investigated the of channels on μOR activation in DRG neurons. Because channels are to several we used the of to our results with we observed that Ca2+ signals were not to inhibition by DAMGO, of whether the neurons were PS-sensitive or not (Figure we TRPV1 channels with μORs in HEK cells, we found evidence for inhibitory action of μORs on TRPV1 channels in the time frame investigated Figure Similarly, we investigated the of μORs to and channels in overexpression again indication of inhibitory action of μORs on these channels (Figure In from the group of TRP channels implicated in noxious and inflammatory only TRPM3 channels a rapid and reversible inhibition during μOR activation. Figure with 2 supplements see all Download asset Open asset Activation of μORs not rapidly inhibit the somatosensory TRP channels and (a) The of DRG neurons reduction of the Ca2+ signals (0.1 μM capsaicin, red trace: n = neurons from cells from two application of 0.3 μM DAMGO that was than the reduction observed during the application of the trace: black trace: n = neurons from cells from the same two (b) Statistical of the inhibition evoked by DAMGO or in the in (a). The dot-like symbols on the right side of the bars indicate inhibition values measured in individual cells. that for both and two cells showing strongly responses during the application of DAMGO or to inhibition values than