Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth.

Glioblastoma (GBM) is the most common malignant primary brain cancer that, despite recent advances in the understanding of its pathogenesis, remains incurable. GBM contains a subpopulation of cells with stem cell-like properties called cancer stem cells (CSCs). Several studies have demonstrated that CSCs are resistant to conventional chemotherapy and radiation thus representing important targets for novel anti-cancer therapies. Proton sensing receptors expressed by CSCs could represent important factors involved in the adaptation of tumours to the extracellular environment. Accordingly, the expression of acid-sensing ion channels (ASICs), proton-gated sodium channels mainly expressed in the neurons of peripheral (PNS) and central nervous system (CNS), has been demonstrated in several tumours and linked to an increase in cell migration and proliferation. In this paper we report that the ASIC3 isoform, usually absent in the CNS and present in the PNS, is enriched in human GBM CSCs while poorly expressed in the healthy human brain. We propose here a novel therapeutic strategy based on the pharmacological activation of ASIC3, which induces a significant GBM CSCs damage while being non-toxic for neurons. This approach might offer a promising and appealing new translational pathway for the treatment of glioblastoma.

Mice lacking ASIC2 and ßENaC are protected from high-fat-diet-induced metabolic syndrome.

Introduction: Degenerin proteins, such as ßENaC and ASIC2, have been implicated in cardiovascular function. However, their role in metabolic syndrome have not been studied. To begin to assess this interaction, we evaluated the impact of a high fat diet (HFD) on mice lacking normal levels of ASIC2 (ASIC2-/-) and ßENaC (ßENaCm/m). Methods: Twenty-week-old male and female mice were placed on a 60% HFD for 12 weeks. Body weight was measured weekly, and body composition by non-invasive ECHO MRI and fasting blood glucose were measured at 0, 4, 8 and 12 weeks. A glucose tolerance test was administered after 12 weeks. Differences between ASIC2-/-/ßENaCm/m and WT groups were compared using independent t-tests or ANOVA where appropriate within each sex. Data are presented as mean ± SEM and ASIC2-/-/ßENaCm/m vs. WT. Results: At 20 weeks of age, ASIC2-/-/ßENaCm/m mice (n=9F/10M) weighed less and gained less weight than WT (n=12F/16M). Total body fat and lean body masses were reduced in female and male ASIC2-/-/ßENaCm/m mice. Total body fat and lean body masses as % control were identical at the end of 12 weeks. Fasting blood glucoses were lower in female and male ASIC2-/-/ßENaCm/m vs. WT mice after 12 weeks HFD. The area under the curve for the glucose tolerance test was reduced in female and tended (p=.079) to decrease in male ASIC2-/-/ßENaCm/m. Plasma leptin and insulin were reduced in female and male ASIC2-/-/ßENaCm/m vs. WT mice. Plasma insulin in female ASIC2-/-/ßENaCm/m mice remained unchanged throughout the HFD period. Liver and liver fat masses, as well as percent liver fat, were reduced in both female and male ASIC2-/-/ßENaCm/m mice after HFD. Plasma triglycerides, cholesterol, LDL- and HDL-cholesterols were markedly improved in male and/or female ASIC2-/-/ßENaCm/m following the HFD. Discussion: These novel findings suggest that loss of ASIC2 and ßENaC offer a significant protection against HFD-induced metabolic syndrome.

A novel experimental mouse model of diabetic nonalcoholic steatohepatitis: A critical role for acid-sensitive Ion Channel 1a.

BACKGROUND: A two-way relationship exists between type 2 diabetes (T2DM) and human nonalcoholic steatohepatitis (NASH). Several diabetic NASH models have the disadvantages of long cycles or inconsistent with the actual incidence of human disease, which would be costly and time-consuming to investigate disease pathogenesis and develop drugs. Therefore, there is an urgent need to establish a diabetic NASH mouse model. METHODS: The combination between Fructose-palmitate-cholesterol diet (FPC) and Streptozotocin (STZ) (FPC+STZ) was used to construct diabetic NASH mouse model. The in vivo effects of silencing acid-sensitive Ion Channel 1a (ASIC1a) were examined with an adeno-associated virus 9 (AAV9) carrying ASIC1a short hairpin RNA (shRNA) in FPC+STZ model. RESULTS: The mice fed with FPC for 12 weeks had insulin resistance, hyperinsulinemia, lipid accumulation, and increased hepatic levels of inflammatory factors. However, it still did not develop remarkable liver fibrosis. Most interestingly, noticeable fibrotic scars were observed in the liver of mice from FPC+STZ group. Furthermore, insulin therapy significantly ameliorated FPC+STZ-induced NASH-related liver fibrosis, indicating that hyperglycemia is of great significance in NASH development and progression. Importantly, ASIC1a was found to be involved in the pathogenesis of diabetic NASH as demonstrated that silencing ASIC1a in HSCs significantly ameliorated FPC+STZ-induced NASH fibrosis. Mechanistically, ASIC1a interacted with Poly Adp-adenosine ribose polymerase (PARP1) to promote HSC activation by inducing autophagy. CONCLUSION: A FPC diet combined with an injection of STZ induces a diabetic NASH mouse model in a shorter period. Targeting ASIC1a may provide a novel therapeutic target for the treatment of diabetic NASH.

ASIC3-activated key enzymes of de novo lipid synthesis supports lactate-driven EMT and the metastasis of colorectal cancer cells.

Acidic microenvironments is a cancer progression driver, unclear core mechanism hinders the discovery of new diagnostic or therapeutic targets. ASIC3 is an extracellular proton sensor and acid-sensitive, but its role in acidic tumor microenvironment of colorectal cancer is not reported. Functional analysis data show that colorectal cancer cells respond to specific concentration of lactate to accelerate invasion and metastasis, and ASIC3 is the main actor in this process. Mechanism reveal de novo lipid synthesis is a regulatory process of ASIC3, down-regulated ASIC3 increases and interacts with ACC1 and SCD1, which are key enzymes in de novo lipid synthesis pathway, this interaction results in increased unsaturated fatty acids, which in turn induce EMT to promote metastasis, and overexpression of ASIC3 reduces acidic TME-enhanced colorectal cancer metastasis. Clinical samples of colorectal cancer also exhibit decreased ASIC3 expression, and low ASIC3 expression is associated with metastasis and stage of colorectal cancer. This study is the first to identify the role of the ASIC3-ACC1/SCD1 axis in acid-enhanced colorectal cancer metastasis. The expression pattern of ASIC3 in colorectal cancer differs significantly from that in other types of cancers, ASIC3 may serve as a novel and reliable marker for acidic microenvironmental in colorectal cancer, and potentially a therapeutic target.

Glutamate acts on acid-sensing ion channels to worsen ischaemic brain injury.

Glutamate is traditionally viewed as the first messenger to activate NMDAR (N-methyl-D-aspartate receptor)-dependent cell death pathways in stroke1,2, but unsuccessful clinical trials with NMDAR antagonists implicate the engagement of other mechanisms3-7. Here we show that glutamate and its structural analogues, including NMDAR antagonist L-AP5 (also known as APV), robustly potentiate currents mediated by acid-sensing ion channels (ASICs) associated with acidosis-induced neurotoxicity in stroke4. Glutamate increases the affinity of ASICs for protons and their open probability, aggravating ischaemic neurotoxicity in both in vitro and in vivo models. Site-directed mutagenesis, structure-based modelling and functional assays reveal a bona fide glutamate-binding cavity in the extracellular domain of ASIC1a. Computational drug screening identified a small molecule, LK-2, that binds to this cavity and abolishes glutamate-dependent potentiation of ASIC currents but spares NMDARs. LK-2 reduces the infarct volume and improves sensorimotor recovery in a mouse model of ischaemic stroke, reminiscent of that seen in mice with Asic1a knockout or knockout of other cation channels4-7. We conclude that glutamate functions as a positive allosteric modulator for ASICs to exacerbate neurotoxicity, and preferential targeting of the glutamate-binding site on ASICs over that on NMDARs may be strategized for developing stroke therapeutics lacking the psychotic side effects of NMDAR antagonists.

iMSC exosome delivers hsa-mir-125b-5p and strengthens acidosis resilience through suppression of ASIC1 protein in cerebral ischemia-reperfusion.

Acid-sensing ion channel 1 (ASIC1) is critical in acidotoxicity and significantly contributes to neuronal death in cerebral stroke. Pharmacological inhibition of ASIC1 has been shown to reduce neuronal death. However, the potential of utilizing exosomes derived from pluripotent stem cells to achieve inhibition of Asic1 remains to be explored. Developing qualified exosome products with precise and potent active ingredients suitable for clinical application is also ongoing. Here, we adopt small RNA-seq to interrogate the miRNA contents in exosomes of pluripotent stem cell induced mesenchymal stem cell (iMSC). RNA-seq was used to compare the oxygen-glucose deprivation-damaged neurons before and after the delivery of exosomes. We used Western blot to quantify the Asic1 protein abundance in neurons before and after exosome treatment. An in vivo test on rats validated the neuroprotective effect of iMSC-derived exosome and its active potent miRNA hsa-mir-125b-5p. We demonstrate that pluripotent stem cell-derived iMSCs produce exosomes with consistent miRNA contents and sustained expression. These exosomes efficiently rescue injured neurons, alleviate the pathological burden, and restore neuron function in rats under oxygen-glucose deprivation stress. Furthermore, we identify hsa-mir-125b-5p as the active component responsible for inhibiting the Asic1a protein and protecting neurons. We validated a novel therapeutic strategy to enhance acidosis resilience in cerebral stroke by utilizing exosomes derived from pluripotent stem cells with specific miRNA content. This holds promise for cerebral stroke treatment with the potential to reduce neuronal damage and improve clinical patient outcomes.

Bone marrow monocytes and macrophages from mice lacking ßENaC and ASIC2 have a reduced chemotactic migration response and polarization.

The monocyte-macrophage system plays an important role in phagocytosis of pathogens and cellular debris following infection or tissue injury in several pathophysiological conditions. We examined ENaC/ASIC subunit transcript expression and the importance of select subunits in migration of bone marrow derived monocytes (freshly isolated) and macrophages (monocytes differentiated in culture). We also examined the effect of select subunit deletion on macrophage phenotype. BM monocytes were harvested from the femurs of male and female WT and KO mice (6-12 weeks of age). Our results show that α, ß, γENaC, and ASIC1-5 transcripts are expressed in BM macrophages and monocytes to varying degrees. At least αENaC, ßENaC, and ASIC2 subunits contribute to chemotactic migration responses in BM monocyte-macrophages. Polarization markers (CD86, soluble TNFα) in BM macrophages from mice lacking ASIC2a plus ßENaC were shifted towards the M1 phenotype. Furthermore, select M1 phenotypic markers were recovered with rescue of ßENaC or ASIC2. Taken together, these data suggest that ßENaC and ASIC2 play an important role in BM macrophage migration and loss of ßENaC and/or ASIC2 partially polarizes macrophages to the M1 phenotype. Thus, targeting ENaC/ASIC expression in BM macrophages may regulate their ability to migrate to sites of injury.

Sensory ASIC3 channel exacerbates psoriatic inflammation via a neurogenic pathway in female mice.

Psoriasis is an immune-mediated skin disease associated with neurogenic inflammation, but the underlying molecular mechanism remains unclear. We demonstrate here that acid-sensing ion channel 3 (ASIC3) exacerbates psoriatic inflammation through a sensory neurogenic pathway. Global or nociceptor-specific Asic3 knockout (KO) in female mice alleviates imiquimod-induced psoriatic acanthosis and type 17 inflammation to the same extent as nociceptor ablation. However, ASIC3 is dispensable for IL-23-induced psoriatic inflammation that bypasses the need for nociceptors. Mechanistically, ASIC3 activation induces the activity-dependent release of calcitonin gene-related peptide (CGRP) from sensory neurons to promote neurogenic inflammation. Botulinum neurotoxin A and CGRP antagonists prevent sensory neuron-mediated exacerbation of psoriatic inflammation to similar extents as Asic3 KO. In contrast, replenishing CGRP in the skin of Asic3 KO mice restores the inflammatory response. These findings establish sensory ASIC3 as a critical constituent in psoriatic inflammation, and a promising target for neurogenic inflammation management.

Revealing molecular determinants governing mambalgin-3 pharmacology at acid-sensing ion channel 1 variants.

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that play a role in neurotransmission and pain sensation. The snake venom-derived peptides, mambalgins, exhibit potent analgesic effects in rodents by inhibiting central ASIC1a and peripheral ASIC1b. Despite their distinct species- and subtype-dependent pharmacology, previous structure-function studies have focussed on the mambalgin interaction with ASIC1a. Currently, the specific channel residues responsible for this pharmacological profile, and the mambalgin pharmacophore at ASIC1b remain unknown. Here we identify non-conserved residues at the ASIC1 subunit interface that drive differences in the mambalgin pharmacology from rat ASIC1a to ASIC1b, some of which likely do not make peptide binding interactions. Additionally, an amino acid variation below the core binding site explains potency differences between rat and human ASIC1. Two regions within the palm domain, which contribute to subtype-dependent effects for mambalgins, play key roles in ASIC gating, consistent with subtype-specific differences in the peptides mechanism. Lastly, there is a shared primary mambalgin pharmacophore for ASIC1a and ASIC1b activity, with certain peripheral peptide residues showing variant-specific significance for potency. Through our broad mutagenesis studies across various species and subtype variants, we gain a more comprehensive understanding of the pharmacophore and the intricate molecular interactions that underlie ligand specificity. These insights pave the way for the development of more potent and targeted peptide analogues required to advance our understating of human ASIC1 function and its role in disease.

Identification of the modulatory Ca2+-binding sites of acid-sensing ion channel 1a.

Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels activated by extracellular acidification. ASICs are involved in learning, fear sensing, pain sensation and neurodegeneration. Increasing the extracellular Ca2+ concentration decreases the H+ sensitivity of ASIC1a, suggesting a competition for binding sites between H+ and Ca2+ ions. Here, we predicted candidate residues for Ca2+ binding on ASIC1a, based on available structural information and our molecular dynamics simulations. With functional measurements, we identified several residues in cavities previously associated with pH-dependent gating, whose mutation reduced the modulation by extracellular Ca2+ of the ASIC1a pH dependence of activation and desensitization. This occurred likely owing to a disruption of Ca2+ binding. Our results link one of the two predicted Ca2+-binding sites in each ASIC1a acidic pocket to the modulation of channel activation. Mg2+ regulates ASICs in a similar way as does Ca2+. We show that Mg2+ shares some of the binding sites with Ca2+. Finally, we provide evidence that some of the ASIC1a Ca2+-binding sites are functionally conserved in the splice variant ASIC1b. Our identification of divalent cation-binding sites in ASIC1a shows how Ca2+ affects ASIC1a gating, elucidating a regulatory mechanism present in many ion channels.

Cholestane-3ß,5α,6ß-Triol Inhibits Acid-Sensing Ion Channels and Reduces Acidosis-Mediated Ischemic Brain Injury.

BACKGROUND: Activation of the acid-sensing ion channels (ASICs) by tissue acidosis, a common feature of brain ischemia, contributes to ischemic brain injury, while blockade of ASICs results in protection. Cholestane-3ß,5α,6ß-triol (Triol), a major cholesterol metabolite, has been demonstrated as an endogenous neuroprotectant; however, the mechanism underlying its neuroprotective activity remains elusive. In this study, we tested the hypothesis that inhibition of ASICs is a potential mechanism. METHODS: The whole-cell patch-clamp technique was used to examine the effect of Triol on ASICs heterogeneously expressed in Chinese hamster ovary cells and ASICs endogenously expressed in primary cultured mouse cortical neurons. Acid-induced injury of cultured mouse cortical neurons and middle cerebral artery occlusion-induced ischemic brain injury in wild-type and ASIC1 and ASIC2 knockout mice were studied to examine the protective effect of Triol. RESULTS: Triol inhibits ASICs in a subunit-dependent manner. In Chinese hamster ovary cells, it inhibits homomeric ASIC1a and ASIC3 without affecting ASIC1ß and ASIC2a. In cultured mouse cortical neurons, it inhibits homomeric ASIC1a and heteromeric ASIC1a-containing channels. The inhibition is use-dependent but voltage- and pH-independent. Structure-activity relationship analysis suggests that hydroxyls at the 5 and 6 positions of the A/B ring are critical functional groups. Triol alleviates acidosis-mediated injury of cultured mouse cortical neurons and protects against middle cerebral artery occlusion-induced brain injury in an ASIC1a-dependent manner. CONCLUSIONS: Our study identifies Triol as a novel ASIC inhibitor, which may serve as a new pharmacological tool for studying ASICs and may also be developed as a potential drug for treating stroke.

Functional expression of the proton sensors ASIC1a, TMEM206, and OGR1 together with BKCa channels is associated with cell volume changes and cell death under strongly acidic conditions in DAOY medulloblastoma cells.

Fast growing solid tumors are frequently surrounded by an acidic microenvironment. Tumor cells employ a variety of mechanisms to survive and proliferate under these harsh conditions. In that regard, acid-sensitive membrane receptors constitute a particularly interesting target, since they can affect cellular functions through ion flow and second messenger cascades. Our knowledge of these processes remains sparse, however, especially regarding medulloblastoma, the most common pediatric CNS malignancy. In this study, using RT-qPCR, whole-cell patch clamp, and Ca2+-imaging, we uncovered several ion channels and a G protein-coupled receptor, which were regulated directly or indirectly by low extracellular pH in DAOY and UW228 medulloblastoma cells. Acidification directly activated acid-sensing ion channel 1a (ASIC1a), the proton-activated Cl- channel (PAC, ASOR, or TMEM206), and the proton-activated G protein-coupled receptor OGR1. The resulting Ca2+ signal secondarily activated the large conductance calcium-activated potassium channel (BKCa). Our analyses uncover a complex relationship of these transmembrane proteins in DAOY cells that resulted in cell volume changes and induced cell death under strongly acidic conditions. Collectively, our results suggest that these ion channels in concert with OGR1 may shape the growth and evolution of medulloblastoma cells in their acidic microenvironment.

Dynamic conformational changes of acid-sensing ion channels in different desensitizing conditions.

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to fast synaptic transmission and have roles in fear conditioning and nociception. Apart from activation at low pH, ASIC1a also undergoes several types of desensitization, including acute desensitization, which terminates activation; steady-state desensitization, which occurs at sub-activating proton concentrations and limits subsequent activation; and tachyphylaxis, which results in a progressive decrease in response during a series of activations. Structural insights from a desensitized state of ASIC1 have provided great spatial detail, but dynamic insights into conformational changes in different desensitizing conditions are largely missing. Here, we use electrophysiology and voltage-clamp fluorometry to follow the functional changes of the pore along with conformational changes at several positions in the extracellular and upper transmembrane domain via cysteine-labeled fluorophores. Acute desensitization terminates activation in wild type, but introducing an N414K mutation in the ß11-12 linker of mouse ASIC1a interfered with this process. The mutation also affected steady-state desensitization and led to pronounced tachyphylaxis. Although the extracellular domain of this mutant remained sensitive to pH and underwent pH-dependent conformational changes, these conformational changes did not necessarily lead to desensitization. N414K-containing channels also remained sensitive to a known peptide modulator that increases steady-state desensitization, indicating that the mutation only reduced, but not precluded, desensitization. Together, this study contributes to our understanding of the fundamental properties of ASIC1a desensitization, emphasizing the complex interplay between the conformational changes of the extracellular domain and the pore during channel activation and desensitization.

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.

The funnel-web spider venom derived single knot peptide Hc3a modulates acid-sensing ion channel 1a desensitisation.

Acid-sensing ion channel 1a (ASIC1a) is a proton-gated channel involved in synaptic transmission, pain signalling, and several ischemia-associated pathological conditions. The spider venom-derived peptides PcTx1 and Hi1a are two of the most potent ASIC1a inhibitors known and have been instrumental in furthering our understanding of the structure, function, and biological roles of ASICs. To date, homologous spider peptides with different pharmacological profiles at ASIC1a have yet to be discovered. Here we report the characterisation of Hc3a, a single inhibitor cystine knot peptide from the Australian funnel-web spider Hadronyche cerberea with sequence similarity to PcTx1. We show that Hc3a has complex pharmacology and binds different ASIC1a conformational states (closed, open, and desensitised) with different affinities, with the most prominent effect on desensitisation. Hc3a slows the desensitisation kinetics of proton-activated ASIC1a currents across multiple application pHs, and when bound directly to ASIC1a in the desensitised conformation promotes current inhibition. The solution structure of Hc3a was solved, and the peptide-channel interaction examined via mutagenesis studies to highlight how small differences in sequence between Hc3a and PcTx1 can lead to peptides with distinct pharmacology. The discovery of Hc3a expands the pharmacological diversity of spider venom peptides targeting ASIC1a and adds to the toolbox of compounds to study the intricacies of ASIC1 gating.

ASIC1 promotes migration and invasion of hepatocellular carcinoma via the PRKACA/AP-1 signaling pathway.

Hepatocellular carcinoma (HCC) exhibits a high mortality rate due to its high invasion and metastatic nature, and the acidic microenvironment plays a pivotal role. Acid-sensing ion channel 1 (ASIC1) is upregulated in HCC tissues and facilitates tumor progression in a pH-dependent manner, while the specific mechanisms therein remain currently unclear. Herein, we aimed to investigate the underlying mechanisms by which ASIC1 contributes to the development of HCC. Using bioinformatics analysis, we found a significant association between ASIC1 expression and malignant transformation of HCC, such as poor prognosis, metastasis and recurrence. Specifically, ASIC1 enhanced the migration and invasion capabilities of Li-7 cells in the in vivo experiment using an HCC lung metastasis mouse model, as well as in the in vitro experiments such as wound healing assay and Transwell assay. Furthermore, our comprehensive gene chip and molecular biology experiments revealed that ASIC1 promoted HCC migration and invasion by activating the PRKACA/AP-1 signaling pathway. Our findings indicate that targeting ASIC1 could have therapeutic potential for inhibiting HCC progression.

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.

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.

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.

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.