Non‐acidic activation of pain‐related Acid‐Sensing Ion Channel 3 by lipids
2016; Springer Nature; Volume: 35; Issue: 4 Linguagem: Inglês
10.15252/embj.201592335
ISSN1460-2075
AutoresSébastien Marra, Romain Clément, Véronique Breuil, A Delaunay, Marine Christin, Valérie Friend, Stéphane Sebille, Christian Cognard, Thierry Ferreira, C. Roux, Liana Euller‐Ziegler, Jacques Noël, Éric Lingueglia, Emmanuel Deval,
Tópico(s)Ion Channels and Receptors
ResumoArticle15 January 2016free access Non-acidic activation of pain-related Acid-Sensing Ion Channel 3 by lipids Sébastien Marra Sébastien Marra CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Romain Ferru-Clément Romain Ferru-Clément CNRS, Laboratoire de Signalisation et Transports Ioniques Membranaires (STIM), ERL 7368, Poitiers Cedex 9, France Université de Poitiers, ERL 7368, Poitiers Cedex 9, France Search for more papers by this author Véronique Breuil Véronique Breuil CHU-Nice, Hôpital l'Archet 1, Nice, France Search for more papers by this author Anne Delaunay Anne Delaunay CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Marine Christin Marine Christin CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Valérie Friend Valérie Friend CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Stéphane Sebille Stéphane Sebille CNRS, Laboratoire de Signalisation et Transports Ioniques Membranaires (STIM), ERL 7368, Poitiers Cedex 9, France Université de Poitiers, ERL 7368, Poitiers Cedex 9, France Search for more papers by this author Christian Cognard Christian Cognard CNRS, Laboratoire de Signalisation et Transports Ioniques Membranaires (STIM), ERL 7368, Poitiers Cedex 9, France Université de Poitiers, ERL 7368, Poitiers Cedex 9, France Search for more papers by this author Thierry Ferreira Thierry Ferreira CNRS, Laboratoire de Signalisation et Transports Ioniques Membranaires (STIM), ERL 7368, Poitiers Cedex 9, France Université de Poitiers, ERL 7368, Poitiers Cedex 9, France Search for more papers by this author Christian Roux Christian Roux CHU-Nice, Hôpital l'Archet 1, Nice, France Search for more papers by this author Liana Euller-Ziegler Liana Euller-Ziegler CHU-Nice, Hôpital l'Archet 1, Nice, France Search for more papers by this author Jacques Noel Jacques Noel CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Eric Lingueglia Eric Lingueglia CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Emmanuel Deval Corresponding Author Emmanuel Deval CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Sébastien Marra Sébastien Marra CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Romain Ferru-Clément Romain Ferru-Clément CNRS, Laboratoire de Signalisation et Transports Ioniques Membranaires (STIM), ERL 7368, Poitiers Cedex 9, France Université de Poitiers, ERL 7368, Poitiers Cedex 9, France Search for more papers by this author Véronique Breuil Véronique Breuil CHU-Nice, Hôpital l'Archet 1, Nice, France Search for more papers by this author Anne Delaunay Anne Delaunay CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Marine Christin Marine Christin CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Valérie Friend Valérie Friend CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Stéphane Sebille Stéphane Sebille CNRS, Laboratoire de Signalisation et Transports Ioniques Membranaires (STIM), ERL 7368, Poitiers Cedex 9, France Université de Poitiers, ERL 7368, Poitiers Cedex 9, France Search for more papers by this author Christian Cognard Christian Cognard CNRS, Laboratoire de Signalisation et Transports Ioniques Membranaires (STIM), ERL 7368, Poitiers Cedex 9, France Université de Poitiers, ERL 7368, Poitiers Cedex 9, France Search for more papers by this author Thierry Ferreira Thierry Ferreira CNRS, Laboratoire de Signalisation et Transports Ioniques Membranaires (STIM), ERL 7368, Poitiers Cedex 9, France Université de Poitiers, ERL 7368, Poitiers Cedex 9, France Search for more papers by this author Christian Roux Christian Roux CHU-Nice, Hôpital l'Archet 1, Nice, France Search for more papers by this author Liana Euller-Ziegler Liana Euller-Ziegler CHU-Nice, Hôpital l'Archet 1, Nice, France Search for more papers by this author Jacques Noel Jacques Noel CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Eric Lingueglia Eric Lingueglia CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Emmanuel Deval Corresponding Author Emmanuel Deval CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France LabEx Ion Channel Science and Therapeutics, Valbonne, France Search for more papers by this author Author Information Sébastien Marra1,2,3, Romain Ferru-Clément4,5, Véronique Breuil6, Anne Delaunay1,2,3, Marine Christin1,2,3, Valérie Friend1,2,3, Stéphane Sebille4,5, Christian Cognard4,5, Thierry Ferreira4,5, Christian Roux6, Liana Euller-Ziegler6, Jacques Noel1,2,3, Eric Lingueglia1,2,3,‡ and Emmanuel Deval 1,2,3,‡ 1CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR 7275, Valbonne, France 2Université de Nice Sophia Antipolis, UMR 7275, Valbonne, France 3LabEx Ion Channel Science and Therapeutics, Valbonne, France 4CNRS, Laboratoire de Signalisation et Transports Ioniques Membranaires (STIM), ERL 7368, Poitiers Cedex 9, France 5Université de Poitiers, ERL 7368, Poitiers Cedex 9, France 6CHU-Nice, Hôpital l'Archet 1, Nice, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33493953418; E-mail: [email protected] The EMBO Journal (2016)35:414-428https://doi.org/10.15252/embj.201592335 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Extracellular pH variations are seen as the principal endogenous signal that triggers activation of Acid-Sensing Ion Channels (ASICs), which are basically considered as proton sensors, and are involved in various processes associated with tissue acidification. Here, we show that human painful inflammatory exudates, displaying non-acidic pH, induce a slow constitutive activation of human ASIC3 channels. This effect is largely driven by lipids, and we identify lysophosphatidylcholine (LPC) and arachidonic acid (AA) as endogenous activators of ASIC3 in the absence of any extracellular acidification. The combination of LPC and AA evokes robust depolarizing current in DRG neurons at physiological pH 7.4, increases nociceptive C-fiber firing, and induces pain behavior in rats, effects that are all prevented by ASIC3 blockers. Lipid-induced pain is also significantly reduced in ASIC3 knockout mice. These findings open new perspectives on the roles of ASIC3 in the absence of tissue pH variation, as well as on the contribution of those channels to lipid-mediated signaling. Synopsis ASIC3 is a proton-sensitive ion channel linked to pain perception. The finding that certain lipids activate ASIC3 in the absence of tissue acidification challenges our view of pain-causing agents and suggests crosstalk between pH fluctuations and lipid signaling. Non-acidic joint effusions from patients with pain constitutively activate ASIC3. The effect is lipid-dependent and exudates contain high levels of lysophosphatidylcholine (LPC) and arachidonic acid (AA). LPC and AA are primarily responsible for ASIC3 activation at resting pH 7.4. LPC and AA evoke ASIC3-dependent pain behaviour in rodents. Introduction Acid-sensing ion channels (ASICs) are depolarizing sodium channels formed by the trimeric association of different subunits numbered from ASIC1 to ASIC3 (for review, see Deval et al, 2010). They are gated by extracellular protons (Waldmann et al, 1997) and are widely expressed throughout the nervous system where they have been involved in different physiological and pathophysiological processes (for reviews, see Noël et al, 2010; Wemmie et al, 2013). Among these processes, pain is particularly interesting since an increasing amount of data identifies ASICs as promising therapeutic targets for the development of new analgesic strategies (Mazzuca et al, 2007; Deval et al, 2008, 2011; Karczewski et al, 2010; Bohlen et al, 2011; Walder et al, 2011; Diochot et al, 2012, 2015; Izumi et al, 2012). ASICs are basically considered as fine sensors of extracellular pH variations, and proton is actually the only endogenous ligand known to directly activate these channels. A non-proton ligand-sensing domain has also been described in ASIC3 (Yu et al, 2010), which contributes to the direct effect of the synthetic compound GMQ (2-guanidine-4-methylquinazoline) and of the arginine metabolite agmatine (Li et al, 2010; Yu et al, 2010). However, agmatine has a very low affinity for the channel (EC50 ~10 mM), suggesting that it is probably not a direct endogenous activator of ASIC3, but rather a physiological modulator of the acid-evoked current. Several other stimuli linked to ischemia and/or inflammation have also been reported to potentiate the acid-induced ASIC3 current, including lactate (Immke & McCleskey, 2001), arachidonic acid (Allen & Attwell, 2002; Smith et al, 2007; Deval et al, 2008), nitric oxide (Cadiou et al, 2007), hypertonicity (Deval et al, 2008; Li et al, 2010), serotonin (Wang et al, 2013), and ATP (Birdsong et al, 2010). We show here that human painful inflammatory exudates that exhibit a physiological non-acidic pH are able to activate ASIC3 channels and that this effect is largely supported by lipids. Lysophosphatidylcholine (LPC) and arachidonic acid (AA), which are co-released following hydrolysis of membrane phospholipids by phospholipase A2 (PLA2) enzymes, are present at high concentrations in these exudates and can directly activate ASIC3 channels at physiological pH 7.4, that is, without any extracellular acidification. LPC is also a strong potentiator of the acid-induced ASIC3 current, similarly to what has been shown for AA (Deval et al, 2008). The combination of LPC and AA forms a potent endogenous signal that induces firing in nociceptive C-fibers and triggers pain responses in animals through an ASIC3-dependent mechanism. These results illustrate the capacity of endogenous lipids to effectively activate ASIC3 in vitro and in vivo, in the absence of any extracellular pH variations to generate pain in rodents and also probably in humans. Results Non-acidic painful human inflammatory exudates activate ASIC3 channels Several studies have reported a role for ASIC3 in mediating pain in animal models of joint inflammation (Ikeuchi et al, 2008; Izumi et al, 2012; Sugimura et al, 2015). We thus tested the effect of crude human inflammatory exudates, collected from patients with joint effusions, on human ASIC3 channels expressed in HEK293 cells (Fig 1). Interestingly, exudates were not acid and two had a pH close to 7.4 (exudates #2 and #13 with pH values of 7.45 ± 0.02 and 7.49 ± 0.01, respectively, see Fig EV1), although the visual analog scale (VAS) for pain was elevated in patients (8/10 and 9/10 for patients #2 and #13, respectively, see Fig EV1). Extracellular applications of these exudates on cells expressing human ASIC3 induced a slow sustained current, but not in non-transfected cells nor in cells expressing human ASIC1a (Fig 1A–C). The exudate-induced current reversed at +59 mV (Fig 1D) and was substantially inhibited by the ASIC3 blocker APETx2 (Diochot et al, 2004) (Fig 1E), confirming the direct activation of human ASIC3 channels in the absence of extracellular acidification. Chelating lipids in these exudates with bovine serum albumin (BSA, see Materials and Methods) significantly decreased their efficiency to activate human ASIC3 current (Fig 1F). Analysis of the total fatty acids (FAs) content of exudates from patients #2 and #13, by transmethylation and gas chromatography experiments (Fig 1G), revealed high levels of palmitic acid (16:0), oleic acid (18:1), stearic acid (18:0), and arachidonic acid (20:4/AA), with concentrations estimated to 441 ± 43, 914 ± 129, 200 ± 27 and 148 ± 42 μM, respectively. As these FAs could be either free or esterified into complex lipids such as phospholipids, we also performed mass spectrometry experiments (Fig 1G, inset) that notably revealed high levels of lysophosphatidylcholine (LPC) in exudates (average concentrations estimated to 113 ± 14, 26 ± 3, 47 ± 6 and 14 ± 1 μM for LPC16:0, LPC18:1, LPC18:0, and LPC20:4, respectively, data from 11 patients, see Fig EV1). High contents of LPC have been similarly reported in synovial fluids from patients with rheumatoid arthritis (Fuchs et al, 2005). Together, these data indicate that painful human inflammatory exudates with a physiological pH close to 7.4 are able to activate a constitutive current generated by human ASIC3 channels and that lipids are involved in this effect. Figure 1. Non-acidic human inflammatory exudates activate recombinant human ASIC3 channels Whole-cell recording experiments performed at −80 mV showing that extracellular application of joint exudate from patient #2 (ex#2, pH = 7.45 ± 0.02) activates an inward sustained current in HEK293 cells transfected with human ASIC3 channels (hA3), but not in non-transfected cells (NT). Functional expression of hA3 is shown by the presence of a typical current in response to extracellular acidification (from pH 7.4 to pH 6.6, insets). Effect of exudate from patient #13 (ex#13, pH = 7.49 ± 0.01) on HEK293 cells expressing either human ASIC3 or ASIC1a channels (insets: currents induced by extracellular acidification from pH 7.4 to pH 6.6). Histograms showing the mean amplitudes of exudate-induced currents (ex#2 and ex#13) measured from hASIC3 (n = 9), hASIC1a (n = 5), or non-transfected cells (n = 5) as illustrated in (A) and (B) (*P < 0.05 and **P < 0.01, Kruskal–Wallis test followed by a Dunn's post hoc test; error bars indicate ± SEM). Current–voltage relationship of the exudate-induced current (ex#2) as shown in (A). The current reverses at +59 mV (inset: current traces recorded at different voltages and at pH 7.4). Effect of APETx2 on ex#2-induced current. The dashed line indicates the zero current level (difference between recordings is due to inhibition by APETx2 of the human ASIC3 alkaline-activated current already present at pH 7.4 (Delaunay et al, 2012). Mean amplitude of the hASIC3 currents induced by either the crude exudate from patient #13 or its delipidated fraction (n = 6 for each condition, **P < 0.01, Mann–Whitney U-test; error bars indicate ± SEM). Left panel: Typical chromatography (GC) analysis of human inflammatory exudate from patient #13 showing the presence of different fatty acids. Right panel: Estimated mean concentrations of palmitic (16:0), oleic (18:1), stearic (18:0), and arachidonic (20:4) acids in exudates from patients #2 and #13 (n = 2 for each fatty acid). Inset shows the estimated mean concentrations of different LPC species (LPC16:0, LPC18:1, LPC18:0, and LPC 20:4) obtained following MS analysis of exudates from 11 patients (n = 11 for each LPC, see also Fig EV1). Error bars indicate ± SEM. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Detailed analysis of exudates from 11 patientsND, not determined. Download figure Download PowerPoint Arachidonic acid (AA) and lysophosphatidylcholine (LPC) activate ASIC3 at pH 7.4 We next tested the effects of the different lipids on ASIC3 channels. The fatty acid AA (20:4, ω6) was already known to strongly potentiate the acid-induced ASIC3 currents (Smith et al, 2007; Deval et al, 2008; Delaunay et al, 2012). We show here that AA (10 μM) was also able to activate a slow sustained ASIC3 current at pH 7.4 (Fig 2A), that is, in the absence of extracellular acidification. In contrast, palmitic acid (16:0 at 10 μM), stearic acid (18:0, 10 μM), or oleic acid (18:1 at 10 μM) failed to activate a similar current when applied at pH 7.4 on cells transfected with human ASIC3 (Fig 2A). Because we also found high levels of LPC in exudates (Fig 1G, inset), we tested the effect of this lysophospholipid on human ASIC3 current at resting pH 7.4. We found that LPC16:0, LPC18:0, or LPC18:1 (10 μM each) had potent activating effects (Fig 2A), and the most efficient activation was obtained with LPC16:0 and LPC18:1. In good agreement with these results, LPC16:0 and LPC18:1 also activated a slow sustained current when applied at pH 7.4 on cells transfected with rat ASIC3, whereas palmitic acid, oleic acid, or phosphatidylcholine containing both palmitic and arachidonic acid (PC 16:0/20:4) had no effect (Fig 2B). Activation of rat ASIC3 by LPC16:0 and LPC18:1 was comparable, while LPC10:0 or LPC18:0 had a much smaller effect (see Fig EV2). We next tested the effect of crude LPC extracted from bovine brain, which mainly contains a mix of LPC16:0, LPC18:0, and LPC18:1 (Fig 2C–E). Crude brain LPC at 10 μM also activated a current at resting pH 7.4 in different cell lines transfected with rat ASIC3, but not in non-transfected cells (F-11, HEK293, and CHO cells, Figs 2C, 2D–E, and EV3, respectively). The activation of ASIC3 by LPC was observed for concentrations > 1 μM, with an estimated EC50 of 4.3 μM (Fig 2D). Hydrolysis of membrane phospholipids by PLA2 enzymes leads to the release of different lysophospholipids, including LPC, but also lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), and lysophosphatidic acid (LPA). LPC was the only lysophospholipid able to significantly activate an ASIC3 current at resting pH 7.4 (Fig 2E). Moreover, the activating effect of LPC was specific for ASIC3 channels since it failed to induce a similar current in cells expressing either ASIC1a, ASIC1b, or ASIC2a channels (Fig 2E, inset). Together, these data indicate that individual application of micromolar (> 1 μM) concentrations of LPC (and most probably also AA), in the extracellular medium and at resting pH 7.4, led to the activation of ASIC3 channels in a slow and sustained manner. The most potent activation was obtained with LPC16:0 and LPC18:1, showing the importance for the effect of the amphipathic properties of LPC molecules and of the length of their lipid chain. Figure 2. Effect of fatty acids and lysophosphatidylcholine on recombinant ASIC3 channelsWhole-cell recordings obtained at −80 mV from HEK293 and F-11 cells expressing either the human or the rat ASIC3 channels (hA3 or rA3) and from non-transfected cells (NT). The number of experiments (n) is indicated above each bar graph and error bars represent ± SEM. Extracellular applications of different fatty acids (20:4/AA, 16:0, 18:0, and 18:1) and lysophosphatidylcholine (LPC16:0, LPC18:0, and LPC18:1) at 10 μM and at pH 7.4 on HEK293 cells expressing human ASIC3 channels (hA3). Histograms that represent the statistical analysis of data are shown in bottom panel, and current densities are measured following 30-s applications of fatty acids or 10-s applications of LPC (*P < 0.05 and ***P < 0.001, Kruskal–Wallis test followed by a Dunn's post hoc test; ###P < 0.001 as compared to effects of the respective fatty acids). Comparison of the effects of palmitic acid (16:0, 10 μM), oleic acid (18:1, 10 μM), LPC16:0 (10 μM), LPC18:1 (10 μM), and phosphatidylcholine (PC) 16:0/20:4 (10 μM) on HEK293 cells expressing rat ASIC3 channels. Current densities are measured following 30-s applications of fatty acids (16:0, 18:1), or 10-s applications of LPC, in the extracellular medium at resting pH 7.4. Typical current trace and statistical analysis of data are shown on the top and bottom, respectively (*P < 0.05 and **P < 0.01, Kruskal–Wallis test followed by a Dunn's post hoc test). Crude LPC extracted from bovine brain elicits a current on F-11 cells expressing rat ASIC3 channels but not on non-transfected cells. Current densities are measured following 30-s applications of 10 μM LPC at resting pH 7.4 (**P < 0.01, Mann–Whitney U-test). Dose-dependent activation of rat ASIC3 channels by crude LPC in HEK293 cells. ASIC3 current densities are measured at resting pH 7.4, following extracellular applications (20 s) of different concentrations of LPC. Inset shows the dose–response curve of the activating effect of LPC fitted with a Boltzmann (IC50 estimated at 4.3 μM). Effects of different lysophospholipids (10 μM each) applied extracellularly for 20 s at resting pH 7.4 on HEK293 cells expressing or not rat ASIC3 channels (rA3 or NT, respectively). Left panel: Typical current traces recorded from ASIC3-transfected cells upon application of lysophosphatidic acid (LPA, upper left) and lysophosphatidylcholine (LPC, lower left). Right panel: Bar graph representing the mean current densities measured following application of LPA, lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), LPC, or lysophosphatidylinositol (LPI) (***P < 0.001 as compared to NT, Mann–Whitney U-test). Inset shows the effect of LPC 10 μM at resting pH 7.4 on cells transfected with either rat ASIC1a, ASIC1b, ASIC2a or ASIC3 channels, or on non-transfected (NT) cells (*P < 0.05 and **P < 0.01, Kruskal–Wallis test followed by a Dunn's post hoc test). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Effects of LPC molecules containing different fatty acid chains on ASIC3-transfected cells at resting pH 7.4Current densities are measured at −80 mV from F-11 cells following 30-s applications of LPC10:0, LPC16:0, LPC18:0, or LPC18:1 at 10 μM (*P < 0.05 Kruskal–Wallis test followed by a Dunn's post hoc test). The number of experiments (n) is indicated below each bar graph, and error bars indicate ± SEM. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Activating effect of LPC on ASIC3 channels expressed in CHO cellsRat ASIC3 (rA3) currents are generated by extracellular application of LPC (crude extract from bovine brain, 10 μM) at resting pH 7.4. Current densities are measured at −80 mV and are compared to the currents induced in non-transfected (NT) cells (*P < 0.05, Mann–Whitney U-test). The number of experiments (n) is indicated below each bar graph, and error bars indicate ± SEM. Download figure Download PowerPoint LPC also potentiates the acid-induced ASIC3 current and it acts by shifting the pH dependence of the channel To further explore the mechanism of action of lipids on ASIC3, we tested the effect of LPC on its acid-induced current. In addition to the activating effect at resting pH 7.4 (Fig 3A, Ibasal), LPC (10 μM) also strongly potentiated the pH 7.0-induced ASIC3 current (Fig 3A). The effect started immediately after its application on the extracellular side of the cells and reached a maximum after 3 min (Fig 3A, bottom panel). Although submicromolar concentrations of LPC did not activate ASIC3 channels at pH 7.4 (Fig 2D), they potentiated the acid-induced ASIC3 current (Fig 3B, inset). The potentiating effect was reversible upon washout (Fig 3B, inset) and was clearly dependent on LPC extracellular concentration. However, the EC50 was not calculated because it was not possible to reach the maximal effect. The efficacy of different lysophospholipids to modulate the pH 7.0-induced ASIC3 current was also tested on transfected cells (see Fig EV4). LPC had the strongest potentiating effect with a ~fivefold increase of the pH 7.0-activated ASIC3 peak current while LPA and LPE had no significant effects. LPS and LPI also displayed a significant but much smaller potentiating effect, with ~1.6-fold increases for both lysophospholipids. LPC acted on the channel by shifting its pH-dependent activation curve toward more alkaline pH values, with no effect on inactivation (Fig 3C), leading to an increase of the ASIC3 window current (Fig 3D), similarly to the effect we previously described for AA (Deval et al, 2008). As a result, LPC is not only a strong potentiator of the acid-induced ASIC3 current, but it also generates a small but constitutive ASIC3 current at resting physiological pH 7.4, that is, in the absence of any extracellular acidification. Figure 3. LPC activates ASIC3 channels at pH 7.4 and potentiates its acid-induced activityEffects of crude LPC extracted from bovine brain on HEK293 and F-11 cells expressing the rat ASIC3 channels. LPC is applied extracellularly in the pH 7.4 bathing solution, and data are obtained from whole-cell patch-clamp experiments performed at −80 mV. The number of experiments (n) is indicated in brackets, dotted lines on traces represent the zero current level, and error bars indicate ± SEM. Effect of LPC (10 μM) on pH 7.0-induced ASIC3 current represented as a function of time. Inset: typical recording illustrating the potentiating effect of LPC on the pH 7.0-induced current recorded in F-11 cells. The curve on the bottom represents current amplitudes measured after LPC application and normalized (I/Ictrl) to those measured in control conditions, that is, before the application of lipid. Note the basal constitutive current induced by LPC at resting pH 7.4 (arrows, Ibasal). Dose–response effect of the potentiation by LPC of the pH 7.0-induced ASIC3 current obtained from HEK293-transfected cells. The percentage of effect is calculated by comparing current amplitudes before and after application of LPC. Inset: typical potentiating effect of LPC (1 μM) on a pH 7.0-induced ASIC3 current showing reversibility upon washout. Note that this concentration is not sufficient to induce the basal constitutive current at pH 7.4 shown in (A). Effect of LPC (10 μM) on the pH-dependent activation and inactivation curves of ASIC3 channels expressed in F-11 cells. Acid-induced ASIC3 currents are normalized to their maximal peak amplitude measured at pH 5.0, and the protocol used to obtain the curves is shown in the inset (data from three different cells). Effect of LPC (10 μM) on the ASIC3 window current measured between pH 8.0 and pH 6.6 from HEK293 cells. Inset: typical current trace obtained in control condition with the arrows (w) indicating the different points at which the window current is measured. Amplitudes of the ASIC3 window current are measured in control condition (without LPC, data from 8 different cells) and after 30-s applications of LPC (data from 6 different cells). Current amplitudes, measured at different external pH in both control and LPC condition, are normalized to the basal current level measured at pH 8.0 without LPC (bottom graph). Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Effects of different lysophospholipids on acid-induced ASIC3 currentComparison of the effects of LPA, LPE, LPS, LPC, and LPI at 10 μM on the pH 7.0-induced current recorded from F-11 cells (each compound is applied for 3 min, *P < 0.05, and **P < 0.01 and ***P < 0.001, Wilcoxon test; ##P < 0.01 and ###P < 0.001, Kruskal–Wallis test followed by a Dunn's post hoc test). The number of experiments (n) is indicated above each bar graph, and error bars indicate ± SEM. Download figure Download PowerPoint These data demonstrate that LPC is the most efficient lysophospholipid acting on ASIC3 channels. It induces an alkaline shift of the pH-dependent activation curve, leading to (i) a strong potentiating effect on the acid (pH 7.0)-induced current and (ii) activa
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