A conserved peptide-binding pocket in HyNaC/ASIC ion channels.

The only known peptide-gated ion channels-FaNaCs/WaNaCs and HyNaCs-belong to different clades of the DEG/ENaC family. FaNaCs are activated by the short neuropeptide FMRFamide, and HyNaCs by Hydra RFamides, which are not evolutionarily related to FMRFamide. The FMRFamide-binding site in FaNaCs was recently identified in a cleft atop the large extracellular domain. However, this cleft is not conserved in HyNaCs. Here, we combined molecular modeling and site-directed mutagenesis and identified a putative binding pocket for Hydra-RFamides in the extracellular domain of the heterotrimeric HyNaC2/3/5. This pocket localizes to only one of the three subunit interfaces, indicating that this trimeric ion channel binds a single peptide ligand. We engineered an unnatural amino acid at the putative binding pocket entrance, which allowed covalent tethering of Hydra RFamide to the channel, thereby trapping the channel in an open conformation. The identified pocket localizes to the same region as the acidic pocket of acid-sensing ion channels (ASICs), which binds peptide ligands. The pocket in HyNaCs is less acidic, and both electrostatic and hydrophobic interactions contribute to peptide binding. Collectively, our results reveal a conserved ligand-binding pocket in HyNaCs and ASICs and indicate independent evolution of peptide-binding cavities in the two subgroups of peptide-gated ion channels.

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.

Acid-sensing ion channels and downstream signalling in cancer cells: is there a mechanistic link?

It is increasingly appreciated that the acidic microenvironment of a tumour contributes to its evolution and clinical outcomes. However, our understanding of the mechanisms by which tumour cells detect acidosis and the signalling cascades that it induces is still limited. Acid-sensing ion channels (ASICs) are sensitive receptors for protons; therefore, they are also candidates for proton sensors in tumour cells. Although in non-transformed tissue, their expression is mainly restricted to neurons, an increasing number of studies have reported ectopic expression of ASICs not only in brain cancer but also in different carcinomas, such as breast and pancreatic cancer. However, because ASICs are best known as desensitizing ionotropic receptors that mediate rapid but transient signalling, how they trigger intracellular signalling cascades is not well understood. In this review, we introduce the acidic microenvironment of tumours and the functional properties of ASICs, point out some conceptual problems, summarize reported roles of ASICs in different cancers, and highlight open questions on the mechanisms of their action in cancer cells. Finally, we propose guidelines to keep ASIC research in cancer on solid ground.

Melatonin alters the excitability of mouse cerebellar granule neurons by inhibiting voltage-gated sodium, potassium, and calcium channels.

Besides its role in the circadian rhythm, the pineal gland hormone melatonin (MLT) also possesses antiepileptogenic, antineoplastic, and cardioprotective properties, among others. The dosages necessary to elicit beneficial effects in these diseases often far surpass physiological concentrations. Although even high doses of MLT are considered to be largely harmless to humans, the possible side effects of pharmacological concentrations are so far not well investigated. In the present study, we report that pharmacological doses of MLT (3 mM) strongly altered the electrophysiological characteristics of cultured primary mouse cerebellar granule cells (CGCs). Using whole-cell patch clamp and ratiometric Ca2+ imaging, we observed that pharmacological concentrations of MLT inhibited several types of voltage-gated Na+ , K+ , and Ca2+ channels in CGCs independently of known MLT-receptors, altering the character and pattern of elicited action potentials (APs) significantly, quickly and reversibly. Specifically, MLT reduced AP frequency, afterhyperpolarization, and rheobase, whereas AP amplitude and threshold potential remained unchanged. The altered biophysical profile of the cells could constitute a possible mechanism underlying the proposed beneficial effects of MLT in brain-related disorders, such as epilepsy. On the other hand, it suggests potential adverse effects of pharmacological MLT concentrations on neurons, which should be considered when using MLT as a pharmacological compound.

Functional characterization of acid-sensing ion channels in the cerebellum-originating medulloblastoma cell line DAOY and in cerebellar granule neurons.

Acid-sensing ion channels (ASICs) are Na+ channels that are almost ubiquitously expressed in neurons of the brain. Functional ASIC1a is also expressed in glioblastoma stem cells, where it might sense the acidic tumor microenvironment. Prolonged acidosis induces cell death in neurons and reduces tumor sphere formation in glioblastoma via activation of ASIC1a. It is currently unknown whether ASICs are expressed and involved in acid-induced cell death in other types of brain tumors. In this study, we investigated ASICs in medulloblastoma, using two established cell lines, DAOY and UW228, as in vitro models. In addition, we characterized ASICs in the most numerous neuron of the brain, the cerebellar granule cell, which shares the progenitor cell with some forms of medulloblastoma. We report compelling evidence using RT-qPCR, western blot and whole-cell patch clamp that DAOY and cerebellar granule cells, but not UW228 cells, functionally express homomeric ASIC1a. Additionally, Ca2+-imaging revealed that extracellular acidification elevated intracellular Ca2+-levels in DAOY cells independently of ASICs. Finally, we show that overexpression of RIPK3, a key component of the necroptosis pathway, renders DAOY cells susceptible to acid-induced cell death via activation of ASIC1a. Our data support the idea that ASIC1a is an important acid sensor in brain tumors and that its activation has potential to induce cell death in tumor cells.

Physiologically relevant acid-sensing ion channel (ASIC) 2a/3 heteromers have a 1:2 stoichiometry.

Acid-sensing ion channels (ASICs) sense extracellular protons and are involved in synaptic transmission and pain sensation. ASIC1a and ASIC3 are the ASIC subunits with the highest proton sensitivity. ASIC2a in contrast has low proton sensitivity but increases the variability of ASICs by forming heteromers with ASIC1a or ASIC3. ASICs are trimers and for the ASIC1a/2a heteromer it has been shown that subunits randomly assemble with a flexible 1:2/2:1 stoichiometry. Both heteromers have almost identical proton sensitivity intermediate between ASIC1a and ASIC2a. Here, we investigated the stoichiometry of the ASIC2a/3 heteromer. Using electrophysiology, we extensively characterized, first, cells expressing ASIC2a and ASIC3 at different ratios, second, concatemeric channels with a fixed subunit stoichiometry, and, third, channels containing loss-of-functions mutations in specific subunits. Our results conclusively show that only ASIC2a/3 heteromers with a 1:2 stoichiometry had a proton-sensitivity intermediate between ASIC2a and ASIC3. In contrast, the proton sensitivity of ASIC2a/3 heteromers with a 2:1 stoichiometry was strongly acid-shifted by more than one pH unit, which suggests that they are not physiologically relevant. Together, our results reveal that the proton sensitivity of the two ASIC2a/3 heteromers is clearly different and that ASIC3 and ASIC1a make remarkably different contributions to heteromers with ASIC2a.

Modulation of Acid-Sensing Ion Channels by Tannic Acid and Green Tea via a Membrane-Mediated Mechanism.

Acid-sensing ion channels (ASICs) are proton-gated ion channels that contribute to pain perception and neurotransmission. Being involved in sensing inflammation and ischemia, ASIC1a and ASIC3 are promising drug targets. Polyphenol tannic acid (TA) as well as green tea can interact with a variety of ion channels, but their effect on ASICs remains unknown. In addition, it is unknown whether they interact with ion channels via a common mechanism. Here, we show that TA is a potent modulator of ASICs. TA inhibited the transient current of rat ASIC3 expressed in HEK cells with an apparent IC50 of 2.2 ± 0.6 µM; it potentiated the sustained current and induced a slowly declining decay current. In addition, it produced an acidic shift of the pH-dependent activation of ASIC3 and inhibited the window current at pH 7.0. Moreover, TA inhibited the transient current of ASIC1a, ASIC1b, and ASIC2a. Pentagalloylglucose that is chemically identical to the central part of TA and a green tea extract both had effects on ASIC3 comparable to TA. TA and green tea inhibited inward currents generated by gramicidin channels, indicating interaction with the membrane. These results show that TA, pentagalloylglucose, and green tea modulate ASICs and identify alteration of the membrane as the potential common mechanism of this modulation. These properties will limit clinical application of these molecules.

Functional analysis in a model sea anemone reveals phylogenetic complexity and a role in cnidocyte discharge of DEG/ENaC ion channels.

Ion channels of the DEG/ENaC family share a similar structure but serve strikingly diverse biological functions, such as Na+ reabsorption, mechanosensing, proton-sensing, chemosensing and cell-cell communication via neuropeptides. This functional diversity raises the question of the ancient function of DEG/ENaCs. Using an extensive phylogenetic analysis across many different animal groups, we found a surprising diversity of DEG/ENaCs already in Cnidaria (corals, sea anemones, hydroids and jellyfish). Using a combination of gene expression analysis, electrophysiological and functional studies combined with pharmacological inhibition as well as genetic knockout in the model cnidarian Nematostella vectensis, we reveal an unanticipated role for a proton-sensitive DEG/ENaC in discharge of N. vectensis cnidocytes, the stinging cells typifying all cnidarians. Our study supports the view that DEG/ENaCs are versatile channels that have been co-opted for diverse functions since their early occurrence in animals and that respond to simple and ancient stimuli, such as omnipresent protons.

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.

Neuropeptides and degenerin/epithelial Na+ channels: a relationship from mammals to cnidarians.

Ion channels of the degenerin (DEG)/epithelial Na+ channel (ENaC) family serve diverse functions ranging from mechanosensation over Na+ reabsorption to H+ sensing and neurotransmission. However, several diverse DEG/ENaCs interact with neuropeptides; some are directly activated, whereas others are modulated by neuropeptides. Two questions arise: does this interaction have a common structural basis and does it have an ancient origin? Current evidence suggests that RFamide neuropeptides activate the FMRFamide-activated Na+ channels (FaNaCs) of invertebrates via binding to a pocket at the external face of their large extracellular domain. It is likely that RFamides might activate DEG/ENaCs from the freshwater polyp Hydra (the HyNaCs) via binding to a similar pocket, although there is not yet any experimental evidence. In contrast, RFamide neuropeptides modulate acid-sensing ion channels (ASICs) from vertebrates via binding to a central cavity enclosed by ß-sheets of the extracellular domain. Dynorphin opioid peptides, for their part, bind to the acidic pocket of ASICs, which might be evolutionarily related to the peptide binding pocket of FaNaCs, but instead of opening the channels they work as antagonists to stabilize its closed state. Moreover, peptides interacting with DEG/ENaCs from animals of different phyla, although having similar sequences, are evolutionarily unrelated to each other. Collectively, it appears that despite a seemingly similar interaction with similar peptides, the interaction of DEG/ENaCs with neuropeptides has diverse structural bases and many origins.

Acidosis induces RIPK1-dependent death of glioblastoma stem cells via acid-sensing ion channel 1a.

Eliciting regulated cell death, like necroptosis, is a potential cancer treatment. However, pathways eliciting necroptosis are poorly understood. It has been reported that prolonged activation of acid-sensing ion channel 1a (ASIC1a) induces necroptosis in mouse neurons. Glioblastoma stem cells (GSCs) also express functional ASIC1a, but whether prolonged activation of ASIC1a induces necroptosis in GSCs is unknown. Here we used a tumorsphere formation assay to show that slight acidosis (pH 6.6) induces necrotic cell death in a manner that was sensitive to the necroptosis inhibitor Nec-1 and to the ASIC1a antagonist PcTx1. In addition, genetic knockout of ASIC1a rendered GSCs resistant to acid-induced reduction in tumorsphere formation, while the ASIC1 agonist MitTx1 reduced tumorsphere formation also at neutral pH. Finally, a 20 amino acid fragment of the ASIC1 C-terminus, thought to interact with the necroptosis kinase RIPK1, was sufficient to reduce the formation of tumorspheres. Meanwhile, the genetic knockout of MLKL, the executive protein in the necroptosis cascade, did not prevent a reduction in tumor sphere formation, suggesting that ASIC1a induced an alternative cell death pathway. These findings demonstrate that ASIC1a is a death receptor on GSCs that induces cell death during prolonged acidosis. We propose that this pathway shapes the evolution of a tumor in its acidic microenvironment and that pharmacological activation of ASIC1a might be a potential new strategy in tumor therapy.

Personalized Medicine Approach in a DCM Patient with LMNA Mutation Reveals Dysregulation of mTOR Signaling.

BACKGROUND: Mutations in the Lamin A/C (LMNA) gene are responsible for about 6% of all familial dilated cardiomyopathy (DCM) cases which tend to present at a young age and follow a fulminant course. METHODS: We report a 47-year-old DCM patient with severely impaired left ventricular ejection fraction and NYHA functional class IV despite optimal heart failure treatment. Whole-exome sequencing revealed an LMNA E161K missense mutation as the pathogenetic cause for DCM in this patient. We generated a patient-specific LMNA-knock in (LMNA-KI) in vitro model using mES cells. RESULTS: Beta adrenergic stimulation of cardiomyocytes derived from LMNA-KI mES cells resulted in augmented mTOR signaling and increased dysregulation of action potentials, which could be effectively prevented by the mTOR-inhibitor rapamycin. A cardiac biopsy confirmed strong activation of the mTOR-signaling pathway in the patient. An off-label treatment with oral rapamycin was initiated and resulted in an improvement in left ventricular ejection fraction (27.8% to 44.5%), NT-BNP (8120 ng/L to 2210 ng/L) and NYHA functional class. CONCLUSION: We have successfully generated the first in vitro model to recapitulate a patient-specific LMNA E161K mutation which leads to a severe form of DCM. The model may serve as a template for individualized and specific treatment of heart failure.

Dynorphin Neuropeptides Decrease Apparent Proton Affinity of ASIC1a by Occluding the Acidic Pocket.

Prolonged acidosis, as it occurs during ischemic stroke, induces neuronal death via acid-sensing ion channel 1a (ASIC1a). Concomitantly, it desensitizes ASIC1a, highlighting the pathophysiological significance of modulators of ASIC1a acid sensitivity. One such modulator is the opioid neuropeptide big dynorphin (Big Dyn) which binds to ASIC1a and enhances its activity during prolonged acidosis. The molecular determinants and dynamics of this interaction remain unclear, however. Here, we present a molecular interaction model showing a dynorphin peptide inserting deep into the acidic pocket of ASIC1a. We confirmed experimentally that the interaction is predominantly driven by electrostatic forces, and using noncanonical amino acids as photo-cross-linkers, we identified 16 residues in ASIC1a contributing to Big Dyn binding. Covalently tethering Big Dyn to its ASIC1a binding site dramatically decreased the proton sensitivity of channel activation, suggesting that Big Dyn stabilizes a resting conformation of ASIC1a and dissociates from its binding site during channel opening.

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

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

The Neuropeptide Nocistatin Is Not a Direct Agonist of Acid-Sensing Ion Channel 1a (ASIC1a).

Acid-sensing ion channels (ASICs) are ionotropic receptors that are directly activated by protons. Although protons have been shown to act as a neurotransmitter and to activate ASICs during synaptic transmission, it remains a possibility that other ligands directly activate ASICs as well. Neuropeptides are attractive candidates for alternative agonists of ASICs, because related ionotropic receptors are directly activated by neuropeptides and because diverse neuropeptides modulate ASICs. Recently, it has been reported that the neuropeptide nocistatin directly activates ASICs, including ASIC1a. Here we show that nocistatin does not directly activate ASIC1a expressed in Xenopus oocytes or CHO cells. Moreover, we show that nocistatin acidifies the bath solution to an extent that can fully explain the previously reported activation by this highly acidic peptide. In summary, we conclude that nocistatin only indirectly activates ASIC1a via acidification of the bath solution.

Efficient expression of a cnidarian peptide-gated ion channel in mammalian cells.

Hydra Na+ channels (HyNaCs) are peptide-gated ion channels of the DEG/ENaC gene family that are directly activated by neuropeptides of the Hydra nervous system. They have previously been successfully characterized in Xenopus oocytes. To establish their expression in mammalian cells, we transiently expressed heteromeric HyNaC2/3/5 in human HEK 293 and monkey COS-7 cells. We found that the expression of HyNaC2/3/5 using native cDNAs was inefficient and that codon optimization strongly increased protein expression and current amplitude in patch-clamp experiments. We used the improved expression of codon-optimized channel subunits to perform Ca2+ imaging and to demonstrate their glycosylation pattern. In summary, we established efficient expression of a cnidarian ion channel in mammalian cell lines.

The mono-ADP-ribosyltransferase ARTD10 regulates the voltage-gated K+ channel Kv1.1 through protein kinase C delta.

BACKGROUND: ADP-ribosylation is a ubiquitous post-translational modification that involves both mono- and poly-ADP-ribosylation. ARTD10, also known as PARP10, mediates mono-ADP-ribosylation (MARylation) of substrate proteins. A previous screen identified protein kinase C delta (PKCδ) as a potential ARTD10 substrate, among several other kinases. The voltage-gated K+ channel Kv1.1 constitutes one of the dominant Kv channels in neurons of the central nervous system and the inactivation properties of Kv1.1 are modulated by PKC. In this study, we addressed the role of ARTD10-PKCδ as a regulator of Kv1.1. RESULTS: We found that ARTD10 inhibited PKCδ, which increased Kv1.1 current amplitude and the proportion of the inactivating current component in HeLa cells, indicating that ARTD10 regulates Kv1.1 in living cells. An inhibitor of ARTD10, OUL35, significantly decreased peak amplitude together with the proportion of the inactivating current component of Kv1.1-containing channels in primary hippocampal neurons, demonstrating that the ARTD10-PKCδ signaling cascade regulates native Kv1.1. Moreover, we show that the pharmacological blockade of ARTD10 increases excitability of hippocampal neurons. CONCLUSIONS: Our results, for the first time, suggest that MARylation by ARTD10 controls neuronal excitability.

Bile acids are potent inhibitors of rat P2X2 receptors.

Extracellular adenosine triphosphate (ATP) regulates a broad variety of physiological functions in a number of tissues partly via ionotropic P2X receptors. Therefore, P2X receptors are promising targets for the development of therapeutically active molecules. Bile acids are cholesterol-derived amphiphilic molecules; their primary function is the facilitation of efficient nutrient fat digestion. However, bile acids have also been shown to serve as signaling molecules and as modulators of different membrane proteins and receptors including ion channels. In addition, some P2X receptors are sensitive to structurally related steroid hormones. In this study, we systematically analyzed whether rat P2X receptors are affected by micromolar concentrations of different bile acids. The taurine-conjugated bile acids TLCA, THDCA, and TCDCA potently inhibited P2X2, whereas other P2X receptors were only mildly affected. Furthermore, stoichiometry and species origin of the P2X receptors affected the modulation by bile acids: in comparison to rat P2X2, the heteromeric P2X2/3 receptor was less potently modulated and the human P2X2 receptor was potentiated by TLCA. In summary, bile acids are a new class of P2X receptor modulators, which might be of physiological relevance.

Screening of 109 neuropeptides on ASICs reveals no direct agonists and dynorphin A, YFMRFamide and endomorphin-1 as modulators.

Acid-sensing ion channels (ASICs) belong to the DEG/ENaC gene family. While ASIC1a, ASIC1b and ASIC3 are activated by extracellular protons, ASIC4 and the closely related bile acid-sensitive ion channel (BASIC or ASIC5) are orphan receptors. Neuropeptides are important modulators of ASICs. Moreover, related DEG/ENaCs are directly activated by neuropeptides, rendering neuropeptides interesting ligands of ASICs. Here, we performed an unbiased screen of 109 short neuropeptides (<20 amino acids) on five homomeric ASICs: ASIC1a, ASIC1b, ASIC3, ASIC4 and BASIC. This screen revealed no direct agonist of any ASIC but three modulators. First, dynorphin A as a modulator of ASIC1a, which increased currents of partially desensitized channels; second, YFMRFamide as a modulator of ASIC1b and ASIC3, which decreased currents of ASIC1b and slowed desensitization of ASIC1b and ASIC3; and, third, endomorphin-1 as a modulator of ASIC3, which also slowed desensitization. With the exception of YFMRFamide, which, however, is not a mammalian neuropeptide, we identified no new modulator of ASICs. In summary, our screen confirmed some known peptide modulators of ASICs but identified no new peptide ligands of ASICs, suggesting that most short peptides acting as ligands of ASICs are already known.