Activation Of Acid-sensing Ion Channels Research Articles

Nocistatin and products of its proteolysis as dual modulators of type 3 acid-sensing ion channels (ASIC3) with an algesic and analgesic effect

The neuropeptide nocistatin (NS) is expressed by cells of the nervous system and neutrophils as part of a precursor protein and can undergo limited proteolysis through stepwise degradation. Previously, it was shown that rat NS (rNS) is able to activate acid-sensing ion channels (ASIC), but this effect correlated with the acidic nature of NS. In this work, we investigated the change in the properties of rNS during its degradation by a comparison of rNS and its two synthesized fragments. We estimated their activity on rat ASIC3 channels expressed in X. laevis oocytes, and the effects in pain tests on mice. We have shown that rNS combines the properties of both positive and negative modulators of ASIC3, which is expressed in the ability to lower the channel’s steady-state desensitization in the pH range 6.8-7.0 and the reduction of the channel’s response to stimuli in the 6.0-6.9 pH range. A shortened analogue (rNSΔ21) (21 amino acid residues (aa) from the N-terminus) retained the effect of the ASIC3 positive modulator only, while the C-terminal pentapeptide (rNSΔ30) retained the ability of the ASIC3 negative modulator only. This tendency was confirmed in animal tests, where rNS and rNSΔ21 induced pain related behavior, but rNSΔ30 showed an analgesic effect. Thus, we have shown the change of the rNS action mode during stepwise degradation, from an algesic molecule through a pain-enhancer to a pain-relief wherefore the final pentapeptide can even be considered as a promising starting point for further drug development.

Nocistatin and Products of Its Proteolysis Are Dual Modulators of Type 3 Acid-Sensing Ion Channels (ASIC3) with Algesic and Analgesic Properties.

The neuropeptide nocistatin (NS) is expressed by the nervous system cells and neutrophils as a part of a precursor protein and can undergo stepwise limited proteolysis. Previously, it was shown that rat NS (rNS) is able to activate acid-sensing ion channels (ASICs) and that this effect correlates with the acidic nature of NS. Here, we investigated changes in the properties of rNS in the course of its proteolytic degradation by comparing the effects of the full-size rNS and its two cleavage fragments on the rat isoform 3 ASICs (ASIC3) expressed in X. laevis oocytes and pain perception in mice. The rNS acted as both positive and negative modulator by lowering the steady-state desensitization of ASIC3 at pH 6.8-7.0 and reducing the channel's response to stimuli at pH 6.0-6.9, respectively. The truncated rNSΔ21 peptide lacking 21 amino acid residues from the N-terminus retained the positive modulatory activity, while the C-terminal pentapeptide (rNSΔ30) acted only as a negative ASIC3 modulator. The effects of the studied peptides were confirmed in animal tests: rNS and rNSΔ21 induced a pain-related behavior, whereas rNSΔ30 showed the analgesic effect. Therefore, we have shown that the mode of rNS action changes during its stepwise degradation, from an algesic molecule through a pain enhancer to a pain reliever (rNSΔ30 pentapeptide), which can be considered as a promising drug candidate.

Protein semisynthesis underscores the role of a conserved lysine in activation and desensitization of acid-sensing ion channels

Acid-sensing ion channels (ASICs) are trimeric ion channels that open a cation-conducting pore in response to proton binding. Excessive ASIC activation during prolonged acidosis in conditions such as inflammation and ischemia is linked to pain and stroke. A conserved lysine in the extracellular domain (Lys211 in mASIC1a) is suggested to play a key role in ASIC function. However, the precise contributions are difficult to dissect with conventional mutagenesis, as replacement of Lys211 with naturally occurring amino acids invariably changes multiple physico-chemical parameters. Here, we study the contribution of Lys211 to mASIC1a function using tandem protein trans-splicing (tPTS) to incorporate non-canonical lysine analogs. We conduct optimization efforts to improve splicing and functionally interrogate semisynthetic mASIC1a. In combination with molecular modeling, we show that Lys211 charge and side-chain length are crucial to activation and desensitization, thus emphasizing that tPTS can enable atomic-scale interrogations of membrane proteins in live cells.

Probing the Proton-Gated ASIC Channels Using Tetraalkylammonium Ions.

The action of tetraalkylammonium ions, from tetrametylammonium (TMA) to tetrapentylammonium (TPtA), on the recombinant and native acid-sensing ion channels (ASICs) was studied using the patch-clamp approach. The responses of ASIC1a, ASIC2a, and native heteromeric ASICs were inhibited by TPtA. The peak currents through ASIC3 were unaffected, whereas the steady-state currents were significantly potentiated. This effect was characterized by an EC50 value of 1.22 ± 0.12 mM and a maximal effect of 3.2 ± 0.5. The effects of TPtA were voltage-independent but significantly decreased under conditions of strong acidification, which caused saturation of ASIC responses. Molecular modeling predicted TPtA binding in the acidic pocket of closed ASICs. Bound TPtA can prevent acidic pocket collapse through a process involving ASIC activation and desensitization. Tetraethylammonium (TEA) inhibited ASIC1a and native ASICs. The effect was independent of the activating pH but decreased with depolarization, suggesting a pore-blocking mechanism.

Reduced Plasma-Membrane Calcium ATPase Activity and Extracellular Acidification Trigger Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction.

At the vertebrate neuromuscular junction (NMJ), presynaptic homeostatic potentiation (PHP) refers to an increase in neurotransmitter release that restores the strength of synaptic transmission following a blockade of nicotinic acetylcholine receptors (nAChRs). Mechanisms informing the presynaptic terminal of the loss of postsynaptic receptivity remain poorly understood. Previous research at the mouse NMJ suggests that extracellular protons may function as a retrograde signal that triggers an upregulation of neurotransmitter output (measured by quantal content, QC) through the activation of acid-sensing ion channels (ASICs). We further investigated the pH-dependency of PHP in an ex-vivo mouse muscle preparation. We observed that increasing the buffering capacity of the perfusion saline with HEPES abolishes PHP and that acidifying the saline from pH 7.4 to pH 7.2-7.1 increases QC, demonstrating the necessity and sufficiency of extracellular acidification for PHP. We then sought to uncover how the blockade of nAChRs leads to the pH decrease. Plasma-membrane calcium ATPase (PMCA), a calcium-proton antiporter, is known to alkalize the synaptic cleft following neurotransmission in a calcium-dependent manner. We hypothesize that since nAChR blockade reduces postsynaptic calcium entry, it also reduces the alkalizing activity of the PMCA, thereby causing acidosis, ASIC activation, and QC upregulation. In line with this hypothesis, we found that pharmacological inhibition of the PMCA with carboxyeosin induces QC upregulation and that this effect requires functional ASICs. We also demonstrated that muscles pre-treated with carboxyeosin fail to generate PHP. These findings suggest that reduced PMCA activity causes presynaptic homeostatic potentiation by activating ASICs at the mouse NMJ.

Identification of Lipid Biomarkers for Chronic Joint Pain Associated with Different Joint Diseases.

Lipids, especially lysophosphatidylcholine LPC16:0, have been shown to be involved in chronic joint pain through the activation of acid-sensing ion channels (ASIC3). The aim of the present study was to investigate the lipid contents of the synovial fluids from controls and patients suffering from chronic joint pain in order to identify characteristic lipid signatures associated with specific joint diseases. For this purpose, lipids were extracted from the synovial fluids and analyzed by mass spectrometry. Lipidomic analyses identified certain choline-containing lipid classes and molecular species as biomarkers of chronic joint pain, regardless of the pathology, with significantly higher levels detected in the patient samples. Moreover, correlations were observed between certain lipid levels and the type of joint pathologies. Interestingly, LPC16:0 levels appeared to correlate with the metabolic status of patients while other choline-containing lipids were more specifically associated with the inflammatory state. Overall, these data point at selective lipid species in synovial fluid as being strong predictors of specific joint pathologies which could help in the selection of the most adapted treatment.

Group II metabotropic glutamate receptor activation attenuates acid-sensing ion channel currents in rat primary sensory neurons

Acid-sensing ion channels (ASICs) play an important role in pain associated with tissue acidification. Peripheral inhibitory group II metabotropic glutamate receptors (mGluRs) have analgesic effects in a variety of pain conditions. Whether there is a link between ASICs and mGluRs in pain processes is still unclear. Herein, we show that the group II mGluR agonist LY354740 inhibited acid-evoked ASIC currents and action potentials in rat dorsal root ganglia neurons. LY354740 reduced the maximum current response to protons, but it did not change the sensitivity of ASICs to protons. LY354740 inhibited ASIC currents by activating group II mGluRs. We found that the inhibitory effect of LY354740 was blocked by intracellular application of the Gi/o protein inhibitor pertussis toxin and the cAMP analogue 8-Br-cAMP and mimicked by the protein kinase A (PKA) inhibitor H-89. LY354740 also inhibited ASIC3 currents in CHO cells coexpressing mGluR2 and ASIC3 but not in cells expressing ASIC3 alone. In addition, intraplantar injection of LY354740 dose-dependently alleviated acid-induced nociceptive behavior in rats through local group II mGluRs. Together, these results suggested that activation of peripheral group II mGluRs inhibited the functional activity of ASICs through a mechanism that depended on Gi/o proteins and the intracellular cAMP/PKA signaling pathway in rat dorsal root ganglia neurons. We propose that peripheral group II mGluRs are an important therapeutic target for ASIC-mediated pain.

Different Involvement of ASIC and TRPA1 in Facial and Hindpaw Allodynia in Nitroglycerin-Induced Peripheral Hypersensitivities in Mice.

The pathophysiological mechanism underlying migraine-associated peripheral hypersensitivity remains unclear. Acid-sensing ion channels (ASICs) and transient receptor potential ankyrin 1 (TRPA1) are known to be causative pathogenic factors of mechanical and cold allodynia, respectively. Here, we sought to investigate their involvement in cold and mechanical allodynia of the face and hindpaws, respectively, in a mouse model of repetitive nitroglycerin (NTG)-induced migraine. NTG (10 mg/kg) was administered to the mice every other day for 9 days, followed 90 min later by HC-030031 (a TRPA1 blocker) or amiloride (a non-selective ASIC blocker). Mechanical or cold sensitivity of the hindpaw and facial regions was quantified using von-Frey filaments or acetone solution, respectively. Immunohistochemistry revealed that c-Fos expression was significantly increased in the trigeminal nucleus caudalis region but not in the spinal cord. Amiloride treatment only reduced NTG-induced hindpaw mechanical allodynia, whereas HC-030031 treatment only improved facial cold allodynia. Interestingly, the number of c-Fos positive cells decreased to a similar level in each drug treatment group. These findings demonstrate that facial cold allodynia and hindpaw mechanical allodynia are differentially mediated by activation of TRPA1 and ASIC, respectively, in mice with repetitive NTG-induced hypersensitivity.

Acid-sensing ion channel 1a regulates the specificity of reconsolidation of conditioned threat responses.

Recent research on altering threat memory has focused on a reconsolidation window. During reconsolidation, threat memories are retrieved and become labile. Reconsolidation of distinct threat memories is synapse dependent, whereas the underlying regulatory mechanism of the specificity of reconsolidation is poorly understood. We designed a unique behavioral paradigm in which a distinct threat memory can be retrieved through the associated conditioned stimulus. In addition, we proposed a regulatory mechanism by which the activation of acid-sensing ion channels (ASICs) strengthens the distinct memory trace associated with the memory reconsolidation to determine its specificity. The activation of ASICs by CO2 inhalation, when paired with memory retrieval, triggers the reactivation of the distinct memory trace, resulting in greater memory lability. ASICs potentiate the memory trace by altering the amygdala-dependent synaptic transmission and plasticity at selectively targeted synapses. Our results suggest that inhaling CO2 during the retrieval event increases the lability of a threat memory through a synapse-specific reconsolidation process.

Suppression of ASIC activity by the activation of A1 adenosine receptors in rat primary sensory neurons

Peripheral A1 adenosine receptor signaling has been shown to have analgesic effects in a variety of pain conditions. However, it is not yet fully elucidated for the precise molecular mechanisms. Acid sensing ion channels (ASICs) are expressed predominantly in nociceptive sensory neurons responding to protons. Given that both A1 adenosine receptors and ASICs are present in dorsal root ganglia (DRG) neurons, we therefore investigated whether there was a cross-talk between the two types of receptors. Herein, electrophysiological recordings showed that the A1 adenosine receptor agonist N6-cyclopentyladenosine (CPA) suppressed acid-induced currents and action potentials, which were mediated by ASICs, in rat DRG neurons. CPA inhibited the maximum response to protons, as shown a downward shift of concentration-response curve for protons. The CPA-induced suppression of ASIC currents was blocked by the A1 adenosine receptor antagonist KW-3902 and also prevented by intracellular application of the Gi/o-protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, and the cAMP analog 8-Br-cAMP. Finally, intraplantar pretreatment of CPA dose-dependently relieved acid-induced nociceptive responses in rats through peripheral A1 adenosine receptors. These results suggested that CPA suppressed ASICs via A1 adenosine receptors and intracellular Gi/o-proteins and cAMP signaling cascades in rat DRG neurons, which was a novel potential mechanism underlying analgesia of peripheral A1 adenosine receptors.

Changes in H+, K+, and Ca2+ Concentrations, as Observed in Seizures, Induce Action Potential Signaling in Cortical Neurons by a Mechanism That Depends Partially on Acid-Sensing Ion Channels.

Acid-sensing ion channels (ASICs) are activated by extracellular acidification. Because ASIC currents are transient, these channels appear to be ideal sensors for detecting the onset of rapid pH changes. ASICs are involved in neuronal death after ischemic stroke, and in the sensation of inflammatory pain. Ischemia and inflammation are associated with a slowly developing, long-lasting acidification. Recent studies indicate however that ASICs are unable to induce an electrical signaling activity under standard experimental conditions if pH changes are slow. In situations associated with slow and sustained pH drops such as high neuronal signaling activity and ischemia, the extracellular K+ concentration increases, and the Ca2+ concentration decreases. We hypothesized that the concomitant changes in H+, K+, and Ca2+ concentrations may allow a long-lasting ASIC-dependent induction of action potential (AP) signaling. We show that for acidification from pH7.4 to pH7.0 or 6.8 on cultured cortical neurons, the number of action potentials and the firing time increased strongly if the acidification was accompanied by a change to higher K+ and lower Ca2+ concentrations. Under these conditions, APs were also induced in neurons from ASIC1a–/– mice, in which a pH of ≤ 5.0 would be required to activate ASICs, indicating that ASIC activation was not required for the AP induction. Comparison between neurons of different ASIC genotypes indicated that the ASICs modulate the AP induction under such changed ionic conditions. Voltage-clamp measurements of the Na+ and K+ currents in cultured cortical neurons showed that the lowering of the pH inhibited Na+ and K+ currents. In contrast, the lowering of the Ca2+ together with the increase in the K+ concentration led to a hyperpolarizing shift of the activation voltage dependence of voltage-gated Na+ channels. We conclude that the ionic changes observed during high neuronal activity mediate a sustained AP induction caused by the potentiation of Na+ currents, a membrane depolarization due to the changed K+ reversal potential, the activation of ASICs, and possibly effects on other ion channels. Our study describes therefore conditions under which slow pH changes induce neuronal signaling by a mechanism involving ASICs.

C-Jun N-Terminal Kinase Post-Translational Regulation of Pain-Related Acid-Sensing Ion Channels 1b and 3.

Neuronal proton-gated acid-sensing ion channels (ASICs) participate in the detection of tissue acidosis, a phenomenon often encountered in painful pathologic diseases. Such conditions often involve in parallel the activation of various signaling pathways such as mitogen activated protein kinases (MAPKs) that ultimately leads to phenotype modifications of sensory neurons. Here, we identify one member of the MAPKs, c-Jun N-terminal kinase (JNK), as a new post-translational positive regulator of ASICs in rodent sensory neurons. Recombinant H+-induced ASIC currents in HEK293 cells are potently inhibited within minutes by the JNK inhibitor SP600125 in a subunit-dependent manner, targeting both rodent and human ASIC1b and ASIC3 subunits (except mouse ASIC3). The regulation by JNK of recombinant ASIC1b- and ASIC3-containing channels (homomers and heteromers) is lost on mutation of a putative phosphorylation site within the intracellular N- and the C-terminal domain of the ASIC1b and ASIC3 subunit, respectively. Moreover, short-term JNK activation regulates the activity of native ASIC1b- and ASIC3-containing channels in rodent sensory neurons and is involved in the rapid potentiation of ASIC activity by the proinflammatory cytokine TNFα. Local JNK activation in vivo in mice induces a short-term potentiation of the acid-induced cutaneous pain in inflammatory conditions that is partially blocked by the ASIC1-specific inhibitor mambalgin-1. Collectively, our data identify pain-related channels as novel physiological JNK substrates in nociceptive neurons and propose JNK-dependent phosphorylation as a fast post-translational mechanism of regulation of sensory-neuron-expressed ASIC1b- and ASIC3-containing channels that may contribute to peripheral sensitization and pain hypersensitivity.SIGNIFICANCE STATEMENT ASICs are a class of excitatory cation channels critical for the detection of tissue acidosis, which is a hallmark of several painful diseases. Previous work in sensory neurons has shown that ASICs containing the ASIC3 or the ASIC1b subunit are important players in different pain models. We combine here functional and pharmacological in vitro and in vivo approaches to demonstrate that the MAP Kinase JNK is a potent post-translational positive regulator, probably via direct phosphorylation, of rodent and human ASIC1b- and ASIC3-containing channels. This JNK-dependent, fast post-translational mechanism of regulation of sensory-neuron-expressed ASICs may contribute to peripheral sensitization and pain hypersensitivity. These data also identify pain-related channels as direct downstream effectors of JNK in nociceptors.

Dexmedetomidine Inhibits ASIC Activity via Activation of α2A Adrenergic Receptors in Rat Dorsal Root Ganglion Neurons.

Dexmedetomidine (DEX), a selective α2 adrenergic receptor (α2-AR) agonist, has been shown to have peripheral analgesic effects in a variety of pain conditions. However, the precise molecular mechanisms have not yet been fully elucidated. Acid sensing ion channels (ASICs) are the major player in pain associated with tissue acidosis. Given that both α2-ARs and ASICs exist in dorsal root ganglia (DRG) neurons, we therefore investigated the effects of DEX on the functional activity of ASICs. Herein, whole-cell patch-clamp recordings demonstrated that DEX suppressed ASIC-mediated and acid-evoked currents and action potentials in dissociated rat DRG neurons. DEX shifted downwards concentration-response curve to protons, with a decrease of 35.83 ± 3.91% in the maximal current response to pH 4.5. DEX-induced inhibition of ASIC currents was blocked by the α2A-AR antagonist BRL44408 in DRG neurons. DEX also inhibited ASIC3 currents in CHO cells co-expressing ASIC3 and α2A-ARs, but not in ASIC3 transfected CHO cells without α2A-ARs expression. DEX-induced inhibition of ASIC currents was mimicked by the protein kinase A inhibitor H-89, and blocked by intracellular application of the Gi/o protein inhibitor pertussis toxin and the cAMP analog 8-Br-cAMP. In addition, peripherally administration of DEX dose-dependently relieved nociceptive responses to intraplantar injection of acetic acid in rats through local α2A-ARs. Our results indicated that DEX inhibited the functional activity of ASICs via α2A-ARs and intracellular Gi/o proteins and cAMP/protein kinase A signaling pathway in rat DRG neurons, which was a novel potential mechanism that probably mediated peripheral analgesia of DEX.

Large Acid-Evoked Currents, Mediated by ASIC1a, Accompany Differentiation in Human Dopaminergic Neurons.

Acid-sensing ion channels (ASICs) are proton-gated Na+ channels. They contribute to synaptic transmission, neuronal differentiation and neurodegeneration. ASICs have been mainly characterized in neurons from mice or rats and our knowledge of their properties in human neurons is scarce. Here, we functionally characterized ASICs in differentiating LUHMES cells, a human mesencephalic cell line with characteristics of dopaminergic neurons. We find that LUHMES cells express functional ASICs, predominantly homomeric ASIC1a. Expression starts early during differentiation with a striking surge in current amplitude at days 4–6 of differentiation, a time point where—based on published data—LUHMES cells start expressing synaptic markers. Peak ASIC expression therefore coincides with a critical period of LUHMES cell differentiation. It was associated with increased excitability, but not paralleled by an increase in ASIC1 mRNA or protein. In differentiating as well as in terminally differentiated LUHMES cells, ASIC activation by slight acidification elicited large currents, action potentials and a rise in cytosolic Ca2+. Applying the ASIC pore blocker diminazene during differentiation reduced the length of neurites, consistent with the hypothesis that ASICs play a critical role in LUHMES cell differentiation. In summary, our study establishes LUHMES cells as a valuable model to study the role of ASICs for neuronal differentiation and potentially also cell death in a human cell line.

TNF-\u03b1 acutely enhances acid-sensing ion channel currents in rat dorsal root ganglion neurons via a p38 MAPK pathway

BackgroundTumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine involved in pain processing and hypersensitivity. It regulates not only the expression of a variety of inflammatory mediators but also the functional activity of some ion channels. Acid-sensing ion channels (ASICs), as key sensors for extracellular protons, are expressed in nociceptive sensory neurons and contribute to pain signaling caused by tissue acidosis. It is still unclear whether TNF-α has an effect on functional activity of ASICs. Herein, we reported that a brief exposure of TNF-α acutely sensitized ASICs in rat dorsal root ganglion (DRG) neurons.MethodsElectrophysiological experiments on rat DRG neurons were performed in vitro and acetic acid induced nociceptive behavior quantified in vitro.ResultsA brief (5min) application of TNF-α rapidly enhanced ASIC-mediated currents in rat DRG neurons. TNF-α (0.1-10 ng/ml) dose-dependently increased the proton-evoked ASIC currents with an EC50 value of 0.12 ± 0.01 nM. TNF-α shifted the concentration-response curve of proton upwards with a maximal current response increase of 42.34 ± 7.89%. In current-clamp recording, an acute application of TNF-α also significantly increased acid-evoked firing in rat DRG neurons. The rapid enhancement of ASIC-mediated electrophysiological activity by TNF-α was prevented by p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190, but not by non-selective cyclooxygenase inhibitor indomethacin, suggesting that p38 MAPK is necessary for this enhancement. Behaviorally, TNF-α exacerbated acid-induced nociceptive behaviors in rats via activation of local p38 MAPK pathway.ConclusionsThese results suggest that TNF-α rapidly enhanced ASIC-mediated functional activity via a p38 MAPK pathway, which revealed a novel peripheral mechanism underlying TNF-α involvement in rapid hyperalgesia by sensitizing ASICs in primary sensory neurons.

Kinetic analysis of ASIC1a delineates conformational signaling from proton-sensing domains to the channel gate

Acid-sensing ion channels (ASICs) are neuronal Na+ channels that are activated by a drop in pH. Their established physiological and pathological roles, involving fear behaviors, learning, pain sensation, and neurodegeneration after stroke, make them promising targets for future drugs. Currently, the ASIC activation mechanism is not understood. Here, we used voltage-clamp fluorometry (VCF) combined with fluorophore-quencher pairing to determine the kinetics and direction of movements. We show that conformational changes with the speed of channel activation occur close to the gate and in more distant extracellular sites, where they may be driven by local protonation events. Further, we provide evidence for fast conformational changes in a pathway linking protonation sites to the channel pore, in which an extracellular interdomain loop interacts via aromatic residue interactions with the upper end of a transmembrane helix and would thereby open the gate.

ASIC Activation Mechanisms Delineated through Genetic Code Expansion

Acid-sensing Ion Channels (ASICs) are a unique class of ion channels that are activated through acidification of the extracellular space. These trimeric sodium-selective ion channels are found in the central and peripheral nervous system, where they are critical players in a myriad of conditions including ischemic cell death, synaptic plasticity, and fear conditioning. As such, determining how these channels function on a molecular level will aid the development of useful pharmacological tools. Currently, ASIC activation is thought to require protonation of acidic side chains in two distinct domains: the acidic pocket and the palm domain.

Inhibition of ASIC-Mediated Currents by Activation of Somatostatin 2 Receptors in Rat Dorsal Root Ganglion Neurons.

Somatostatin (SST) and its analogues like octreotide (OCT) have analgesic effect on a variety of pain through peripheral SST receptors (SSTRs). However, the precise molecular mechanisms have not yet been fully elucidated. This research aimed to identify possible antinociceptive mechanisms, showing functional links of the SSTR2 and acid-sensing ion channels (ASICs). Herein, we reported that OCT inhibited the electrophysiological activity of ASICs in rat dorsal root ganglia (DRG) neurons. OCT concentration-dependently decreased the peak amplitude of acid-evoked inward currents, which were mediated by ASICs. OCT shifted concentration-response curve to protons downwards, with a decrease of 36.53 ± 5.28% in the maximal current response to pH4.5 in the presence of OCT. OCT inhibited ASIC-mediated currents through SSTR2, since the inhibition was blocked by Cyn 154806, a specific SSTR2 antagonist. The OCT inhibition of ASIC-mediated currents was mimicked by H-89, a membrane-permeable inhibitor of PKA, and reversed by internal treatment of an adenylyl cyclase activator forskolin or 8-Br-cAMP. OCT also decreased the number of action potentials induced by acid stimuli through SSTR2. Finally, peripheral administration of 20μM OCT, but not 2μM OCT, significantly relieved nociceptive responses to intraplantar injection of acetic acid in rats. This occurred through local activation of SSTR2 in the injected hindpaw and was reversed following co-application of Cyn 154806. Our results indicate that activation SSTR2 by OCT can inhibit the activity of ASICs via an intracellular cAMP and PKA signaling pathway in rat DRG neurons. These observations demonstrate a cross-talk between ASICs and SSTR2 in peripheral sensory neurons, which was a novel peripheral analgesic mechanism of SST and its analogues.

Activating Acid-Sensing Ion Channels with Photoacid Generators.

Acid-sensing ion channels (ASICs), present in both central and peripheral neurons, respond to changes in extracellular protons. They play important roles in many symptoms and diseases, such as pain, ischemic stroke and neurodegenerative diseases. Herein, we report a novel approach to activate ASICs with the precision of light using organic photoacid generators (PAGs), which are molecules that release H+ upon light illumination, and have been recently used in biomedical studies. The PAGs showed low toxicity in dark conditions. Under LED light illumination, ASICs activation and consequent calcium ion influx was monitored and analysed by fluorescence microscopy, and showed a strong light-dependent response. This approach allows the activation of ASICs with the precision of light, and may be valuable to help better elucidate the molecular mechanism of ASICs and unveil their roles in physiology, pathophysiology, and behaviour.

Acid-sensing ion channel 1a mediates acid-induced pyroptosis through calpain-2/calcineurin pathway in rat articular chondrocytes.

The pyroptosis is a causative agent of rheumatoid arthritis, a systemic autoimmune disease merged with degenerative articular cartilage. Nevertheless, the precise mechanism of extracellular acidosis on chondrocyte pyroptosis is largely unclear. Acid-sensing ion channels (ASICs) belong to an extracellular H+ -activated cation channel family. Accumulating evidence has highlighted activation of ASICs induced by extracellular acidosis upregulate calpain and calcineurin expression in arthritis. In the present study, to investigate the expression and the role of acid-sensing ion channel 1a (ASIC1a), calpain, calcineurin, and NLRP3 inflammasome proteins in regulating acid-induced articular chondrocyte pyroptosis, primary rat articular chondrocytes were subjected to different pH, different time, and different treatments with or without ASIC1a, calpain-2, and calcineurin, respectively. Initially, the research results showed that extracellular acidosis-induced the protein expression of ASIC1a in a pH- and time-dependent manner, and the messenger RNA and protein expressions of calpain, calcineurin, NLRP3, apoptosis-associated speck-like protein, and caspase-1 were significantly increased in a time-dependent manner. Furthermore, the inhibition of ASIC1a, calpain-2, or calcineurin, respectively, could decrease the cell death accompanied with the decreased interleukin-1β level, and the decreased expression of ASIC1a, calpain-2, calcineurin, and NLRP3 inflammasome proteins. Taken together, these results indicated the activation of ASIC1a induced by extracellular acidosis could trigger pyroptosis of rat articular chondrocytes, the mechanism of which might partly be involved with the activation of calpain-2/calcineurin pathway.