Acid-sensing Ion Channels Channels Research Articles

4-(Azolyl)-Benzamidines as a Novel Chemotype for ASIC1a Inhibitors.

Acid-sensing ion channels (ASICs) play a key role in the perception and response to extracellular acidification changes. These proton-gated cation channels are critical for neuronal functions, like learning and memory, fear, mechanosensation and internal adjustments like synaptic plasticity. Moreover, they play a key role in neuronal degeneration, ischemic neuronal injury, seizure termination, pain-sensing, etc. Functional ASICs are homo or heterotrimers formed with (ASIC1-ASIC3) homologous subunits. ASIC1a, a major ASIC isoform in the central nervous system (CNS), possesses an acidic pocket in the extracellular region, which is a key regulator of channel gating. Growing data suggest that ASIC1a channels are a potential therapeutic target for treating a variety of neurological disorders, including stroke, epilepsy and pain. Many studies were aimed at identifying allosteric modulators of ASIC channels. However, the regulation of ASICs remains poorly understood. Using all available crystal structures, which correspond to different functional states of ASIC1, and a molecular dynamics simulation (MD) protocol, we analyzed the process of channel inactivation. Then we applied a molecular docking procedure to predict the protein conformation suitable for the amiloride binding. To confirm the effect of its sole active blocker against the ASIC1 state transition route we studied the complex with another MD simulation run. Further experiments evaluated various compounds in the Enamine library that emerge with a detectable ASIC inhibitory activity. We performed a detailed analysis of the structural basis of ASIC1a inhibition by amiloride, using a combination of in silico approaches to visualize its interaction with the ion pore in the open state. An artificial activation (otherwise, expansion of the central pore) causes a complex modification of the channel structure, namely its transmembrane domain. The output protein conformations were used as a set of docking models, suitable for a high-throughput virtual screening of the Enamine chemical library. The outcome of the virtual screening was confirmed by electrophysiological assays with the best results shown for three hit compounds.

Dual Modulator of ASIC Channels and GABAA Receptors from Thyme Alters Fear-Related Hippocampal Activity.

Acid-sensing ion channels (ASICs) are proton-gated ion channels that mediate nociception in the peripheral nervous system and contribute to fear and learning in the central nervous system. Sevanol was reported previously as a naturally-occurring ASIC inhibitor from thyme with favorable analgesic and anti-inflammatory activity. Using electrophysiological methods, we found that in the high micromolar range, the compound effectively inhibited homomeric ASIC1a and, in sub- and low-micromolar ranges, positively modulated the currents of α1β2γ2 GABAA receptors. Next, we tested the compound in anxiety-related behavior models using a targeted delivery into the hippocampus with parallel electroencephalographic measurements. In the open field, 6 µM sevanol reduced both locomotor and θ-rhythmic activity similar to GABA, suggesting a primary action on the GABAergic system. At 300 μM, sevanol markedly suppressed passive avoidance behavior, implying alterations in conditioned fear memory. The observed effects could be linked to distinct mechanisms involving GABAAR and ASIC1a. These results elaborate the preclinical profile of sevanol as a candidate for drug development and support the role of ASIC channels in fear-related functions of the hippocampus.

The role of acid sensing ion channels in the cardiovascular function.

Acid Sensing Ion Channels (ASIC) are proton sensors involved in several physiological and pathophysiological functions including synaptic plasticity, sensory systems and nociception. ASIC channels have been ubiquitously localized in neurons and play a role in their excitability. Information about ASIC channels in cardiomyocyte function is limited. Evidence indicates that ASIC subunits are expressed in both, plasma membrane and intracellular compartments of mammalian cardiomyocytes, suggesting unrevealing functions in the cardiomyocyte physiology. ASIC channels are expressed in neurons of the peripheral nervous system including the nodose and dorsal root ganglia (DRG), both innervating the heart, where they play a dual role as mechanosensors and chemosensors. In baroreceptor neurons from nodose ganglia, mechanosensation is directly associated with ASIC2a channels for detection of changes in arterial pressure. ASIC channels expressed in DRG neurons have several roles in the cardiovascular function. First, ASIC2a/3 channel has been proposed as the molecular sensor of cardiac ischemic pain for its pH range activation, kinetics and the sustained current. Second, ASIC1a seems to have a critical role in ischemia-induced injury. And third, ASIC1a, 2 and 3 are part of the metabolic component of the exercise pressure reflex (EPR). This review consists of a summary of several reports about the role of ASIC channels in the cardiovascular system and its innervation.

Distribution of acid-sensing ion channel subunits in human sensory neurons contrasts with that in rodents.

Acid-sensing ion channels (ASICs) play a critical role in nociception in human sensory neurons. Four genes (ASIC1, ASIC2, ASIC3, and ASIC4) encoding multiple subunits through alternative splicing have been identified in humans. Real time-PCR experiments showed strong expression of three subunits ASIC1, ASIC2, and ASIC3 in human dorsal root ganglia; however, their detailed expression pattern in different neuronal populations has not been investigated yet. In the current study, using an in situ hybridization approach (RNAscope), we examined the presence of ASIC1, ASIC2, and ASIC3 mRNA in three subpopulations of human dorsal root ganglia neurons. Our results revealed that ASIC1 and ASIC3 were present in the vast majority of dorsal root ganglia neurons, while ASIC2 was only expressed in less than half of dorsal root ganglia neurons. The distribution pattern of the three ASIC subunits was the same across the three populations of dorsal root ganglia neurons examined, including neurons expressing the REarranged during Transfection (RET) receptor tyrosine kinase, calcitonin gene-related peptide, and a subpopulation of nociceptors expressing Transient Receptor Potential Cation Channel Subfamily V Member 1. These results strongly contrast the expression pattern of Asics in mice since our previous study demonstrated differential distribution of Asics among the various subpopulation of dorsal root ganglia neurons. Given the distinct acid-sensitivity and activity dynamics among different ASIC channels, the expression differences between human and rodents should be taken under consideration when evaluating the translational potential and efficiency of drugs targeting ASICs in rodent studies.

Development of ASIC1a ligand-gated ion channel drug screening assays across multiple automated patch clamp platforms.

Human acid-sensing ion channels (ASIC) are ligand-gated ionotropic receptors expressed widely in peripheral tissues as well as sensory and central neurons and implicated in detection of inflammation, tissue injury, and hypoxia-induced acidosis. This makes ASIC channels promising targets for drug discovery in oncology, pain and ischemia, and several modulators have progressed into clinical trials. We describe the use of hASIC1a as a case study for the development and validation of low, medium and high throughput automated patch clamp (APC) assays suitable for the screening and mechanistic profiling of new ligands for this important class of ligand-gated ion channel. Initial efforts to expand on previous manual patch work describing an endogenous hASIC1a response in HEK cells were thwarted by low current expression and unusual pharmacology, so subsequent work utilized stable hASIC1a CHO cell lines. Ligand-gated application protocols and screening assays on the Patchliner, QPatch 48, and SyncroPatch 384 were optimized and validated based on pH activation and nM-μM potency of reference antagonists (e.g., Amiloride, Benzamil, Memantine, Mambalgin-3, A-317567, PcTx1). By optimizing single and stacked pipette tip applications available on each APC platform, stable pH-evoked currents during multiple ligand applications enabled cumulative EC50 and IC50 determinations with minimized receptor desensitization. Finally, we successfully demonstrated for the first time on an APC platform the ability to use current clamp to implement the historical technique of input resistance tracking to measure ligand-gated changes in membrane conductance on the Patchliner platform.

Pain and nociception bioinspiration for the development of a micellar-based screening test for antinociceptive drugs

Pain is a signal of harmful stimuli generated by the nociceptive system through the activation of receptors or channels present on the surface of the nociceptors. For example, the acid-sensing ion channels (ASICs) are activated by minor pH variations related to many different disorders. Drugs that cease the stimulus (and pain) antagonize this process. The treatment of pain is a serious public health issue and because of that, there has been a huge effort to develop new antinociceptive drugs. The main objective of this article was to develop a screening test bioinspired by ASIC channels to accelerate the development of new antinociceptive drugs and reduce the number of experiments conducted in animals. For that, a micellar dispersion of polystyrene-b-poly (acrylic acid) (PS-b-PAA) was tested encapsulating p-nitrophenyl butyrate (p-NFB) in the presence of lipase isolated from Pseudomonas fluorescens. When the p-NFB is released, the lipase hydrolyses it to the corresponding phenol, which can be detected in a clear analytical response. The system was pH-responsive between pH 7.4 and 7.0, and the signal was suppressed by ibuprofen as expected for an ASICs bioinspired system. The developed system seems to be effective for screening new drugs in the physiological pH range, proved to be stable, with associated low cost, and is easy to use.

Single Subcutaneous Injection of Lysophosphatidyl-Choline Evokes ASIC3-Dependent Increases of Spinal Dorsal Horn Neuron Activity.

Lysophosphatidyl-choline (LPC), a member of the phospholipid family, is an emerging player in pain. It is known to modulate different pain-related ion channels, including Acid-Sensing Ion Channel 3 (ASIC3), a cationic channel mainly expressed in peripheral sensory neurons. LPC potentiates ASIC3 current evoked by mild acidifications, but can also activate the channel at physiological pH. Very recently, LPC has been associated to chronic pain in patients suffering from fibromyalgia or osteoarthritis. Accordingly, repetitive injections of LPC within mouse muscle or joint generate both persistent pain-like and anxiety-like behaviors in an ASIC3-dependent manner. LPC has also been reported to generate acute pain behaviors when injected intraplantarly in rodents. Here, we explore the mechanism of action of a single cutaneous injection of LPC by studying its effects on spinal dorsal horn neurons. We combine pharmacological, molecular and functional approaches including in vitro patch clamp recordings and in vivo recordings of spinal neuronal activity. We show that a single cutaneous injection of LPC exclusively affects the nociceptive pathway, inducing an ASIC3-dependent sensitization of nociceptive fibers that leads to hyperexcitabilities of both high threshold (HT) and wide dynamic range (WDR) spinal neurons. ASIC3 is involved in LPC-induced increase of WDR neuron’s windup as well as in WDR and HT neuron’s mechanical hypersensitivity, and it participates, together with TRPV1, to HT neuron’s thermal hypersensitivity. The nociceptive input induced by a single LPC cutaneous rather induces short-term sensitization, contrary to previously described injections in muscle and joint. If the effects of peripheral LPC on nociceptive pathways appear to mainly depend on peripheral ASIC3 channels, their consequences on pain may also depend on the tissue injected. Our findings contribute to a better understanding of the nociceptive signaling pathway activated by peripheral LPC via ASIC3 channels, which is an important step regarding the ASIC3-dependent roles of this phospholipid in acute and chronic pain conditions.

Lysophosphatidylcholine 16:0 mediates chronic joint pain associated to rheumatic diseases through acid-sensing ion channel 3.

Rheumatic diseases are often associated to debilitating chronic pain, which remains difficult to treat and requires new therapeutic strategies. We had previously identified lysophosphatidylcholine (LPC) in the synovial fluids from few patients and shown its effect as a positive modulator of acid-sensing ion channel 3 (ASIC3) able to induce acute cutaneous pain in rodents. However, the possible involvement of LPC in chronic joint pain remained completely unknown. Here, we show, from 2 independent cohorts of patients with painful rheumatic diseases, that the synovial fluid levels of LPC are significantly elevated, especially the LPC16:0 species, compared with postmortem control subjects. Moreover, LPC16:0 levels correlated with pain outcomes in a cohort of osteoarthritis patients. However, LPC16:0 do not appear to be the hallmark of a particular joint disease because similar levels are found in the synovial fluids of a second cohort of patients with various rheumatic diseases. The mechanism of action was next explored by developing a pathology-derived rodent model. Intra-articular injections of LPC16:0 is a triggering factor of chronic joint pain in both male and female mice, ultimately leading to persistent pain and anxiety-like behaviors. All these effects are dependent on ASIC3 channels, which drive sufficient peripheral inputs to generate spinal sensitization processes. This study brings evidences from mouse and human supporting a role for LPC16:0 via ASIC3 channels in chronic pain arising from joints, with potential implications for pain management in osteoarthritis and possibly across other rheumatic diseases.

Paradoxical Potentiation of Acid-Sensing Ion Channel 3 (ASIC3) by Amiloride via Multiple Mechanisms and Sites Within the Channel

Acid-Sensing Ion Channels (ASICs) are proton-gated sodium-selective cation channels that have emerged as metabolic and pain sensors in peripheral sensory neurons and contribute to neurotransmission in the CNS. These channels and their related degenerin/epithelial sodium channel (DEG/ENaC) family are often characterized by their sensitivity to amiloride inhibition. However, amiloride can also cause paradoxical potentiation of ASIC currents under certain conditions. Here we characterized and investigated the determinants of paradoxical potentiation by amiloride on ASIC3 channels. While inhibiting currents induced by acidic pH, amiloride potentiated sustained currents at neutral pH activation. These effects were accompanied by alterations in gating properties including (1) an alkaline shift of pH-dependent activation, (2) inhibition of pH-dependent steady-state desensitization (SSD), (3) prolongation of desensitization kinetics, and (4) speeding of recovery from desensitization. Interestingly, extracellular Ca2+ was required for paradoxical potentiation and it diminishes the amiloride-induced inhibition of SSD. Site-directed mutagenesis within the extracellular non-proton ligand-sensing domain (E79A, E423A) demonstrated that these residues were critical in mediating the amiloride-induced inhibition of SSD. However, disruption of the purported amiloride binding site (G445C) within the channel pore blunted both the inhibition and potentiation of amiloride. Together, our results suggest that the myriad of modulatory and blocking effects of amiloride are the result of a complex competitive interaction between amiloride, Ca2+, and protons at probably more than one site in the channel.

Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction

At the vertebrate neuromuscular junction (NMJ), presynaptic homeostatic potentiation (PHP) refers to the upregulation of neurotransmitter release via an increase in quantal content (QC) when the postsynaptic nicotinic acetylcholine receptors (nAChRs) are partially blocked. The mechanism of PHP has not been completely worked out. In particular, the identity of the presumed retrograde signal is still a mystery. We investigated the role of acid-sensing ion channels (ASICs) and extracellular protons in mediating PHP at the mouse NMJ. We found that blocking AISCs using benzamil, psalmotoxin-1 (PcTx1), or mambalgin-3 (Mamb3) prevented PHP. Likewise, extracellular acidification from pH 7.4 to 7.2 triggered a significant, reversable increase in QC and this increase could be prevented by PcTx1. Interestingly, an acidic saline (pH 7.2) also precluded the subsequent induction of PHP. Using immunofluorescence we observed ASIC2a and ASIC1 subunits at the NMJ. Our results indicate that protons and ASIC channels are involved in activating PHP at the mouse NMJ. We speculate that the partial blockade of nAChRs leads to a modest decrease in the pH of the synaptic cleft (∼0.2 pH units) and this activates ASIC channels on the presynaptic nerve terminal.

Cell-Type-Specific Expression Pattern of Proton-Sensing Receptors and Channels in Pituitary Gland

In mammalian cells, extracellular protons act as orthosteric and allosteric ligands for multiple receptors and channels. The aim of this study is to identify proton sensors in the rat pituitary gland. qRT-PCR analysis indicated the expression of G-protein-coupled receptor 68 gene (Gpr68) and acid-sensing ion channel (ASIC) genes Asic1, Asic2, and Asic4 in anterior pituitary cells and Asic1 and Asic2 in immortalized GH3 pituitary cells. Asic1a and Asic2b were the dominant splice isoforms. Single anterior pituitary cell RNA sequencing and immunocytochemical analysis showed that nonexcitable folliculostellate cells express GPR68 gene and protein, whereas excitable secretory cells express ASIC genes and proteins. Asic1 was detected in all secretory cell types, Asic2 in gonadotrophs, thyrotrophs, and somatotrophs, and Asic4 in lactotrophs. Extracellular acidification activated two types of currents in a concentration-dependent manner: a fast-developing, desensitizing current with an estimated EC50-value of pH 6.7 and a slow-developing, non-desensitizing current that required a higher proton concentration for activation. The desensitizing current was abolished by removal of bath sodium and application of amiloride, a blocker of ASIC channels, whereas the non-desensitizing current was amiloride insensitive and voltage dependent. Activation of both currents increased the excitability of secretory pituitary cells, consistent with their potential physiological relevance in control of voltage-gated calcium influx and calcium-dependent cellular functions.

Upregulation of ASIC1a channels in an in vitro model of Fabry disease

Neuropathic pain is one of the key features of the classical phenotype of Fabry disease (FD). Acid sensing ion channels (ASICs) are H+-gated cation channels, which belong to the epithelial sodium channel/DeGenerin superfamily, sensitive to the diuretic drug Amiloride. Molecular cloning has identified several distinct ASIC subunits. In particular the ASIC1a subunit has been associated to pain and its upregulation has been documented in animal models of pain. We analyzed the expression of ASIC1a channels in cellular models that mimic the accumulation of glycosphingolipids in FD (FD-GLs) like Gb3, and LysoGb3. We used mouse primary neurons from brain cortex and hippocampus -supraspinal structures that accumulate FD-GLs-, as well as HEK293 cells. Incubation with Gb3, lysoGb3 and the inhibitor (1-deoxy-galactonojirymicin, DJG) of the enzyme α-galactosidase A (Gla) lead to the upregulation of ASIC1a channels. In addition, activation of ASIC1a results in the activation of the MAPK ERK pathway, a signaling pathway associated with pain. Moreover, accumulation of glycosphingolipids results in activation of ERK, an effect that was prevented by blocking ASIC1a channels with the specific blocker Psalmotoxin. Our results suggest that FD-GLs accumulation and triggering of the ERK pathway via ASIC channels might be involved in the mechanism responsible for pain in FD, thus providing a new therapeutic target for pain relief treatment.

Heterologous expression of biologically active Mambalgin-1 peptide as a new potential anticancer, using a PVX-based viral vector in Nicotiana benthamiana.

Mambalgin-1 is a peptide that acts as a potent analgesic through inhibiting acid-sensing ion channels (ASIC) in nerve cells. Research has shown that ASIC channels are involved in the proliferation and growth of cancer cells; therefore, Mambalgin-1 can be a potential anti-cancer by inhibiting these channels. In the present study, the Nicotiana benthamiana codon optimized Mambalgin-1 gene was synthesized and cloned in PVX (potato virus X) viral vector. The two cultures of Agrobacterium containing Mambalgin-1 and P19 silencing suppressor genes were co-agroinfiltrated into N. benthamiana leaves. Five days post infiltration, the production of recombinant Mambalgin-1 was determined by western blotting. For biological activity, MTT (3(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide) was analyzed for the cytotoxicity recombinant Mambalgin-1 from the transformed plants on nervous (SH-SY5Y) and breast (MCF7) cancer cells. The results showed that the plants expressing open reading frame of Mambalgin-1 showed recombinant 7.4 kDa proteins in the entire plant extract. In the MTT test, it was found that Mambalgin-1 had cytotoxic effects on SH-SY5Y cancer cells, yet no effects on MCF7 cancer cells were observed. According to the results, the expression of the biologically active recombinant Mambalgin-1 in the transformed plant leaves was confirmed and Mambalgin-1 can also have anti-cancer (inhibition of ASIC channels) potential along with its already known analgesic effect.Electronic supplementary materialThe online version of this article (doi:10.1007/s11240-020-01838-x) contains supplementary material, which is available to authorized users.

Machaerium hirtum (Vell.) Stellfeld Alleviates Acute Pain and Inflammation: Potential Mechanisms of Action.

Machaerium hirtum (Vell.) Stellfeld (Fabaceae) known in Brazil as “jacaranda de espinho” or “espinheira santa nativa” is a medicinal plant commonly used in folk medicine to treat ulcers, cough and diarrhea. This study aimed to investigate the anti-inflammatory and antinociceptive effects of hydroalcoholic extracts from M. hirtum twig (HEMh) using in vivo experimental models of nociception through the involvement of transient receptor potential channels, acid-sensing ion channel (ASIC), nitrergic, opioidergic, glutamatergic, and supraspinal pathways. Our results revealed an antinociceptive effect of HEMh mediated by the opioidergic, l-arginine-nitric oxide and glutamate systems, as well as by interactions with TRPA1/ASIC channels. The anti-inflammatory effect of HEMh evaluated with a xylene-induced ear edema and by the involvement of arachidonic acid and prostaglandin E2 (PGE2) showed involvement of the COX pathway, based on observed decreases in PGE2 levels. A phytochemical investigation of the HEMh led to the isolation of α-amyrin, β-amyrin, allantoin, apigenin-7-methoxy-6-C-β-d-glucopyranoside, and apigenin-6-C-β-d-glucopyranosyl-8-C-β-d-xylopyranoside. In conclusion, the acute oral administration of HEMh inhibits the nociceptive behavioral response in animals through the nitrergic, opioid, glutamatergic pathways, and by inhibition of the TRPA1 and ASIC channels, without causing locomotor dysfunction. In addition, its anti-inflammatory effect is associated with the COX pathway and decreased PGE2 levels.

Bilirubin enhances the activity of ASIC channels to exacerbate neurotoxicity in neonatal hyperbilirubinemia in mice.

Neonatal hyperbilirubinemia is a common clinical condition that can lead to brain encephalopathy, particularly when concurrent with acidosis due to infection, ischemia, and hypoxia. The prevailing view is that acidosis increases the permeability of the blood-brain barrier to bilirubin and exacerbates its neurotoxicity. In this study, we found that the concentration of the cell death marker, lactate dehydrogenase (LDH) in cerebrospinal fluid (CSF), is elevated in infants with both hyperbilirubinemia and acidosis and showed stronger correlation with the severity of acidosis rather than increased bilirubin concentration. In mouse neonatal neurons, bilirubin exhibits limited toxicity but robustly potentiates the activity of acid-sensing ion channels (ASICs), resulting in increases in intracellular Ca2+ concentration, spike firings, and cell death. Furthermore, neonatal conditioning with concurrent hyperbilirubinemia and hypoxia-induced acidosis promoted long-term impairments in learning and memory and complex sensorimotor functions in vivo, which are largely attenuated in ASIC1a null mice. These findings suggest that targeting acidosis and ASICs may attenuate neonatal hyperbilirubinemia complications.

Acid-sensing ion channels (ASICs) influence excitability of stellate neurons in the mouse cochlear nucleus.

Acid-sensing ion channels (ASICs) are voltage-independent and proton-gated channels. In this study, we aimed to test the hypothesis whether ASICs might be involved in modifying the excitability of stellate cells in the cochlear nucleus (CN). We determined gene expressions of ASIC1, ASIC2 and ASIC3 in the CN of BALB/mice. ASIC currents in stellate cells were characterized by using whole-cell patch-clamp technique. In the voltage-clamp experiments, inward currents were recorded upon application of 2-[N-Morpholino ethanesulfonic acid]-normal artificial cerebrospinal fluid (MES-aCSF), whose pH 50 was 5.84. Amiloride inhibited the acid-induced currents in a dose-dependent manner. Inhibition of the ASIC currents by extracellular Ca2+ and Pb2+ (10μM) was significant evidence for the existence of homomeric ASIC1a subunits. ASIC currents were increased by 20% upon extracellular application of Zn2+ (300μM) (p < 0.05, n = 13). In current-clamp experiments, application of MES-aCSF resulted in the depolarization of stellate cells. The results show that the ASIC currents in stellate cells of the cochlear nucleus are carried largely by the ASIC1a and ASIC2a channels. ASIC channels affect the excitability of the stellate cells and therefore they appear to have a role in the processing of auditory information.

Synaptic signals mediated by protons and acid-sensing ion channels.

Extracellular pH changes may constitute significant signals for neuronal communication. During synaptic transmission, changes in pH in the synaptic cleft take place. Its role in the regulation of presynaptic Ca2+ currents through multivesicular release in ribbon-type synapses is a proven phenomenon. In recent years, protons have been recognized as neurotransmitters that participate in neuronal communication in synapses of several regions of the CNS such as amygdala, nucleus accumbens, and brainstem. Protons are released by nerve stimulation and activate postsynaptic acid-sensing ion channels (ASICs). Several types of ASIC channels are expressed in the peripheral and central nervous system. The influx of Ca2+ through some subtypes of ASICs, as a result of synaptic transmission, agrees with the participation of ASICs in synaptic plasticity. Pharmacological and genetical inhibition of ASIC1a results in alterations in learning, memory, and phenomena like fear and cocaine-seeking behavior. The recognition of endogenous molecules, such as arachidonic acid, cytokines, histamine, spermine, lactate, and neuropeptides, capable of inhibiting or potentiating ASICs suggests the existence of mechanisms of synaptic modulation that have not yet been fully identified and that could be tuned by new emerging pharmacological compounds with potential therapeutic benefits.

Modulation of acid sensing ion channel dependent protonergic neurotransmission at the mouse calyx of Held

Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases. It has been reported that homomeric ASIC-1a channels are expressed in neurons of the medial nucleus of the trapezoid body (MNTB) of the auditory system in the CNS. During synaptic transmission, acidification of the synaptic cleft presumably due to the co-release of neurotransmitter and H+ from synaptic vesicles activates postsynaptic ASIC-1a channels in mice up to 3 weeks old. This generates synaptic currents (ASIC1a-SCs) that add to the glutamatergic excitatory postsynaptic currents (EPSCs). Here we report that neuromodulators like histamine and natural products like lactate and spermine potentiate ASIC1a-SCs in an additive form such that excitatory ASIC synaptic currents as well as the associated calcium influx become significantly large and physiologically relevant. We show that ASIC1a-SCs enhanced by endogenous neuromodulators are capable of supporting synaptic transmission in the absence of glutamatergic EPSCs. Furthermore, at high frequency stimulation (HFS), ASIC1a-SCs contribute to diminish short term depression (STD) and their contribution is even more relevant at early stages of development. Since ASIC channels are present in almost all types of neurons and synaptic vesicles content is acid, the participation of protons in synaptic transmission and its potentiation by endogenous substances could be a general phenomenon across the central nervous system. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Opioid-Mediated Modulation of Acid-Sensing Ion Channel Currents in Adult Rat Sensory Neurons.

Muscle ischemia, associated with peripheral artery disease (PAD), leads to the release of proinflammatory mediators that decrease extracellular pH and trigger the activation of proton-activated acid-sensing ion channels (ASIC). Claudication pain, linked with low blood flow, can be partially relieved by endogenous opioid peptide release. However, we previously reported that sustained ASIC currents in dorsal root ganglion (DRG) neurons were enhanced by naturally occurring endomorphin-1 and -2 opioid peptides, indicating a role of opioid involvement in hyperalgesia. The present study examined whether clinically employed synthetic (fentanyl, remifentanil) and the semisynthetic opioid (oxycodone) would also potentiate sustained ASIC currents, which arise from ASIC3 channel isoforms. Here, we show that exposure of each opioid to DRG neurons resulted in potentiation of the sustained ASIC currents. On the other hand, the potentiation was not observed in DRG neurons from ASIC3 knockout rats. Further, the enhancement of the ASIC currents was resistant to pertussis toxin treatment, suggesting that Gα i/Gα o G-proteins are not involved. Additionally, the potentiation of sustained ASIC currents was greater in DRG neurons isolated from rats with ligated femoral arteries (a model of PAD). The effect of all three opioids on the transient ASIC peak current was mixed (increase, decrease, no effect). The inhibitory action appears to be mediated by the presence of ASIC1 isoform, while the potentiating effect is primarily due to ASIC3 isoform expression. These findings reveal that, under certain conditions, these three opioids can increase ASIC channel activity, possibly giving rise to opioid-induced hyperalgesia.

Expression of ASIC3 in the Trigeminal Nucleus Caudalis Plays a Role in a Rat Model of Recurrent Migraine.

Acid-sensing ion channel 3 (ASIC3) is abundant in the trigeminal nervous system and is most sensitive to a slight pH decrease. Recent studies have indicated that ASIC3 in the peripheral trigeminal ganglia is likely involved in the pathogenesis of migraine pain. However, it is unclear whether this receptor plays a role in recurrent migraine, namely, migraine chronicity. Here, we aimed to investigate the role of ASIC3 in an animal model of recurrent migraine (RM). In this study, we established a rat model of RM through repeated administration of inflammatory soup (IS) onto the dura. Then, we tested the mechanical pain thresholds of the face and hindpaws by von Frey filaments. qRT-PCR, Western blot and immunofluorescence labelling were used to detect the expression and localization of ASIC3 in the trigeminal nucleus caudalis (TNC). The protein levels of calcitonin gene-related peptide (CGRP), its receptor component receptor activity modifying protein 1 (RAMP1) and c-Fos were analysed following treatment with the ASIC3 inhibitor APETx2 and activator 2-guanidine-4-methylquinazoline (GMQ). We found decreased pain thresholds after repeated dural inflammatory stimulation, which suggested the establishment of an RM model. Based on this model, we observed elevated expression of ASIC3 in the TNC group compared to that in the Sham group. ASIC3 was primarily expressed in neurons but not in astrocytes of the TNC. Moreover, APETx2 attenuated tactile allodynia and significantly decreased the expression of c-Fos, CGRP and RAMP1, while GMQ aggravated these effects compared to those observed in the IS + vehicle group. These findings indicate a critical role of ASIC3 channels in the pathophysiology of RM, and ASIC3 might represent a potential therapeutic target to prevent the progression of migraine.