Complex alterations in inflammatory pain and analgesic sensitivity in young and ageing female rats: involvement of ASIC3 and Nav1.8 in primary sensory neurons.

OBJECTIVE AND DESIGN: Our aim was to determine an age-dependent role of Nav1.8 and ASIC3 in dorsal root ganglion (DRG) neurons in a rat pre-clinical model of long-term inflammatory pain. METHODS: We compared 6 and 24 months-old female Wistar rats after cutaneous inflammation. We used behavioral pain assessments over time, qPCR, quantitative immunohistochemistry, selective pharmacological manipulation, ELISA and in vitro treatment with cytokines. RESULTS: Older rats exhibited delayed recovery from mechanical allodynia and earlier onset of spontaneous pain than younger rats after inflammation. Moreover, the expression patterns of Nav1.8 and ASIC3 were time and age-dependent and ASIC3 levels remained elevated only in aged rats. In vivo, selective blockade of Nav1.8 with A803467 or of ASIC3 with APETx2 alleviated mechanical and cold allodynia and also spontaneous pain in both age groups with slightly different potency. Furthermore, in vitro IL-1ß up-regulated Nav1.8 expression in DRG neurons cultured from young but not old rats. We also found that while TNF-α up-regulated ASIC3 expression in both age groups, IL-6 and IL-1ß had this effect only on young and aged neurons, respectively. CONCLUSION: Inflammation-associated mechanical allodynia and spontaneous pain in the elderly can be more effectively treated by inhibiting ASIC3 than Nav1.8.

Inhibition of ASIC1a Improves Behavioral Recovery after Stroke.

Stroke continues to be a leading cause of death and long-term disabilities worldwide, despite extensive research efforts. The failure of multiple clinical trials raises the need for continued study of brain injury mechanisms and novel therapeutic strategies for ischemic stroke. The contribution of acid-sensing ion channel 1a (ASIC1a) to neuronal injury during the acute phase of stroke has been well studied; however, the long-term impact of ASIC1a inhibition on stroke recovery has not been established. The present study sought to bridge part of the translational gap by focusing on long-term behavioral recovery after a 30 min stroke in mice that had ASIC1a knocked out or inhibited by PcTX1. The neurological consequences of stroke in mice were evaluated before and after the stroke using neurological deficit score, open field, and corner turn test over a 28 d period. ASIC1a knock-out and inhibited mice showed improved neurological scores more quickly than wild-type control and vehicle-injected mice after the stroke. ASIC1a knock-out mice also recovered from mobility deficits in the open field test more quickly than wild-type mice, while PcTX1-injected mice did not experience significant mobility deficits at all after the stroke. In contrast to vehicle-injected mice that showed clear-sidedness bias in the corner turn test after stroke, PcTX1-injected mice never experienced significant-sidedness bias at all. This study supports and extends previous work demonstrating ASIC1a as a potential therapeutic target for the treatment of ischemic stroke.

Redistribution of ASIC1a channels triggered by IL-6: Potential role of ASIC1a in neuroinflammation.

Cytokines, particularly IL-6, play a crucial role in modulating immune responses in the central nervous system (CNS). Elevated IL-6 levels have been observed in neuroinflammatory conditions, as well as in the sera and brains of patients with neurodegenerative diseases such as Parkinson's, Huntington's, Multiple Sclerosis, and Alzheimer's. Additionally, alterations in regional brain pH have been noted in these conditions. Acid-sensing ion channels (ASICs), including ASIC1a, activated by low pH levels, are highly abundant in the CNS and have recently been associated with various neurological disorders. Our study examined the impact of IL-6 on ASIC1a channels in cell cultures, demonstrating IL-6-induced the redistribution of cytosolic ASIC1a channels to the cell membrane. This redistribution was accompanied by increased ASIC1a current amplitude upon activation, as well as elevated levels of phosphorylated CaMKII and ERK kinases. Additionally, we observed posttranslational modifications on the ASIC1a channel itself. These findings provide insight into a potential link between inflammatory processes and neurodegenerative mechanisms, highlighting ASIC1a channels as promising therapeutic targets in these conditions.

Genetic exploration of roles of acid-sensing ion channel subtypes in neurosensory mechanotransduction including proprioception.

Although acid-sensing ion channels (ASICs) are proton-gated ion channels responsible for sensing tissue acidosis, accumulating evidence has shown that ASICs are also involved in neurosensory mechanotransduction. However, in contrast to Piezo ion channels, evidence of ASICs as mechanically gated ion channels has not been found using conventional mechanoclamp approaches. Instead, ASICs are involved in the tether model of mechanotransduction, with the channels gated via tethering elements of extracellular matrix and intracellular cytoskeletons. Methods using substrate deformation-driven neurite stretch and micropipette-guided ultrasound were developed to reveal the roles of ASIC3 and ASIC1a, respectively. Here we summarize the evidence supporting the roles of ASICs in neurosensory mechanotransduction in knockout mouse models of ASIC subtypes and provide insight to further probe their roles in proprioception.

Molecular Basis for Mambalgin-2 Interaction with Heterotrimeric α-ENaC/ASIC1a/γ-ENaC Channels in Cancer Cells.

Cancer progression is characterized by microenvironmental acidification. Tumor cells adapt to low environmental pH by activating acid-sensing trimeric ion channels of the DEG/ENaC family. The α-ENaC/ASIC1a/γ-ENaC heterotrimeric channel is a tumor-specific acid-sensing channel, and its targeting can be considered a new strategy for cancer therapy. Mambalgin-2 from the Dendroaspis polylepis venom inhibits the α-ENaC/ASIC1a/γ-ENaC heterotrimer more effectively than the homotrimeric ASIC1a channel, initially proposed as the target of mambalgin-2. Although the molecular basis of such mambalgin selectivity remained unclear. Here, we built the models of the complexes of mambalgin-2 with the α-ENaC/ASIC1a/γ-ENaC and ASIC1a channels, performed MD and predicted the difference in the binding modes. The importance of the 'head' loop region of mambalgin-2 for the interaction with the hetero-, but not with the homotrimeric channel was confirmed by site-directed mutagenesis and electrophysiology. A new mode of allosteric regulation of the ENaC channels by linking the thumb domain of the ASIC1a subunit with the palm domain of the γ-ENaC subunit was proposed. The data obtained provide new insights into the regulation of various types of acid-sensing ion channels and the development of new strategies for cancer treatment.

Photomodulation of the ASIC1a acidic pocket destabilizes the open state.

Acid-sensing ion channels (ASICs) are important players in detecting extracellular acidification throughout the brain and body. ASICs have large extracellular domains containing two regions replete with acidic residues: the acidic pocket, and the palm domain. In the resting state, the acidic pocket is in an expanded conformation but collapses in low pH conditions as the acidic side chains are neutralized. Thus, extracellular acidification has been hypothesized to collapse the acidic pocket that, in turn, ultimately drives channel activation. However, several observations run counter to this idea. To explore how collapse or mobility of the acidic pocket is linked to channel gating, we employed two distinct tools. First, we incorporated the photocrosslinkable noncanonical amino acids (ncAAs) 4-azido-L-phenylalanine (AzF) or 4-benzoyl-L-phenylalanine (BzF) into several positions in the acidic pocket. At both E315 and Y318, AzF incorporation followed by UV irradiation led to right shifts in pH response curves and accelerations of desensitization and deactivation, consistent with restrictions of acidic pocket mobility destabilizing the open state. Second, we reasoned that because Cl- ions are found in the open and desensitized structures but absent in the resting state structures, Cl- substitution would provide insight into how stability of the pocket is linked to gating. Anion substitution resulted in faster deactivation and desensitization, consistent with the acidic pocket regulating the stability of the open state. Taken together, our data support a model where acidic pocket collapse is not essential for channel activation. Rather, collapse of the acidic pocket influences the stability of the open state of the pore.

Function and phylogeny support the independent evolution of an ASIC-like Deg/ENaC channel in the Placozoa.

ASIC channels are bilaterian proton-gated sodium channels belonging to the large and functionally-diverse Deg/ENaC family that also includes peptide- and mechanically-gated channels. Here, we report that the non-bilaterian invertebrate Trichoplax adhaerens possesses a proton-activated Deg/ENaC channel, TadNaC2, with a unique combination of biophysical features including tachyphylaxis like ASIC1a, reduced proton sensitivity like ASIC2a, biphasic macroscopic currents like ASIC3, as well as low sensitivity to the Deg/ENaC channel blocker amiloride and Ca2+ ions. Structural modeling and mutation analyses reveal that TadNaC2 proton gating is different from ASIC channels, lacking key molecular determinants, and involving unique residues within the palm and finger regions. Phylogenetic analysis reveals that a monophyletic clade of T. adhaerens Deg/ENaC channels, which includes TadNaC2, is phylogenetically distinct from ASIC channels, instead forming a clade with BASIC channels. Altogether, this work suggests that ASIC-like channels evolved independently in T. adhaerens and its phylum Placozoa. Our phylogenetic analysis also identifies several clades of uncharacterized metazoan Deg/ENaC channels, and provides phylogenetic evidence for the existence of Deg/ENaC channels outside of Metazoa, present in the gene data of select unicellular heterokont and filasterea-related species.

ASIC1a affects hypothalamic signaling and regulates the daily rhythm of body temperature in mice.

The body temperature of mice is higher at night than during the day. We show here that global deletion of acid-sensing ion channel 1a (ASIC1a) results in lower body temperature during a part of the night. ASICs are pH sensors that modulate neuronal activity. The deletion of ASIC1a decreased the voluntary activity at night of mice that had access to a running wheel but did not affect their spontaneous activity. Daily rhythms of thyrotropin-releasing hormone mRNA in the hypothalamus and of thyroid-stimulating hormone ß mRNA in the pituitary, and of prolactin mRNA in the hypothalamus and pituitary were suppressed in ASIC1a-/- mice. The serum thyroid hormone levels were however not significantly changed by ASIC1a deletion. Our findings indicate that ASIC1a regulates activity and signaling in the hypothalamus and pituitary. This likely leads to the observed changes in body temperature by affecting the metabolism or energy expenditure.

Acid-sensing ion channel 1a exacerbates renal ischemia-reperfusion injury through the NF-κB/NLRP3 inflammasome pathway.

Ischemia-reperfusion injury (IRI) is the main cause of acute kidney injury (AKI), and there is no effective therapy. Microenvironmental acidification is generally observed in ischemic tissues. Acid-sensing ion channel 1a (ASIC1a) can be activated by a decrease in extracellular pH which mediates neuronal IRI. Our previous study demonstrated that, ASIC1a inhibition alleviates renal IRI. However, the underlying mechanisms have not been fully elucidated. In this study, we determined that renal tubule-specific deletion of ASIC1a in mice (ASIC1afl/fl/CDH16cre) attenuated renal IRI, and reduced the expression of NLRP3, ASC, cleaved-caspase-1, GSDMD-N, and IL-1ß. Consistent with these in vivo results, inhibition of ASIC1a by the specific inhibitor PcTx-1 protected HK-2 cells from hypoxia/reoxygenation (H/R) injury, and suppressed H/R-induced NLRP3 inflammasome activation. Mechanistically, the activation of ASIC1a by either IRI or H/R induced the phosphorylation of NF-κB p65, which translocates to the nucleus and promotes the transcription of NLRP3 and pro-IL-1ß. Blocking NF-κB by treatment with BAY 11-7082 validated the roles of H/R and acidosis in NLRP3 inflammasome activation. This further confirmed that ASIC1a promotes NLRP3 inflammasome activation, which requires the NF-κB pathway. In conclusion, our study suggests that ASIC1a contributes to renal IRI by affecting the NF-κB/NLRP3 inflammasome pathway. Therefore, ASIC1a may be a potential therapeutic target for AKI. KEY MESSAGES: Knockout of ASIC1a attenuated renal ischemia-reperfusion injury. ASIC1a promoted the NF-κB pathway and NLRP3 inflammasome activation. Inhibition of the NF-κB mitigated the NLRP3 inflammasome activation induced by ASIC1a.

Involvement of ASIC3 and Substance P in Therapeutic Ultrasound-Mediated Analgesia in Mouse Models of Fibromyalgia.

Therapeutic ultrasound (tUS) is widely used in chronic muscle pain control. However, its analgesic molecular mechanism is still not known. Our objective is to reveal the mechanism of the tUS-induced analgesia in mouse models of fibromyalgia. We applied tUS in mice that have developed chronic hyperalgesia induced by intramuscular acidification and determined the tUS frequency at 3 MHz, dosage at 1 W/cm2 (measured output as 6.3 mW/cm2) and 100% duty cycle for 3 minutes having the best analgesic effect. Pharmacological and genetic approaches were used to probe the molecular determinants involved in tUS-mediated analgesia. A second mouse model of fibromyalgia induced by intermittent cold stress was further used to validate the mechanism underlying the tUS-mediated analgesia. The tUS-mediated analgesia was abolished by a pretreatment of NK1 receptor antagonist-RP-67580 or knockout of substance P (Tac1-/-). Besides, the tUS-mediated analgesia was abolished by ASIC3-selective antagonist APETx2 but not TRPV1-selective antagonist capsazepine, suggesting a role for ASIC3. Moreover, the tUS-mediated analgesia was attenuated by ASIC3-selective nonsteroid anti-inflammation drugs (NSAIDs)-aspirin and diclofenac but not by ASIC1a-selective ibuprofen. We next validated the antinociceptive role of substance P signaling in the model induced by intermittent cold stress, in which tUS-mediated analgesia was abolished in mice lacking substance P, NK1R, Asic1a, Asic2b, or Asic3 gene. tUS treatment could activate ASIC3-containing channels in muscle afferents to release substance P intramuscularly and exert an analgesic effect in mouse models of fibromyalgia. NSAIDs should be cautiously used or avoided in the tUS treatment. PERSPECTIVE: Therapeutic ultrasound showed analgesic effects against chronic mechanical hyperalgesia in the mouse model of fibromyalgia through the signaling pathways involving substance P and ASIC3-containing ion channels in muscle afferents. NSAIDs should be cautiously used during tUS treatment.

Peripheral and central employment of acid-sensing ion channels during early bilaterian evolution.

Nervous systems are endowed with rapid chemosensation and intercellular signaling by ligand-gated ion channels (LGICs). While a complex, bilaterally symmetrical nervous system is a major innovation of bilaterian animals, the employment of specific LGICs during early bilaterian evolution is poorly understood. We therefore questioned bilaterian animals' employment of acid-sensing ion channels (ASICs), LGICs that mediate fast excitatory responses to decreases in extracellular pH in vertebrate neurons. Our phylogenetic analysis identified an earlier emergence of ASICs from the overarching DEG/ENaC (degenerin/epithelial sodium channel) superfamily than previously thought and suggests that ASICs were a bilaterian innovation. Our broad examination of ASIC gene expression and biophysical function in each major bilaterian lineage of Xenacoelomorpha, Protostomia, and Deuterostomia suggests that the earliest bilaterian ASICs were probably expressed in the periphery, before being incorporated into the brain as it emerged independently in certain deuterostomes and xenacoelomorphs. The loss of certain peripheral cells from Ecdysozoa after they separated from other protostomes likely explains their loss of ASICs, and thus the absence of ASICs from model organisms Drosophila and Caenorhabditis elegans. Thus, our use of diverse bilaterians in the investigation of LGIC expression and function offers a unique hypothesis on the employment of LGICs in early bilaterian evolution.

Most animals on Earth, from worms to chimpanzees, belong to a group known as the bilaterians. Despite their rich variety of shapes and lifestyles, all these creatures share similarities ­ in particular, a complex nervous system where neurons can quickly relay electric signals. This is made possible by a class of proteins, known as ligand-gated ion channels, which are studded through the membrane of cells. There, they help neurons efficiently communicate with each other by converting external chemical information into internal electrical signals. Yet despite their importance, how and when these proteins have evolved remains poorly understood. Marti-Solans et al. decided to explore this question by focusing on acid-sensing ion channels, a family which often forms the linchpin of bilaterian neural networks. They examined when these proteins first evolved (that is, in which putative ancestral animals) and where in the body. To do so, they combed through genetic data from all major bilaterian lineages as well as from non-biletarian groups; this included previously unexplored datasets that give insight into the type of cells in which a particular gene is active. The analyses revealed that the channels are specific to bilaterians, but that they appeared earlier than previously thought, being present in the very first members of this group. However, at this stage, the proteins were mainly located in cells at the periphery of the body rather than in those from emerging neural circuits. This suggests that the channels were co-opted by nerve cells later on, when the nervous systems became more complex. The proteins being initially located in cells at the outer edge of the body could also explain why they are absent in bilaterian creatures such as fruit flies and nematode worms; these animals all belong to a lineage where growth takes place by shedding their external layers. Acid-sensing ion channels are an important group of potential drug targets, often being implicated in pain and diseases of the nervous system. The work of Marti-Solans et al. offers an insight into the diversity of roles these proteins can play in the body, demonstrating once again how evolution can repurpose the same biophysical functions to serve a range of needs inside an organism.

Metabolic Characterization and Glyceraldehyde-3-Phosphate Dehydrogenase-Dependent Regulation of Epithelial Sodium Channels in hPheo1 Wild-type and SDHB Knockdown Cells.

Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited curative treatment options outside of surgical resection. Patients with mutations in succinate dehydrogenase subunit B (SDHB) are at an increased risk of malignant and aggressive disease. As cation channels are associated with tumorigenesis, we studied the expression and activity of cation channels from the Degenerin superfamily in a progenitor cell line derived from a human PCC. hPheo1 wild-type (WT) and SDHB knockdown (KD) cells were studied to investigate whether epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) are regulated by the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). First, we performed targeted metabolomic studies and quantified changes in glycolysis pathway intermediates and citric acid cycle intermediates using hPheo1 WT cells and SDHB KD cells. Next, we performed protein biochemistry and electrophysiology studies to characterize the protein expression and activity, respectively, of these ion channels. Our western blot experiments show both ENaC alpha and ASIC1/2 are expressed in both hPheo1 WT and SDHB KD cells, with lower levels of a cleaved 60 kDa form of ENaC in SDHB KD cells. Single-channel patch clamp studies corroborate these results and further indicate channel activity is decreased in SDHB KD cells. Additional experiments showed a more significant decreased membrane potential in SDHB KD cells, which were sensitive to amiloride compared to WT cells. We provide evidence for the differential expression and activity of ENaC and ASIC hybrid channels in hPheo1 WT and SDHB KD cells, providing an important area of investigation in understanding SDHB-related disease.

The diverse functions of the DEG/ENaC family: linking genetic and physiological insights.

The DEG/ENaC family of ion channels was defined based on the sequence similarity between degenerins (DEG) from the nematode Caenorhabditis elegans and subunits of the mammalian epithelial sodium channel (ENaC), and also includes a diverse array of non-voltage-gated cation channels from across animal phyla, including the mammalian acid-sensing ion channels (ASICs) and Drosophila pickpockets. ENaCs and ASICs have wide ranging medical importance; for example, ENaCs play an important role in respiratory and renal function, and ASICs in ischaemia and inflammatory pain, as well as being implicated in memory and learning. Electrophysiological approaches, both in vitro and in vivo, have played an essential role in establishing the physiological properties of this diverse family, identifying an array of modulators and implicating them in an extensive range of cellular functions, including mechanosensation, acid sensation and synaptic modulation. Likewise, genetic studies in both invertebrates and vertebrates have played an important role in linking our understanding of channel properties to function at the cellular and whole animal/behavioural level. Drawing together genetic and physiological evidence is essential to furthering our understanding of the precise cellular roles of DEG/ENaC channels, with the diversity among family members allowing comparative physiological studies to dissect the molecular basis of these diverse functions.

Intoxicating effects of alcohol depend on acid-sensing ion channels.

Persons at risk for developing alcohol use disorder (AUD) differ in their sensitivity to acute alcohol intoxication. Alcohol effects are complex and thought to depend on multiple mechanisms. Here, we explored whether acid-sensing ion channels (ASICs) might play a role. We tested ASIC function in transfected CHO cells and amygdala principal neurons, and found alcohol potentiated currents mediated by ASIC1A homomeric channels, but not ASIC1A/2 A heteromeric channels. Supporting a role for ASIC1A in the intoxicating effects of alcohol in vivo, we observed marked alcohol-induced changes on local field potentials in basolateral amygdala, which differed significantly in Asic1a-/- mice, particularly in the gamma, delta, and theta frequency ranges. Altered electrophysiological responses to alcohol in mice lacking ASIC1A, were accompanied by changes in multiple behavioral measures. Alcohol administration during amygdala-dependent fear conditioning dramatically diminished context and cue-evoked memory on subsequent days after the alcohol had cleared. There was a significant alcohol by genotype interaction. Context- and cue-evoked memory were notably worse in Asic1a-/- mice. We further examined acute stimulating and sedating effects of alcohol on locomotor activity, loss of righting reflex, and in an acute intoxication severity scale. We found loss of ASIC1A increased the stimulating effects of alcohol and reduced the sedating effects compared to wild-type mice, despite similar blood alcohol levels. Together these observations suggest a novel role for ASIC1A in the acute intoxicating effects of alcohol in mice. They further suggest that ASICs might contribute to intoxicating effects of alcohol and AUD in humans.

ASIC1a involves the acid-mediated activation of pancreatic stellate cells associated with autophagy induction.

The acidic tumor microenvironment (TME) of pancreatic cancer affects the physiological function of pancreatic stellate cells (PSCs), which in turn promotes cancer progression. Acid-sensing ion channel 1a (ASIC1a) is responsible for acidosis-related physiopathological processes. In this study, we investigated the effect of acid exposure on the activation and autophagy of PSCs, and the role of ASIC1a in these events. The results showed that acidic medium upregulated the expression of ASIC1a, induced PSCs activation and autophagy, which can be suppressed by inhibiting ASIC1a using PcTx1 or ASIC1a knockdown, suggesting that ASIC1a involves these two processes. In addition, the acid-induced activation of PSCs was impaired after the application of autophagy inhibitor alone or in combination with ASIC1a siRNA, meaning a connection between autophagy and activation. Collectively, our study provides evidence for the involvement of ASIC1a in the acid-caused PSCs activation, which may be associated with autophagy induction.

Aggressive migration in acidic pH of a glioblastoma cancer stem cell line in vitro is independent of ASIC and KCa3.1 ion channels, but involves phosphoinositide 3-kinase.

The microenvironment of proliferative and aggressive tumours, such as the brain tumour glioblastoma multiforme (GBM), is often acidic, hypoxic, and nutrient deficient. Acid-sensing ion channels (ASICs) are proton-sensitive Na+ channels that have been proposed to play a role in pH sensing and in modulation of cancer cell migration. We previously reported that primary glioblastoma stem cells (GSCs), which grow as multicellular tumour spheroids, express functional ASIC1a and ASIC3, whereas ASIC2a is downregulated in GSCs. Using a 2.5D migration assay, here we report that acidic pH dramatically increased migration of GSCs of the pro-neural subtype. Pharmacological blockade as well as CRISPR-Cas9-mediated gene knock-out of ASIC1a or stable overexpression of ASIC2a, however, revealed that neither ASIC1a nor ASIC3, nor downregulation of ASIC2a, mediated the aggressive migration at acidic pH. Therefore, we tested the role of two other proteins previously implicated in cancer cell migration: the Ca2+-activated K+ channel KCa3.1 (KCNN4) and phosphoinositide 3-kinase (PI3K). While pharmacological blockade of KCa3.1 did also not affect migration, blockade of PI3K decreased migration at acidic pH to control levels. In summary, our study reveals a strongly enhanced migration of GSCs at acidic pH in vitro and identifies PI3K as an important mediator of this effect.

Acid-sensing Ion Channels: Implications for Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) is the leading cause of lower back pain and seriously affects the quality of life of patients. The intervertebral disc (IVD) is an environment of hypoxia, ischemia, acidity, and hypertonicity. Matrix acidity potentially negatively affects gene expression, activity, proliferation, and apoptosis of IVD cells. Acid-sensing ion channels (ASICs) are a group of proton-gated ion channels that play important roles in physiological and pathological conditions. The distribution of ASICs in the nucleus pulposus (NP), annulus fibrosus, cartilage endplate, and nucleus pulposus mesenchymal stem cells (NP-MSCs), as well as the special functions of ASIC1a and ASIC3, show that ASICs play an important role in IDD. In this review, we comprehensively discuss the roles of ASICs in the development and basic pathology of IDD and their potential relevance as therapeutic targets. A deeper understanding of the roles of ASICs in these processes may provide novel therapeutic targets for IDD prevention and treatment.

Estrogen antagonizes ASIC1a-induced chondrocyte mitochondrial stress in rheumatoid arthritis.

BACKGROUND: Destruction of articular cartilage and bone is the main cause of joint dysfunction in rheumatoid arthritis (RA). Acid-sensing ion channel 1a (ASIC1a) is a key molecule that mediates the destruction of RA articular cartilage. Estrogen has been proven to have a protective effect against articular cartilage damage, however, the underlying mechanisms remain unclear. METHODS: We treated rat articular chondrocytes with an acidic environment, analyzed the expression levels of mitochondrial stress protein HSP10, ClpP, LONP1 by q-PCR and immunofluorescence staining. Transmission electron microscopy was used to analyze the mitochondrial morphological changes. Laser confocal microscopy was used to analyze the Ca2+, mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) level. Moreover, ASIC1a specific inhibitor Psalmotoxin 1 (Pctx-1) and Ethylene Glycol Tetraacetic Acid (EGTA) were used to observe whether acid stimulation damage mitochondrial function through Ca2+ influx mediated by ASIC1a and whether pretreatment with estrogen could counteract these phenomena. Furthermore, the ovariectomized (OVX) adjuvant arthritis (AA) rat model was treated with estrogen to explore the effect of estrogen on disease progression. RESULTS: Our results indicated that HSP10, ClpP, LONP1 protein and mRNA expression and mitochondrial ROS level were elevated in acid-stimulated chondrocytes. Moreover, acid stimulation decreased mitochondrial membrane potential and damaged mitochondrial structure of chondrocytes. Furthermore, ASIC1a specific inhibitor PcTx-1 and EGTA inhibited acid-induced mitochondrial abnormalities. In addition, estrogen could protect acid-stimulated induced mitochondrial stress by regulating the activity of ASIC1a in rat chondrocytes and protects cartilage damage in OVX AA rat. CONCLUSIONS: Extracellular acidification induces mitochondrial stress by activating ASIC1a, leading to the damage of rat articular chondrocytes. Estrogen antagonizes acidosis-induced joint damage by inhibiting ASIC1a activity. Our study provides new insights into the protective effect and mechanism of action of estrogen in RA.

Calcium regulates acid-sensing ion channel 3 activation by competing with protons in the channel pore and at an allosteric binding site.

The extracellular Ca2+ concentration changes locally under certain physiological and pathological conditions. Such variations affect the function of ion channels of the nervous system and consequently also neuronal signalling. We investigated here the mechanisms by which Ca2+ controls the activity of acid-sensing ion channel (ASIC) 3. ASICs are neuronal, H+-gated Na+ channels involved in several physiological and pathological processes, including the expression of fear, learning, pain sensation and neurodegeneration after ischaemic stroke. It was previously shown that Ca2+ negatively modulates the ASIC pH dependence. While protons are default activators of ASIC3, this channel can also be activated at pH7.4 by the removal of the extracellular Ca2+. Two previous studies concluded that low pH opens ASIC3 by displacing Ca2+ ions that block the channel pore at physiological pH. We show here that an acidic residue, distant from the pore, together with pore residues, controls the modulation of ASIC3 by Ca2+. Our study identifies a new regulatory site in ASIC3 and demonstrates that ASIC3 activation involves an allosteric mechanism together with Ca2+ unbinding from the channel pore. We provide a molecular analysis of a regulatory mechanism found in many ion channels.

Acid sensor ASIC1a induces synovial fibroblast proliferation via Wnt/ß-catenin/c-Myc pathway in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia and progressive joint destruction in the middle and late stages. Notably, activated rheumatoid arthritis synovial fibroblasts (RASFs) exhibit tumor-like features, including an increased proliferation rate that largely contributes to pannus formation and joint destruction. Our previous studies have demonstrated that acid-sensing ion channel 1a (ASIC1a) was highly expressed in RASFs, and acidic microenvironment of synovial fluid in patients with RA can activate ASIC1a to promote synovial inflammation, leading to the progression of RA. However, the role and possible mechanism of ASIC1a in RASF proliferation remains unclear. The present study aimed to investigate the effect of ASIC1a activation upon acidosis on RASF proliferation and its molecular mechanism in vivo and in vitro. The results of in vitro experiments showed that activation of ASIC1a upon acidosis promoted the proliferation of RASFs, which could be attenuated by the specific ASIC1a inhibitor Psalmotoxin-1 (PcTx-1) or specific siRNA for ASIC1a. Mechanistically, Wnt/ß-catenin/c-Myc signaling pathway was involved in ASIC1a-induced RASF proliferation. The results of in vivo experiments indicated that intra-articular injection of PcTx-1 reduced synovial hyperplasia and ameliorated cartilage degradation in rats with adjuvant arthritis (AA). Collectively, these results suggest that activation of ASIC1a upon acidosis promotes RASF proliferation, and the mechanism may be related to Wnt/ß-catenin/c-Myc pathway.