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.

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.

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.

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.

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.

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.

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.

Identification of a cono-RFamide from the venom of Conus textile that targets ASIC3 and enhances muscle pain.

Acid-sensing ion channels (ASICs) are proton-gated Na+ channels that are expressed throughout the nervous system. ASICs have been implicated in several neuronal disorders, like ischemic stroke, neuronal inflammation, and pathological pain. Several toxins from venomous animals have been identified that target ASICs with high specificity and potency. These toxins are extremely useful in providing protein pharmacophores and to characterize function and structure of ASICs. Marine cone snails contain a high diversity of toxins in their venom such as conotoxins, which are short polypeptides stabilized by disulfide bonds, and conopeptides, which have no or only one disulfide bond. Whereas conotoxins selectively target specific neuronal proteins, mainly ion channels, the targets of conopeptides are less well known. Here, we perform an in vitro screen of venoms from 18 cone snail species to identify toxins targeting ASICs. We identified a small conopeptide of only four amino acids from the venom of Conus textile that strongly potentiated currents of ASIC3, which has a specific role in the pain pathway. This peptide, RPRFamide, belongs to the subgroup of cono-RFamides. Electrophysiological characterization of isolated dorsal root ganglion (DRG) neurons revealed that RPRFamide increases their excitability. Moreover, injection of the peptide into the gastrocnemius muscle strongly enhanced acid-induced muscle pain in mice that was abolished by genetic inactivation of ASIC3. In summary, we identified a conopeptide that targets the nociceptor-specific ion channel ASIC3.

Dual signaling of Wamide myoinhibitory peptides through a peptide-gated channel and a GPCR in Platynereis.

Neuropeptides commonly signal by metabotropic GPCRs. In some mollusks and cnidarians, RFamide neuropeptides mediate fast ionotropic signaling by peptide-gated ion channels that belong to the DEG/ENaC family. Here we describe a neuropeptide system with a dual mode of signaling by both a peptide-gated ion channel and a GPCR. We identified and characterized a peptide-gated channel in the marine annelid Platynereis dumerilii that is specifically activated by Wamide myoinhibitory peptides derived from the same proneuropeptide. The myoinhibitory peptide-gated ion channel (MGIC) belongs to the DEG/ENaC family and is paralogous to RFamide-gated ion channels. Platynereis myoinhibitory peptides also activate a previously described GPCR, MAG. We measured the potency of all Wamides on both MGIC and MAG and identified peptides that preferentially activate one or the other receptor. Analysis of a single-cell transcriptome resource indicates that MGIC and MAG signal in distinct target neurons. The identification of a Wamide-gated ion channel suggests that peptide-gated channels are more diverse and widespread in animals than previously appreciated. The possibility of neuropeptide signaling by both ionotropic and metabotropic receptors to different target cells in the same organism highlights an additional level of complexity in peptidergic signaling networks.-Schmidt, A., Bauknecht, P., Williams, E. A., Augustinowski, K., Gründer, S., Jékely, G. Dual signaling of Wamide myoinhibitory peptides through a peptide-gated channel and a GPCR in Platynereis.

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.

Modulation of DEG/ENaCs by Amphiphiles Suggests Sensitivity to Membrane Alterations.

The bile acid-sensitive ion channel is activated by amphiphilic substances such as bile acids or artificial detergents via membrane alterations; however, the mechanism of membrane sensitivity of the bile acid-sensitive ion channel is not known. It has also not been systematically investigated whether other members of the degenerin/epithelial Na+ channel (DEG/ENaC) gene family are affected by amphiphilic compounds. Here, we show that DEG/ENaCs ASIC1a, ASIC3, ENaC, and the purinergic receptor P2X2 are modulated by a large number of different, structurally unrelated amphiphilic substances, namely the detergents N-lauroylsarcosine, Triton X-100, and ß-octylglucoside; the fenamate flufenamic acid; the antipsychotic drug chlorpromazine; the natural phenol resveratrol; the chili pepper compound capsaicin; the loop diuretic furosemide; and the antiarrythmic agent verapamil. We determined the modification of membrane properties using large-angle x-ray diffraction experiments on model lipid bilayers, revealing that the amphiphilic compounds are positioned in a characteristic fashion either in the lipid tail group region or in the lipid head group region, demonstrating that they perturbed the membrane structure. Collectively, our results show that DEG/ENaCs and structurally related P2X receptors are modulated by diverse amphiphilic molecules. Furthermore, they suggest alterations of membrane properties by amphiphilic compounds as a mechanism contributing to modulation.

The Conorfamide RPRFa Stabilizes the Open Conformation of Acid-Sensing Ion Channel 3 via the Nonproton Ligand-Sensing Domain.

Acid-sensing ion channel 3 (ASIC3) is a proton-gated Na+ channel with important roles in pain. ASIC3 quickly desensitizes in less than a second, limiting its capacity to sense sustained acidosis during pain. RFamide neuropeptides are modulators of ASIC3 that slow its desensitization and induce a variable sustained current. The molecular mechanism of slowed desensitization and the RFamide binding site on ASIC3 are unknown. RPRFamide, a RFamide from the venom of a cone snail, has a comparatively high affinity for ASIC3 and strongly slows its desensitization. Here we show that covalent binding of a UV-sensitive RPRFamide variant to ASIC3 prevents desensitization, suggesting that RPRFamide has to unbind from ASIC3 before it can desensitize. Moreover, we show by in silico docking to a homology model of ASIC3 that a cavity in the lower palm domain, which is also known as the nonproton ligand-sensing domain, is a potential binding site of RPRFamide. Finally, using extensive mutagenesis of residues lining the nonproton ligand-sensing domain, we confirm that this domain is essential for RPRFamide modulation of ASIC3. As comparative analysis of ASIC crystal structures in the open and in the desensitized conformation suggests that the lower palm domain contracts during desensitization, our results collectively suggest that RPRFamide, and probably also other RFamide neuropeptides, bind to the nonproton ligand-sensing domain to stabilize the open conformation of ASIC3.

New techniques, applications and perspectives in neuropeptide research.

Neuropeptides are one of the most diverse classes of signaling molecules and have attracted great interest over the years owing to their roles in regulation of a wide range of physiological processes. However, there are unique challenges associated with neuropeptide studies stemming from the highly variable molecular sizes of the peptides, low in vivo concentrations, high degree of structural diversity and large number of isoforms. As a result, much effort has been focused on developing new techniques for studying neuropeptides, as well as novel applications directed towards learning more about these endogenous peptides. The areas of importance for neuropeptide studies include structure, localization within tissues, interaction with their receptors, including ion channels, and physiological function. Here, we discuss these aspects and the associated techniques, focusing on technologies that have demonstrated potential in advancing the field in recent years. Most identification and structural information has been gained by mass spectrometry, either alone or with confirmations from other techniques, such as nuclear magnetic resonance spectroscopy and other spectroscopic tools. While mass spectrometry and bioinformatic tools have proven to be the most powerful for large-scale analyses, they still rely heavily on complementary methods for confirmation. Localization within tissues, for example, can be probed by mass spectrometry imaging, immunohistochemistry and radioimmunoassays. Functional information has been gained primarily from behavioral studies coupled with tissue-specific assays, electrophysiology, mass spectrometry and optogenetic tools. Concerning the receptors for neuropeptides, the discovery of ion channels that are directly gated by neuropeptides opens up the possibility of developing a new generation of tools for neuroscience, which could be used to monitor neuropeptide release or to specifically change the membrane potential of neurons. It is expected that future neuropeptide research will involve the integration of complementary bioanalytical technologies and functional assays.

Diminazene Is a Slow Pore Blocker of Acid-Sensing Ion Channel 1a (ASIC1a).

Acid-sensing ion channels (ASICs) are neuronal receptors for extracellular protons. They contribute to the excitatory postsynaptic current and to the detection of painful acidosis. Moreover, they are activated during peripheral inflammation and acidosis associated with various neuronal disorders, such as stroke and neuroinflammation, rendering them interesting drug targets. Diminazene aceturate is a small-molecule inhibitor of ASICs with a reported apparent affinity in the low micromolar range, making it an interesting lead compound. It was reported that diminazene accelerates desensitization of ASICs, which was, however, not explained mechanistically. Furthermore, a binding site in a groove of the extracellular domain was proposed but not experimentally verified. In this study, we revisited the mechanism of inhibition by diminazene and its binding site on ASIC1a, the ASIC subunit with the greatest importance in the central nervous system. We show that diminazene slowly blocks ASIC1a, leading to the apparent acceleration of desensitization and underestimating its potency; we show that diminazene indeed has a submicromolar potency at ASIC1a (IC50 0.3 µM). Moreover, we show that the inhibition is voltage-dependent and competes with that by amiloride, a pore blocker of ASICs. Finally, we identify by molecular docking a binding site in the ion pore that we confirm by site-directed mutagenesis. In summary, our results show that diminazene blocks ASIC1a by a slow open-channel block and suggest that diminazene is an interesting lead compound for high-affinity blockers of ASICs.

A Cytosolic Amphiphilic α-Helix Controls the Activity of the Bile Acid-sensitive Ion Channel (BASIC).

The bile acid-sensitive ion channel (BASIC) is a member of the degenerin/epithelial Na+ channel (Deg/ENaC) family of ion channels. It is mainly found in bile duct epithelial cells, the intestinal tract, and the cerebellum and is activated by alterations of its membrane environment. Bile acids, one class of putative physiological activators, exert their effect by changing membrane properties, leading to an opening of the channel. The physiological function of BASIC, however, is unknown. Deg/ENaC channels are characterized by a trimeric subunit composition. Each subunit is composed of two transmembrane segments, which are linked by a large extracellular domain. The termini of the channels protrude into the cytosol. Many Deg/ENaC channels contain regulatory domains and sequence motifs within their cytosolic domains. In this study, we show that BASIC contains an amphiphilic α-helical structure within its N-terminal domain. This α-helix binds to the cytosolic face of the plasma membrane and stabilizes a closed state. Truncation of this domain renders the channel hyperactive. Collectively, we identify a cytoplasmic domain, unique to BASIC, that controls channel activity via membrane interaction.

Acid-sensing ion channel (ASIC) 1a/2a heteromers have a flexible 2:1/1:2 stoichiometry.

Acid-sensing ion channels (ASICs) are widely expressed proton-gated Na(+) channels playing a role in tissue acidosis and pain. A trimeric composition of ASICs has been suggested by crystallization. Upon coexpression of ASIC1a and ASIC2a in Xenopus oocytes, we observed the formation of heteromers and their coexistence with homomers by electrophysiology, but could not determine whether heteromeric complexes have a fixed subunit stoichiometry or whether certain stoichiometries are preferred over others. We therefore imaged ASICs labeled with green and red fluorescent proteins on a single-molecule level, counted bleaching steps from GFP and colocalized them with red tandem tetrameric mCherry for many individual complexes. Combinatorial analysis suggests a model of random mixing of ASIC1a and ASIC2a subunits to yield both 2:1 and 1:2 ASIC1a:ASIC2a heteromers together with ASIC1a and ASIC2a homomers.

Peptide-gated ion channels and the simple nervous system of Hydra.

Neurons either use electrical or chemical synapses to communicate with each other. Transmitters at chemical synapses are either small molecules or neuropeptides. After binding to their receptors, transmitters elicit postsynaptic potentials, which can either be fast and transient or slow and longer lasting, depending on the type of receptor. Fast transient potentials are mediated by ionotropic receptors and slow long-lasting potentials by metabotropic receptors. Transmitters and receptors are well studied for animals with a complex nervous system such as vertebrates and insects, but much less is known for animals with a simple nervous system like Cnidaria. As cnidarians arose early in animal evolution, nervous systems might have first evolved within this group and the study of neurotransmission in cnidarians might reveal an ancient mechanism of neuronal communication. The simple nervous system of the cnidarian Hydra extensively uses neuropeptides and, recently, we cloned and functionally characterized an ion channel that is directly activated by neuropeptides of the Hydra nervous system. These results demonstrate the existence of peptide-gated ion channels in Hydra, suggesting they mediate fast transmission in its nervous system. As related channels are also present in the genomes of the cnidarian Nematostella, of placozoans and of ctenophores, it should be considered that the early nervous systems of cnidarians and ctenophores have co-opted neuropeptides for fast transmission at chemical synapses.

Pentafluorosulfanyl-containing flufenamic acid analogs: Syntheses, properties and biological activities.

Pentafluorosulfanyl-containing analogs of flufenamic acid have been synthesized in high yields. Computationally, pKa, LogP and LogD values have been determined. Initial bioactivity studies reveal effects as ion channel modulators and inhibitory activities on aldo-keto reductase 1C3 (AKR1C3) as well as COX-1 and COX-2.

The comprehensive analysis of DEG/ENaC subunits in Hydra reveals a large variety of peptide-gated channels, potentially involved in neuromuscular transmission.

BACKGROUND: It is generally the case that fast transmission at neural synapses is mediated by small molecule neurotransmitters. The simple nervous system of the cnidarian Hydra, however, contains a large repertoire of neuropeptides and it has been suggested that neuropeptides are the principal transmitters of Hydra. An ion channel directly gated by Hydra-RFamide neuropeptides has indeed been identified in Hydra - the Hydra Na+ channel (HyNaC) 2/3/5, which is expressed at the oral side of the tentacle base. Hydra-RFamides are more widely expressed, however, being found in neurons of the head and peduncle region. Here, we explore whether further peptide-gated HyNaCs exist, where in the animal they are expressed, and whether they are all gated by Hydra-RFamides. RESULTS: We report molecular cloning of seven new HyNaC subunits - HyNaC6 to HyNaC12, all of which are members of the DEG/ENaC gene family. In Xenopus oocytes, these subunits assemble together with the four already known subunits into thirteen different ion channels that are directly gated by Hydra-RFamide neuropeptides with high affinity (up to 40 nM). In situ hybridization suggests that HyNaCs are expressed in epitheliomuscular cells at the oral and the aboral side of the tentacle base and at the peduncle. Moreover, diminazene, an inhibitor of HyNaCs, delayed tentacle movement in live Hydra. CONCLUSIONS: Our results show that Hydra has a large variety of peptide-gated ion channels that are activated by a restricted number of related neuropeptides. The existence and expression pattern of these channels, and behavioral effects induced by channel blockers, suggests that Hydra co-opted neuropeptides for fast neuromuscular transmission.