Skip to main content
Download PDF

ASIC3 Channels Integrate Agmatine and Multiple Inflammatory Signals through the Nonproton Ligand Sensing Domain

Molecular Pain volume 6, Article number: 88 (2010) Cite this article

Abstract

Background

Acid-sensing ion channels (ASICs) have long been known to sense extracellular protons and contribute to sensory perception. Peripheral ASIC3 channels represent natural sensors of acidic and inflammatory pain. We recently reported the use of a synthetic compound, 2-guanidine-4-methylquinazoline (GMQ), to identify a novel nonproton sensing domain in the ASIC3 channel, and proposed that, based on its structural similarity with GMQ, the arginine metabolite agmatine (AGM) may be an endogenous nonproton ligand for ASIC3 channels.

Results

Here, we present further evidence for the physiological correlation between AGM and ASIC3. Among arginine metabolites, only AGM and its analog arcaine (ARC) activated ASIC3 channels at neutral pH in a sustained manner similar to GMQ. In addition to the homomeric ASIC3 channels, AGM also activated heteromeric ASIC3 plus ASIC1b channels, extending its potential physiological relevance. Importantly, the process of activation by AGM was highly sensitive to mild acidosis, hyperosmolarity, arachidonic acid (AA), lactic acid and reduced extracellular Ca2+. AGM-induced ASIC3 channel activation was not through the chelation of extracellular Ca2+ as occurs with increased lactate, but rather through a direct interaction with the newly identified nonproton ligand sensing domain. Finally, AGM cooperated with the multiple inflammatory signals to cause pain-related behaviors in an ASIC3-dependent manner.

Conclusions

Nonproton ligand sensing domain might represent a novel mechanism for activation or sensitization of ASIC3 channels underlying inflammatory pain-sensing under in vivo conditions.

Background

Acid-sensing ion channels (ASICs) represent a new subgroup of the epithelial sodium channel/degenerin (ENaC/DEG) family of ion channels. To date, functional cloning studies revealed four genes that give rise to at least six ASIC informs (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4) [1]. These isoforms, which are composed of cytosolic N and C termini, two transmembrane helices, and a disulfide-rich, multi-domain extracellular region, can associate into homo- or heterotrimers [1, 2]. ASICs are amiloride-sensitive voltage-independent cationic channels that are activated by a decrease in extracellular pH [3]. Protons trigger a transient inward current that desensitizes rapidly in all forms of ASICs except ASIC3, which displays a sustained current that does not fully desensitize despite prolonged exposure to acidic extracellular pH [4–7]. ASIC3 is predominantly expressed in sensory neurons and has been shown to be a sensor of acidic and primary inflammatory pain [8, 9]. In addition to protons, a synthetic compound, 2-guanidine-4-methylquinazoline (GMQ) has been found to activate ASIC3 channels at physiologically normal pH in a sustained manner [10]. Furthermore, GMQ acts at a site on the ASIC3 that is separate from the known proton binding sites [10]. The identification of this nonproton ligand sensing domain argues that natural ligands beyond protons may activate ASICs under physiological conditions.

Arginine is one of the most versatile amino acids in mammals and has multiple metabolic fates. Not only is it metabolically interconvertible with the amino acids proline and glutamate, but it also serves as a precursor for synthesis of protein, nitric oxide (NO), agmatine (AGM), polyamines, ornithine, and urea [11]. Among these, AGM and polyamines (including spermine, spermidine, and putrescine) are positively charged at physiological pH and thus can interact electrostatically with negatively charged nucleic acids and proteins, including receptors and ion channels. For example, extracellular AGM binds to imidazoline receptors [12, 13], and blocks N-methyl-D-aspartate (NMDA) receptors [14] and other ligand- or voltage-gated cation channels [15–17]. However, intracellular spermine and spermidine contribute to the rectification of inward rectifier K+ channels [18, 19] and certain types of glutamate receptors [20, 21]. Furthermore, polyamines block the Transient Receptor Potential Melastatin (TRPM) channels, TRPM4 [22] and TRPM7 [23], and serve as potent ligands for the capsaicin receptor Transient Receptor Potential Vanilloid Type 1 (TRPV1) [24] and the calcium-sensing receptor [25], a G-protein-coupled receptor that contributes to the regulation of calcium homeostasis. In addition, spermine produces complex effects on NMDA receptors, with either stimulating activity [26, 27] or inducing a voltage-dependent blockade [28]. Interestingly, spermine shifts the steady-state desensitization of ASIC1a channels to more acidic pH conditions [29] and contributes significantly to ischemic neuronal injury through enhancing ASIC1a activity [30].

Furthermore, peripheral AGM and polyamines are implicated in inflammation and pain signaling. Levels of AGM and polyamines are increased during infection, trauma, and cancer [31, 32]. In a previous study, we have shown that extracellular AGM and its structural analog ARC (Figure 1A) activate ASIC3 at neutral pH [10]. In this study, we further showed that AGM-induced activation of ASIC3 is profoundly potentiated by mild acidosis, hyperosmolarity, increased arachidonic acid (AA), or reduced extracellular Ca2+, conditions that occur during inflammation and many other pathophysiological processes [8, 33–39]. Furthermore, AGM cooperated with the multiple inflammatory signals to cause pain-related behaviors in an ASIC3-dependent manner.

Figure 1
figure 1

Effects of Arginine and Its Metabolites on ASIC3 Channels. (A) Chemical structure of GMQ, arcaine (ARC), arginine and its metabolites. (B) Summary (periphery) of and sample traces (inset) showing the effects of compounds on ASIC3 channels at neutral pH. Each compound was applied at a concentration of 1 mM. The first above trace represents the baseline level without drug application (zero level), followed by the current induced by AGM, ARC, and GMQ, respectively. Data points are means ± S.E.M. of four to six measurements normalized to GMQ-induced response. **p < 0.001 vs. baseline level.

Full size image

Results & Discussion

AGM but Not Polyamines Activates ASIC3 Channels

The fact that AGM directly activates ASIC3 channels [10] prompted us to look for the effects of polyamines and other arginine metabolites [11] (Figure 1A). Conventional whole-cell patch clamp recordings were performed in Chinese Hamster Ovary (CHO) cell lines transiently expressing ASIC3 tagged with GFP to measure the functional activation of ASIC3 channels under voltage clamp conditions. At a concentration of 1 mM, only AGM and its analog ARC (Figure 1B), but not polyamines (including spermine, spermidine, and putrescine), nor L-arginine, nor L-ornithine, were able to activate the ASIC3 channel at pH 7.4. The resistance of ASIC3 to polyamines differs from the modulation of ASIC1a by spermine [29, 30] and the modulation of TRPV1 by polyamines [24], arguing for multifunctional roles of arginine metabolites in inflammatory responses [40]. Therefore, as two major sensors for inflammatory pain, ASIC3 and TRPV1 sense different arginine metabolites (AGM of ASIC3 vs. polyamines of TRPV1, respectively).

AGM is widely and unevenly distributed in mammalian tissues [41]. Observed first in the rat brain, AGM was shown later that its concentration in the brain (2.40 ng/g) is lower than in others, such as stomach (71.00 ng/g), intestine, spleen and liver (5.63 ng/g) [42]. The relative low concentration of AGM in normal tissues together with the relative low potency of AGM on ASIC3 channels (3.3 ± 0.3% of the GMQ's response, Figure 1B) argue for the negligible contribution of AGM under physiological conditions. However, AGM is detectable in human plasma and higher concentrations have been observed in depressed patients [43]. Similarly, levels of peripheral AGM and polyamines are elevated during infection, trauma, and cancer [31, 32]. Interestingly, AGM evokes ASIC3-dependent pain-behavior in mice [10], suggesting that AGM-ASIC3 interaction may become functionally relevant under pathological conditions.

Synergic Effect of AGM and Mild Acidosis on ASIC3 Channels

Pain conditions are associated with multiple inflammatory signals (mild acidosis, hyperosmolarity, increased arachidonic acid, or reduced extracellular Ca2+). To understand the functional relevance of AGM-ASIC3 interaction, we examined the pH dependence of AGM action by applying graded pH to the CHO cell expressing ASIC3 channels, in the presence or absence of AGM (Figure 2A). We found that when the typical biphasic ASIC3 responses were evoked by a pH reduction, AGM dramatically enhanced the sustained component (Figure 2A, C), without altering the peak component (Figure 2A, B). The effect was most evident under mild acidosis conditions (pH 7.2~6.8). This enhancement could be simply the summation of two independent currents induced by AGM and mild acidosis, respectively. To address this issue, we explored the interaction between AGM and pH 7.0 more specifically (Figure 2D-F). We found that pH 7.0 significantly potentiated AGM response and the potentiation was always more than additive with pH reduction regardless the sequence of agonist application (Figure 2D, E), a characteristic that is opposite to the accelerated ASIC desensitization caused by pretreatment with acidic solution [36]. This potentiation occurred only under the simultaneous presence of two ligands (i.e., H+ and AGM) (Figure 2D, E, panels I and III), suggesting coincident detection of proton and nonproton ligands by ASIC3 channels in vivo. Remarkably, a significant potentiation was observed even when AGM was applied at lower concentrations (~100 μM, Figure 2F). The inability of AGM pretreatment to enhance ASIC3 response to mild acidosis (Figure 2D, E, panel II) suggests a novel mechanism underlying the observed synergy between H+ and AGM that differs from the allosteric effect of tarantula toxin psalmotoxin 1 (PcTX1) on ASIC1 channels [44, 45].

Figure 2
figure 2

Mild Acidosis Potentiates AGM-Induced ASIC3 Channel Activation. (A) Representative traces showing currents induced by acid with graded pH as indicated in the absence (black) or presence (grey) of AGM (1 mM). (B, C) Pooled data as shown in (A) illustrating pH-dependent interaction between AGM- and acid-induced transient (B) or sustained (C) inward currents. Each point is the mean ± S.E.M. of four to five measurements and the solid (black) or dashed (grey) lines are fits to the Hill equation. *p < 0.05, represents the significant difference of current amplitude in the absence or presence of AGM (1 mM). The pH at half maximal activation (pH50) values are 6.74 ± 0.02 (n = 3.6 ± 0.4) and 6.81 ± 0.02 (n = 3.8 ± 0.6) in the absence or presence of AGM, respectively. (D-F) Synergistic interaction between AGM (1 mM) and mild acidosis (pH 7.0). 'I', 'II', and 'III' in (D) indicate co-, pre-, and pre + co-administrations of AGM (1 mM) and mild acid (pH 7.0) as also represented in (E), respectively. The synergistic interaction between AGM and mild acidosis (pH 7.0) is suggested by the two-way ANOVA analysis (p < 0.0001). (F) Concentration-dependence of AGM under mild acidosis (pH 7.0). AGM was co-applied as shown in protocol I of (D). Data points are means ± S.E.M. of four to five measurements normalized to pH 7.0-induced currents (control, dashed line). Expected value is the linear summation of normalized currents induced by pH 7.0 and AGM individually. **p < 0.001.

Full size image

It is well known that peripheral pH falls to < 7 during inflammation, infection, ischemia, hematomas, and exercise [1]. Moreover, such acidosis is well recognized to activate nociceptors and to produce pain in humans that can be attenuated by the ENaC/DEG inhibitor amiloride [46–48]. Additionally, inflammatory mediators, such as nerve growth factor (NGF), serotonin (5-HT), interleukin-1, bradykinin, and brain-derived neurotrophic factor (BDNF) can stimulate ASIC3 transcription, which perhaps contributes to the pain-enhancing effects of these mediators [49, 50]. Thus, ASIC3 channels seem to act as a major inflammatory pain integrator [8, 9]. Considering that both AGM production [31, 32] and ASIC3 expression [51, 52] are increased during inflammation, the positive synergy between H+ and AGM in activating ASIC3 channels adds a new level of complexity to the molecular events that can lead to inflammatory pain. The dramatic enhancement under pH 7.2-6.8 (Figure 2C) is reminiscent of a previous observation reporting sustained 'window' current through ASIC3 channels at modest pH changes presumably contributing to myocardial ischemia [36]. Whether AGM regulates cardiac pain-sensing [36] and other forms of muscle pain [9, 52, 53] awaits further investigations.

Synergy between AGM and Hyperosmolarity

In inflamed or injured tissues, multiple mediators meet in the interstitial fluid and form an inflammatory exudate, the content of which is acidic [34] and hyperosmotic [37]. Previous studies have shown that hyperosmolarity increases neuronal excitability in DRG neurons [8], affecting preferentially the sustained component of ASIC3 currents. These previous studies promoted us to examine the synergy among hyperosmolarity, acidosis, and AGM. ASIC3-expressing CHO cells were exposed to AGM and mild acidosis in the absence or presence of hyperosmolarity (Figure 3). The hyperosmolarity (600 mosmol kg-1 with mannitol) itself did not induce any detectable current (Figure 3A) but significantly potentiated the ASIC3 currents evoked by a pH reduction to 7.0 (Figure 3), an effect previously observed in rat DRG neurons or an ASIC3 expressing F-11 DRG cell line [8]. Similarly, the current induced by AGM was markedly enhanced by hyperosmolarity (Figure 3). Interestingly, AGM caused further increase over the current induced by the combination of pH 7.0 and hyperosmolarity (Figure 3), suggesting that AGM, mild acidosis, and hyperosmolarity act synergically to facilitate ASIC3 opening, which may explain the enhanced sensory neuronal excitability under conditions of inflammation [8] or cardiac ischemia [36].

Figure 3
figure 3

Hyperosmolarity Potentiates AGM-Evoked ASIC3 Activation. (A) Typical traces showing a lack of response to hyperosmolarity (600 mosmolkg-1 with mannitol, H-Osm), and currents induced by mild acid (pH 7.0), mild acid with hyperosmolarity (H-Osm + pH 7.0), AGM (1 mM), AGM (1 mM) with hyperosmolarity (H-Osm + AGM), AGM (1 mM) plus mild acid (pH 7.0 + AGM), and AGM (1 mM) with the combined mild acid and hyperosmolarity (H-Osm + pH 7.0 + AGM). (B) Pooled data as shown in (A). Data points are means ± S.E.M. of five to six measurements normalized to pH 7.0-induced current (dashed line). *p < 0.05, **p < 0.001, demonstrate the significant difference of current amplitude in the absence or presence of hyperosmolarity; & p < 0.05, demonstrates the significant difference of current amplitude in the absence or presence of mild acid (pH 7.0); @ p < 0.05, demonstrates the significant difference of current amplitude in the absence or presence of AGM (1 mM).

Full size image

Synergy between AGM and Arachidonic Acid

Next, we tested arachidonic acid (AA), a pro-inflammatory and ischemic factor which enhances ASIC currents induced by acid and increases neuronal excitability [8, 54, 55]. At the physiological normal pH, AA induced negligible currents from ASIC3-expressing CHO cells (Figure 4A). As expected, AA significantly potentiated AGM (1 mM)-induced currents (Figure 4). The relatively slow developing kinetics of the AGM current following addition of AA (Figure 4A) presumably reflects the slow onset of AA effect which requires several minutes to be fully established [8]. Therefore, AGM not only activates ASIC3 by itself at normal pH (Figure 1; Ref. [10]), but also exerts positive cooperative effect when administrated with other inflammatory factors such as mild acidosis, hyperosmolarity, and AA, further strengthening the notion that ASIC3 channels act as a multiple sensor to integrate diverse signals present in the pathophysiological environment [8, 9].

Figure 4
figure 4

Arachidonic Acid (AA) Potentiates AGM-Induced ASIC3 Channel Activation. (A) Representative traces showing currents induced by AA (10 μM, dark grey), AGM (1 mM) in the absence (black) or presence (light grey) of AA (10 μM). (B) Pooled data as shown in (A). Data points are means ± S.E.M. of five measurements normalized to AGM (1 mM)-induced current (dashed line). Expected value is the linear summation of normalized currents induced by AA (10 μM) and AGM (1 mM) individually. **p < 0.001.

Full size image

AGM, Ca2+, and ASIC3 Channel Activation

We also investigated the sensitivity of AGM-induced currents to alterations of extracellular Ca2+, which has marked effect on the GMQ response [10]. As shown in Figure 5A, B, the presence of 10 mM extracellular Ca2+ completely abolished the AGM currents, whereas reducing Ca2+ significantly potentiated AGM-induced currents. Low Ca2+ itself evoked a significant inward current (Figure 5A, left panel), consistent with a previous report [56]. Thus, similar to GMQ [10], AGM-ASIC3 interaction is highly sensitive to altered extracellular Ca2+. The extracellular Ca2+ concentration can decrease from a resting value of around 1.2-1.8 mM to values as low as 0.08 mM under certain conditions [38], suggesting that the signaling cascade induced by AGM-ASIC3 interaction might be markedly amplified under such conditions. Moreover, lactate produced by anaerobic metabolism reduces extracellular Ca2+ concentration and results in the enhancement of the acid-induced ASIC currents in ischemia-sensing neurons [39]. Likewise, AGM-evoked currents were increased about 3-folds when lactate and AGM were co-applied (Figure 5C, D). These results suggest that ASIC3 channels may be gated in vivo by the combined actions of reduced extracellular Ca2+, mild acidosis, and AGM through synergic interactions reported here.

Figure 5
figure 5

Reduced Extracellular Ca2+ Potentiates AGM-Induced ASIC3 Channel Activation. (A) Representative traces showing currents induced by reduced extracellular Ca2+ (left) and 1 mM AGM applied under low Ca2+ (right). (B) Pooled data as shown in (A). Data points are means ± S.E.M. of four to five measurements normalized to currents evoked with 0.01 mM Ca2+ (dashed line). Expected value is the linear summation of normalized currents induced by reduced Ca2+ and 1 mM AGM under low Ca2+ conditions individually. *p < 0.05, **p < 0.001. The two-way ANOVA analysis suggests the synergetic interaction between AGM and Ca2+ reduction (p < 0.01). (C) Representative traces showing currents induced by lactate (15 mM, keeping the pH neutral, light grey), AGM (1 mM) in the absence (black) or presence (dark grey) of 15 mM lactate. (D) Pooled data as shown in (C). Data points are means ± S.E.M. of five measurements normalized to AGM (1 mM)-induced currents (dashed line). Expected value is the linear summation of normalized currents induced by lactate (15 mM) and AGM (1 mM) individually. **p < 0.001.

Full size image

It is, however, possible that AGM-induced ASIC3 channel activation was through the chelation of extracellular Ca2+ as observed with lactate [39, 56] at neutral pH. To clarify this possibility, we recorded ASIC3 response to AGM in Ca2+-free external solution. We found that AGM activated ASIC3 channels regardless the presence or absence of extracellular Ca2+ (data not shown). That AGM activates ASIC3 channels independent of Ca2+ chelation is consistent with the notion that AGM modulates ASIC3 activation via novel mechanisms (Figure 2D, E).

Critical Role of the Nonproton Ligand Sensing Domain

Next we asked how AGM activates ASIC3 channels in a manner similar to GMQ, given the difference in their structural flexibility (linear AGM vs. circular GMQ with a heterocycle, Figure 1A) [10]. In a previous study, we have shown that the nonproton ligand sensing domain plays a critical role in mediating AGM and GMQ effects on ASIC3 [10]. While its critical role for GMQ was supported by the fact that covalently linking circular GMQ or TNB to C79 activated ASIC3E79C channels (with the GMQ-dimer or DTNB treatment) [10], the role of the same site for the linear AGM was not established. For comparison, we tested 2-aminoethyl-methanethiosulfonate (MTSEA), a linear thiol-reactive compound on ASIC3E79C channels, in which the residue Glu79 was replaced by a cysteine, thus mimicking AGM-E79 interaction (Figure 6A). Bath application of 0.2 mM MTSEA persistently activated ASIC3E79C channels at pH 7.4 (Figure 6B, D). By contrast, MTSEA (0.2 mM) was ineffective in CHO cells expressing wild-type (WT, data not shown) or ASIC3E423C channels (Figure 6B). Interestingly, 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET, 0.5 mM), a MTS reagent in which the amino group is replaced by a trimethylamine, failed to induce any detectable currents in ASIC3E79C-expressing CHO cells (Figure 6C), suggesting that the amino group in AGM plays an essential role in activating ASIC3 channels, as has been shown in GMQ-ASIC3 interaction [10]. Together, these data strongly support that activation of ASIC3 by AGM relies on polar, steric, and electrostatic interactions with the nonproton ligand sensing domain in the channel [10], regardless whether the ligand is linear and flexible (i.e. AGM) or circular and rigid (i.e. GMQ).

Figure 6
figure 6

Effects of MTS Reagents on ASIC3E79C Channels at Neutral pH. (A) Models showing the structural similarity between MTSEA and AGM in activating ASIC3. (B) Sample traces showing effects of MTSEA (0.2 mM) on ASIC3E79C (upper trace) and ASIC3E423C mutated channels (lower traces) at pH 7.4. Similar results were obtained from other four measurements. (C) Sample traces showing a lack of effects of MTSET (0.5 mM) on ASIC3E79C. (D) Maximal currents induced in ASIC3E79C by MTSEA (0.2 mM, 2 min) and MTSET (0.5 mM, 5-10 min). Data points are means ± S.E.M. of five measurements normalized to pH 5.0-induced peak currents. **p < 0.001 vs. baseline level. #indicates no detectable changes upon exposure to MTSET (0.5 mM, 5-10 min).

Full size image

ASIC Subunit Specificity

Most ASIC-like acid-evoked currents in DRG neurons are mediated by heteromers of ASIC3, -2, and -1 [57]. To address ASIC subunit specificity, we recorded AGM or ARC responses in CHO cells expressing different combinations of ASIC subunits (Figure 7). Similar to GMQ [10], none of the homomeric channels ASIC1a, ASIC1b, or ASIC2a were activated by either AGM or ARC (Figure 7A, C). However, heteromeric ASIC3 plus ASIC1b channels responded to AGM and ARC (Figure 7B, D) in a manner similar to homomeric ASIC3 channels (Figure 7A, C). On the other hand, heteromeric combinations of ASIC3 plus ASIC1a, ASIC2a, or ASIC2b were insensitive to these ligands (Figure 7B, D). This subunit specificity together with the restricted distribution pattern of ASIC3 [9] suggests that an AGM-dependent regulatory pathway most likely occurs in peripheral tissues expressing homomeric ASIC3 and/or heteromeric ASIC3 + 1b channels [8, 36, 58]. Alternatively, AGM may act as a co-agonist sensitizing the apparently-unresponsive heteromeric ASIC3 channels (i.e., ASIC3 + 1a, 2a, or 2b) (Figure 7) to protons. In addition, according to a recent report [59], AGM may also act on the ASIC3 channels through coincident detection of multiple ligands (i.e., AGM, H+, and other factors such as ATP) by cross-activating an as yet unidentified ion channel. In addition, the emergence of new ASIC isoforms [60] adds an additional possibility underlying the ASIC3-dependent pain-behavior induced by AGM in vivo [10].

Figure 7
figure 7

Subunit Specificity of Agmatine (AGM) and Arcaine (ARC) in CHO Cells Transfected with One or Two Different ASIC Subunits. (A, B) Representative traces showing the response of AGM (1 mM) or ARC (1 mM) on CHO cells transfected with one (A) or two (B) different ASIC subunits. (C, D) Polled data as shown in (A, B). AGM (1 mM) and ARC (1 mM) directly activate ASIC3 homomeric channels (A, C) and heteromeric ASIC3 + 1b channels (B, D) but not ASIC1a nor ASIC1b nor ASIC2a homomeric channels (A, C), nor heteromeric ASIC3 + 1a, ASIC3 + 2a, ASIC3 + 2b channels (B, D) at the neutral pH (pH = 7.4). Data are means ± S.E.M. n = 3-5. **p < 0.001 vs. baseline level.

Full size image

ASIC3 Channels Integrate Multiple Inflammatory Signals in vivo

Finally, to gain insights into the pathophysiological relevance of ASIC3-dependent integration of AGM and multiple inflammatory signals, we performed in vivo pain-related behavioral tests [10] following the injection of AGM and hyperosmolarity, arachidonic acid (AA), or lactate into the right hindpaw of asic3+/+ and asic3-/- mice. We measured the total time the animals spent licking the injected paw during a 30-min period. As shown previously, control asic3+/+ mice showed a significant increase in paw-licking time after AGM (10 mM) injection compared to saline-injected controls [10]. In consideration of the high AGM concentration used, we re-evaluated the paradigms by injecting 1 mM AGM (Figure 8). Similarly, asic3+/+ mice showed a significant increase in paw-licking time after AGM (1 mM) injection, though less intense than that observed following 10 mM AGM injection [10]. As expected, the reaction of asic3-/- mice to AGM was significantly reduced (Figure 8). When AGM (1 mM) was co-applied with hyperosmolarity (H-Osm, 600 mosmol kg-1 with mannitol), AA (10 μM), or lactate (15 mM, keeping the pH neutral), the injection elicited more intense response in asic3+/+ mice while failed to elicit the comparable response in asic3-/- mice. Interestingly, asic3+/+ mice showed a significant increase in paw-licking time following the treatment of H-Osm (600 mosmol kg-1 with mannitol), AA (10 μM), or lactate (15 mM) alone compared to saline-injected controls. These behavioral responses were similarly reduced in asic3-/- mice (Figure 8), supporting an essential role of ASIC3 in sensing multiple inflammatory signals [9], including AGM.

Figure 8
figure 8

AGM Causes Pain-Related Behaviors via ASIC3 Channels. Pain-related behavior as determined by the time spent for paw licking following saline or AGM (1 mM) injection (10 μl) in the absence (white) or presence (grey) of hyperosmolarity (H-Osm, 600 mosmolkg-1 with mannitol), AA (10 μM), or lactate (15 mM, keeping the pH neutral). Data are means ± S.E.M. n = 6-9. *p < 0.05 versus saline; # p < 0.05, asic3-/- versus asic3+/+; & p < 0.05.

Full size image

Conclusions

ASIC3 channels sense extracellular protons and nonproton ligands, including the endogenous molecule AGM, which is a metabolite of arginine. In this study, we extended the previous finding that ASIC3 can be activated by small molecules with basic groups such as GMQ, AGM, and ARC by uncovering the functional interactions of AGM with multiple inflammatory factors such as hyperosmolarity, arachidonic acid, and lactate. Cysteine modification with a linear thiol-reactive compound that mimics AGM binding induces ASIC3 opening in a sustained manner similar to AGM, supporting the critical role of the newly identified nonproton ligand sensing domain. In vivo tests using both asic3+/+ and asic3-/- mice revealed that AGM cooperates with the multiple inflammatory signals to cause pain-related behaviors in an ASIC3-dependent manner. Thus, the present findings suggest a new mechanism for activation or sensitization of ASIC3 channels underlying inflammatory pain-sensing under in vivo conditions.

Methods

Cell Culture and Transfection

All constructs were expressed in CHO cells as described previously [10]. In brief, CHO cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The cells were maintained in F12 medium (INVITROGEN) supplemented with 1 mM L-glutamine, 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. Transient transfection of CHO cells was carried out using Lipofectamine™2000 (INVITROGEN). Electrophysiological measurements were performed 24-48 h after transfection.

Solutions and Drugs

The ionic composition of the incubation solution (SS, see Figures 2E, F and 6B) was (mM): 150 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, aerated with 95% O2/5% CO2 to a final pH of 7.4. The standard external solution contained (mM): 150 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, and 10 glucose, buffered to various pH values with either 10 mM HEPES, pH 6.0-7.4, or 10 mM MES, pH < 6.0. For the Na+-free medium (Figure 6B), Na+ was substituted with equimolar N-Methyl-D-glucamine (NMDG). The patch pipette internal solution for whole-cell patch recording was (mM): 120 KCl, 30 NaCl, 1 MgCl2, 0.5 CaCl2, 5 EGTA, 2 Mg-ATP, and 10 HEPES. The internal solution was adjusted to pH 7.2 with Tris-base. The osmolarities of all these solutions were maintained at 300-325 mOsm (Advanced Instrument, Norwood, MA). Hyperosmotic (H-Osm) conditions were obtained by adding mannitol to the standard external solution (or saline) as indicating in the text.

Solutions with different composition were applied using a rapid application technique termed the "Y-tube" method throughout the experiments [10]. This system allows a complete exchange of external solution surrounding a cell within 20 ms.

Site-Directed Mutagenesis

The cDNA of rat ASIC3 was subcloned into the pEGFPC3 vector (Promega Corporation, Madison, WI, U.S.A.). Each mutant was generated with the QuikChange® mutagenesis kit (Stratagene, La Jolla, CA) in accordance with the manufacturer's protocol using high-performance-liquid-chromatography-purified or PAGE-purified oligonucleotide primers (Sigma-Genosys, The Woodlands, TX). Individual mutations were verified by DNA sequence analysis, and the predicted amino acid sequences were determined by computer analysis.

Electrophysiology

The electrophysiological recordings were performed using the conventional whole-cell patch recording configuration under voltage clamp condition. Patch pipettes were pulled from glass capillaries with an outer diameter of 1.5 mm on a two-stage puller (PP-830, Narishige Co., Ltd., Tokyo, Japan). The resistance between the recording electrode filled with pipette solution and the reference electrode was 3-5 MΩ. Membrane currents were measured using a patch clamp amplifier (Axon 700A, Axon Instruments, Foster City, CA) and were sampled and analyzed using a Digidata 1320A interface and a computer running the Clampex and Clampfit software (version 8.0.1, Axon Instruments). In most experiments, 70-90% of the series resistance was compensated. Unless otherwise noted, the membrane potential was held at -60 mV throughout the experiment under voltage clamp conditions. All the experiments were carried out at room temperature (23 ± 2 °C).

Pain-Related Behavioral Assays

Animals were acclimatized for 30 min before experiments. A total volume of 10 μl solution (in 0.9% NaCl) containing either saline (0.9% NaCl only), or AGM (1 mM), or hyperosmolarity (H-Osm, 600 mosmol kg-1 with mannitol), or AA (10 μM), or lactate (15 mM), or AGM + H-Osm, or AGM + AA, or AGM + lactate was injected intraplantarly using a 30G needle and paw-licking behavior was quantified for 30 min [10].

Data Analysis

Results were expressed as means ± S.E.M. Unless otherwise noted, statistical comparisons were made with the Student's t test.*, or &, or @, p < 0.05 or **p < 0.001 was considered significantly different. To test the synergic interaction between two factors (i.e., mild acidosis and AGM, or AGM and Ca2+ reduction) on ASIC3 currents, additional two-way ANOVA analyses were made (Figures 2 and 5, p < 0.05 was considered significant). Concentration-response relationships for pH-dependent activation of ASIC3 channels were obtained by measuring currents in response to acidic solutions with graded pH values. Each acidic solution was tested on at least three CHO cells and all results used to generate a concentration-response relationship were from the same group. The data were fit to the Hill equation: I/I max =1/[1+(EC 50/[Ligand])n], where I is the normalized current at a given pH, I max is the maximum normalized current, EC 50 is the concentration of proton yielding a current that is half of the maximum, and n is the Hill coefficient.

Abbreviations

5-HT:

serotonin

AA:

arachidonic acid

AGM:

agmatine

ARC:

arcaine

ASIC:

acid-sensing ion channel

BDNF:

brain-derived neurotrophic factor

CHO:

Chinese Hamster Ovary

DRG:

dorsal root ganglion

ENaC/DEG:

epithelial sodium channel/degenerin

GFP:

green fluorescent protein

GMQ:

2-guanidine-4-methylquinazoline

H-Osm:

Hyperosmolarity

MTSEA:

2-aminoethyl-methanethiosulfonate

MTSET:

2-(trimethylammonium)ethyl methanethiosulfonate

NGF:

nerve growth factor

NMDA:

N-methyl-D-aspartate

NO:

nitric oxide

PcTX1:

psalmotoxin 1

TRPM:

Transient Receptor Potential Melastatin

TRPV1:

Transient Receptor Potential Vanilloid Type 1

WT:

wild-type.

References

  1. Wemmie JA, Price MP, Welsh MJ: Acid-sensing ion channels: advances, questions and therapeutic opportunities. Trends Neurosci 2006, 29: 578–586. 10.1016/j.tins.2006.06.014

    Article  CAS  PubMed  Google Scholar 

  2. Jasti J, Furukawa H, Gonzales EB, Gouaux E: Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH. Nature 2007, 449: 316–323. 10.1038/nature06163

    Article  CAS  PubMed  Google Scholar 

  3. Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski M: A proton-gated cation channel involved in acid-sensing. Nature 1997, 386: 173–177. 10.1038/386173a0

    Article  CAS  PubMed  Google Scholar 

  4. Babinski K, Le KT, Seguela P: Molecular cloning and regional distribution of a human proton receptor subunit with biphasic functional properties. J Neurochem 1999, 72: 51–57. 10.1046/j.1471-4159.1999.0720051.x

    Article  CAS  PubMed  Google Scholar 

  5. de Weille JR, Bassilana F, Lazdunski M, Waldmann R: Identification, functional expression and chromosomal localisation of a sustained human proton-gated cation channel. FEBS Lett 1998, 433: 257–260. 10.1016/S0014-5793(98)00916-8

    Article  CAS  PubMed  Google Scholar 

  6. Waldmann R, Bassilana F, de Weille J, Champigny G, Heurteaux C, Lazdunski M: Molecular cloning of a non-inactivating proton-gated Na+ channel specific for sensory neurons. J Biol Chem 1997, 272: 20975–20978. 10.1074/jbc.272.34.20975

    Article  CAS  PubMed  Google Scholar 

  7. Salinas M, Lazdunski M, Lingueglia E: Structural elements for the generation of sustained currents by the acid pain sensor ASIC3. J Biol Chem 2009, 284: 31851–31859. 10.1074/jbc.M109.043984

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Deval E, Noel J, Lay N, Alloui A, Diochot S, Friend V, Jodar M, Lazdunski M, Lingueglia E: ASIC3, a sensor of acidic and primary inflammatory pain. EMBO J 2008, 27: 3047–3055. 10.1038/emboj.2008.213

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Li WG, Xu TL: ASIC3 channels in multimodal sensory perception. ACS Chem Neurosci, in press.

  10. Yu Y, Chen Z, Li WG, Cao H, Feng EG, Yu F, Liu H, Jiang H, Xu TL: A nonproton ligand sensor in the acid-sensing ion channel. Neuron 2010, 68: 61–72. 10.1016/j.neuron.2010.09.001

    Article  CAS  PubMed  Google Scholar 

  11. Morris SM Jr: Arginine metabolism: boundaries of our knowledge. J Nutr 2007, 137: 1602S-1609S.

    CAS  PubMed  Google Scholar 

  12. Li G, Regunathan S, Barrow CJ, Eshraghi J, Cooper R, Reis DJ: Agmatine: an endogenous clonidine-displacing substance in the brain. Science 1994, 263: 966–969. 10.1126/science.7906055

    Article  CAS  PubMed  Google Scholar 

  13. Reis DJ, Regunathan S: Agmatine: an endogenous ligand at imidazoline receptors may be a novel neurotransmitter in brain. J Auton Nerv Syst 1998, 72: 80–85. 10.1016/S0165-1838(98)00091-5

    Article  CAS  PubMed  Google Scholar 

  14. Yang XC, Reis DJ: Agmatine selectively blocks the N-methyl-D-aspartate subclass of glutamate receptor channels in rat hippocampal neurons. J Pharmacol Exp Ther 1999, 288: 544–549.

    CAS  PubMed  Google Scholar 

  15. Loring RH: Agmatine acts as an antagonist of neuronal nicotinic receptors. Br J Pharmacol 1990, 99: 207–211.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Li Q, Yin JX, He RR: Effect of agmatine on L-type calcium current in rat ventricular myocytes. Acta Pharmacol Sin 2002, 23: 219–224.

    CAS  PubMed  Google Scholar 

  17. Weng XC, Gai XD, Zheng JQ, Li J: Agmatine blocked voltage-gated calcium channel in cultured rat hippocampal neurons. Acta Pharmacol Sin 2003, 24: 746–750.

    CAS  PubMed  Google Scholar 

  18. Lopatin AN, Makhina EN, Nichols CG: Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature 1994, 372: 366–369. 10.1038/372366a0

    Article  CAS  PubMed  Google Scholar 

  19. Ficker E, Taglialatela M, Wible BA, Henley CM, Brown AM: Spermine and spermidine as gating molecules for inward rectifier K+ channels. Science 1994, 266: 1068–1072. 10.1126/science.7973666

    Article  CAS  PubMed  Google Scholar 

  20. Kamboj SK, Swanson GT, Cull-Candy SG: Intracellular spermine confers rectification on rat calcium-permeable AMPA and kainate receptors. J Physiol 1995,486(Pt 2):297–303.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Bowie D, Mayer ML: Inward rectification of both AMPA and kainate subtype glutamate receptors generated by polyamine-mediated ion channel block. Neuron 1995, 15: 453–462. 10.1016/0896-6273(95)90049-7

    Article  CAS  PubMed  Google Scholar 

  22. Nilius B, Prenen J, Voets T, Droogmans G: Intracellular nucleotides and polyamines inhibit the Ca2+-activated cation channel TRPM4b. Pflugers Arch 2004, 448: 70–75. 10.1007/s00424-003-1221-x

    Article  CAS  PubMed  Google Scholar 

  23. Kerschbaum HH, Kozak JA, Cahalan MD: Polyvalent cations as permeant probes of MIC and TRPM7 pores. Biophys J 2003, 84: 2293–2305. 10.1016/S0006-3495(03)75035-8

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Ahern GP, Wang X, Miyares RL: Polyamines are potent ligands for the capsaicin receptor TRPV1. J Biol Chem 2006, 281: 8991–8995. 10.1074/jbc.M513429200

    Article  CAS  PubMed  Google Scholar 

  25. Quinn SJ, Ye CP, Diaz R, Kifor O, Bai M, Vassilev P, Brown E: The Ca2+-sensing receptor: a target for polyamines. Am J Physiol 1997, 273: C1315–1323.

    CAS  PubMed  Google Scholar 

  26. Rock DM, Macdonald RL: The polyamine spermine has multiple actions on N-methyl-D-aspartate receptor single-channel currents in cultured cortical neurons. Mol Pharmacol 1992, 41: 83–88.

    CAS  PubMed  Google Scholar 

  27. Zhang L, Zheng X, Paupard MC, Wang AP, Santchi L, Friedman LK, Zukin RS, Bennett MV: Spermine potentiation of recombinant N-methyl-D-aspartate receptors is affected by subunit composition. Proc Natl Acad Sci USA 1994, 91: 10883–10887. 10.1073/pnas.91.23.10883

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Rock DM, MacDonald RL: Spermine and related polyamines produce a voltage-dependent reduction of N-methyl-D-aspartate receptor single-channel conductance. Mol Pharmacol 1992, 42: 157–164.

    CAS  PubMed  Google Scholar 

  29. Babini E, Paukert M, Geisler HS, Grunder S: Alternative splicing and interaction with di- and polyvalent cations control the dynamic range of acid-sensing ion channel 1 (ASIC1). J Biol Chem 2002, 277: 41597–41603. 10.1074/jbc.M205877200

    Article  CAS  PubMed  Google Scholar 

  30. Duan B, Wang YZ, Yang T, Chu XP, Yu Y, Huang Y, Cao H, Hansen J, Simon RP, Zhu MX, et al.: Extracellular spermine exacerbates ischemic neuronal injury through sensitization of ASIC1a channels to extracellular acidosis. J Neurosci, in press.

  31. Zhang M, Wang H, Tracey KJ: Regulation of macrophage activation and inflammation by spermine: a new chapter in an old story. Crit Care Med 2000, 28: N60–66. 10.1097/00003246-200004001-00007

    Article  CAS  PubMed  Google Scholar 

  32. Sastre M, Galea E, Feinstein D, Reis DJ, Regunathan S: Metabolism of agmatine in macrophages: modulation by lipopolysaccharide and inhibitory cytokines. Biochem J 1998,330(Pt 3):1405–1409.

    CAS  PubMed Central  PubMed  Google Scholar 

  33. Cobbe SM, Poole-Wilson PA: The time of onset and severity of acidosis in myocardial ischaemia. J Mol Cell Cardiol 1980, 12: 745–760. 10.1016/0022-2828(80)90077-2

    Article  CAS  PubMed  Google Scholar 

  34. Steen KH, Steen AE, Reeh PW: A dominant role of acid pH in inflammatory excitation and sensitization of nociceptors in rat skin, in vitro. J Neurosci 1995, 15: 3982–3989.

    CAS  PubMed  Google Scholar 

  35. Issberner U, Reeh PW, Steen KH: Pain due to tissue acidosis: a mechanism for inflammatory and ischemic myalgia? Neurosci Lett 1996, 208: 191–194. 10.1016/0304-3940(96)12576-3

    Article  CAS  PubMed  Google Scholar 

  36. Yagi J, Wenk HN, Naves LA, McCleskey EW: Sustained currents through ASIC3 ion channels at the modest pH changes that occur during myocardial ischemia. Circ Res 2006, 99: 501–509. 10.1161/01.RES.0000238388.79295.4c

    Article  CAS  PubMed  Google Scholar 

  37. Vakili C, Ruiz-Ortiz F, Burke JF: Chemical and osmolar changes of interstitial fluid in acute inflammatory states. Surg Forum 1970, 21: 227–228.

    CAS  PubMed  Google Scholar 

  38. Nicholson C, Bruggencate GT, Steinberg R, Stockle H: Calcium modulation in brain extracellular microenvironment demonstrated with ion-selective micropipette. Proc Natl Acad Sci USA 1977, 74: 1287–1290. 10.1073/pnas.74.3.1287

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  39. Immke DC, McCleskey EW: Lactate enhances the acid-sensing Na+ channel on ischemia-sensing neurons. Nat Neurosci 2001, 4: 869–870. 10.1038/nn0901-869

    Article  CAS  PubMed  Google Scholar 

  40. Satriano J: Arginine pathways and the inflammatory response: interregulation of nitric oxide and polyamines: review article. Amino Acids 2004, 26: 321–329. 10.1007/s00726-004-0078-4

    Article  CAS  PubMed  Google Scholar 

  41. Grillo MA, Colombatto S: Metabolism and function in animal tissues of agmatine, a biogenic amine formed from arginine. Amino Acids 2004, 26: 3–8. 10.1007/s00726-003-0030-z

    Article  CAS  PubMed  Google Scholar 

  42. Raasch W, Regunathan S, Li G, Reis DJ: Agmatine is widely and unequally distributed in rat organs. Ann N Y Acad Sci 1995, 763: 330–334. 10.1111/j.1749-6632.1995.tb32419.x

    Article  CAS  PubMed  Google Scholar 

  43. Halaris A, Piletz JE: Relevance of imidazoline receptors and agmatine to psychiatry: a decade of progress. Ann N Y Acad Sci 2003, 1009: 1–20. 10.1196/annals.1304.001

    Article  CAS  PubMed  Google Scholar 

  44. Chen X, Kalbacher H, Grunder S: The tarantula toxin psalmotoxin 1 inhibits acid-sensing ion channel (ASIC) 1a by increasing its apparent H+ affinity. J Gen Physiol 2005, 126: 71–79. 10.1085/jgp.200509303

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Chen X, Kalbacher H, Grunder S: Interaction of acid-sensing ion channel (ASIC) 1 with the tarantula toxin psalmotoxin 1 is state dependent. J Gen Physiol 2006, 127: 267–276. 10.1085/jgp.200509409

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Steen KH, Reeh PW: Sustained graded pain and hyperalgesia from harmless experimental tissue acidosis in human skin. Neurosci Lett 1993, 154: 113–116. 10.1016/0304-3940(93)90184-M

    Article  CAS  PubMed  Google Scholar 

  47. Ugawa S, Ueda T, Ishida Y, Nishigaki M, Shibata Y, Shimada S: Amiloride-blockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors. J Clin Invest 2002, 110: 1185–1190.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  48. Jones NG, Slater R, Cadiou H, McNaughton P, McMahon SB: Acid-induced pain and its modulation in humans. J Neurosci 2004, 24: 10974–10979. 10.1523/JNEUROSCI.2619-04.2004

    Article  CAS  PubMed  Google Scholar 

  49. Voilley N, de Weille J, Mamet J, Lazdunski M: Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors. J Neurosci 2001, 21: 8026–8033.

    CAS  PubMed  Google Scholar 

  50. Mamet J, Baron A, Lazdunski M, Voilley N: Proinflammatory mediators, stimulators of sensory neuron excitability via the expression of acid-sensing ion channels. J Neurosci 2002, 22: 10662–10670.

    CAS  PubMed  Google Scholar 

  51. Ikeuchi M, Kolker SJ, Sluka KA: Acid-sensing ion channel 3 expression in mouse knee joint afferents and effects of carrageenan-induced arthritis. J Pain 2009, 10: 336–342. 10.1016/j.jpain.2008.10.010

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  52. Yiangou Y, Facer P, Smith JA, Sangameswaran L, Eglen R, Birch R, Knowles C, Williams N, Anand P: Increased acid-sensing ion channel ASIC-3 in inflamed human intestine. Eur J Gastroenterol Hepatol 2001, 13: 891–896. 10.1097/00042737-200108000-00003

    Article  CAS  PubMed  Google Scholar 

  53. Sluka KA, Radhakrishnan R, Benson CJ, Eshcol JO, Price MP, Babinski K, Audette KM, Yeomans DC, Wilson SP: ASIC3 in muscle mediates mechanical, but not heat, hyperalgesia associated with muscle inflammation. Pain 2007, 129: 102–112. 10.1016/j.pain.2006.09.038

    Article  PubMed Central  PubMed  Google Scholar 

  54. Smith ES, Cadiou H, McNaughton PA: Arachidonic acid potentiates acid-sensing ion channels in rat sensory neurons by a direct action. Neuroscience 2007, 145: 686–698. 10.1016/j.neuroscience.2006.12.024

    Article  CAS  PubMed  Google Scholar 

  55. Allen NJ, Attwell D: Modulation of ASIC channels in rat cerebellar Purkinje neurons by ischaemia-related signals. J Physiol 2002, 543: 521–529. 10.1113/jphysiol.2002.020297

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  56. Immke DC, McCleskey EW: Protons open acid-sensing ion channels by catalyzing relief of Ca2+ blockade. Neuron 2003, 37: 75–84. 10.1016/S0896-6273(02)01130-3

    Article  CAS  PubMed  Google Scholar 

  57. Benson CJ, Xie J, Wemmie JA, Price MP, Henss JM, Welsh MJ, Snyder PM: Heteromultimers of DEG/ENaC subunits form H+-gated channels in mouse sensory neurons. Proc Natl Acad Sci USA 2002, 99: 2338–2343. 10.1073/pnas.032678399

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  58. Xie J, Price MP, Wemmie JA, Askwith CC, Welsh MJ: ASIC3 and ASIC1 mediate FMRFamide-related peptide enhancement of H+-gated currents in cultured dorsal root ganglion neurons. J Neurophysiol 2003, 89: 2459–2465. 10.1152/jn.00707.2002

    Article  CAS  PubMed  Google Scholar 

  59. Birdsong WT, Fierro L, Williams FG, Spelta V, Naves LA, Knowles M, Marsh-Haffner J, Adelman JP, Almers W, Elde RP, McCleskey EW: Sensing muscle ischemia: coincident detection of acid and ATP via interplay of two ion channels. Neuron 2010, 68: 739–749. 10.1016/j.neuron.2010.09.029

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  60. Hoagland EN, Sherwood TW, Lee KG, Walker CJ, Askwith CC: Identification of a calcium permeable human acid-sensing ion channel 1 transcript variant. J Biol Chem, in press.

Download references

Acknowledgements

We thank all groups that provided us with ASIC cDNAs, Drs. John A. Wemmie, Margaret P. Price, and Michael J. Welsh (University of Iowa, Iowa City, IA) for providing ASIC3 knockout mice. We also thank Dr. James Celentano for helpful comments on the manuscript. This study was supported by grants from the National Natural Science Foundation of China (Nos. 30830035, 30700145), the National Basic Research Program of China (No. 2011CBA00408), and the Shanghai Municipal Government (09XD1404900).

Author information

Author notes

    Authors and Affiliations

    1. Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China

      Wei-Guang Li, Ye Yu, Zhu-Dan Zhang, Hui Cao & Tian-Le Xu

    2. Graduate School of Chinese Academy of Sciences, Shanghai, 200031, China

      Wei-Guang Li & Zhu-Dan Zhang

    Authors
    1. Wei-Guang Li
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Ye Yu
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Zhu-Dan Zhang
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Hui Cao
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Tian-Le Xu
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Tian-Le Xu.

    Additional information

    Competing interests

    The authors declare that they have no competing interests.

    Authors' contributions

    WGL, YY, and TLX designed the project. WGL, YY, and ZDZ performed cell culture, patch-clamp recording, behavior tests, and data analysis. ZDZ and HC did mutations. WGL and TLX wrote the manuscript. All authors read and approved the final manuscript.

    Wei-Guang Li, Ye Yu contributed equally to this work.

    Authors’ original submitted files for images

    Rights and permissions

    Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Reprints and permissions

    About this article

    Cite this article

    Li, WG., Yu, Y., Zhang, ZD. et al. ASIC3 Channels Integrate Agmatine and Multiple Inflammatory Signals through the Nonproton Ligand Sensing Domain. Mol Pain 6, 88 (2010). https://doi.org/10.1186/1744-8069-6-88

    Download citation

    • Received:

    • Accepted:

    • Published:

    • DOI: https://doi.org/10.1186/1744-8069-6-88

    Keywords

    Molecular Pain

    ISSN: 1744-8069

    Contact us