Definition

Acid-sensing ion channels (ASICs) are non-voltage-gated sodium channels transiently activated by extracellular protons, selective for sodium, and belong to the epithelial sodium channel (ENaC)/degenerin (DEG) family of ion channels. The members of this ion channel superfamily share a similar topology with short intracellular amino and carboxy termini and two membrane-spanning domains connected by a large extracellular domain. The ASIC ion channel group consists of four genes encoding at least six ASIC subunits including 1a, 1b, 2a, 2b, 3, and 4.

Basic Characteristics

Channel family, structural organization and function

Channel Family

The ENaC/DEG superfamily of cation channels encompasses more than 60 members including the ASICs (Wemmie et al. 2006, 2013; Kellenberger and Schild 2015, 2002; Stockand et al. 2008). ASICs have been cloned in the mid-1990s based on sequence homology to ENaC and DEGs. Extracellular acidification opens ASICs only transiently, because of rapid desensitization, indicating that ASICs can exist in the three functional states closed, open, and desensitized (Waldmann et al. 1997; Carattino 2011; Kellenberger and Schild 2015) (Fig. 1c). ASICs are expressed in all vertebrates and responsible for acid-evoked currents in many neurons of the nervous system.

Fig. 1
figure 1

ASIC structure and function. (a) Structural model of a single ASIC subunit (left) and of a channel trimer (right), based on the crystal structure of chicken ASIC1a opened by mit-toxin (Baconguis et al. 2014). The subdomains are color-coded in one subunit and labeled. (b) Typical ASIC1a current trace, obtained with two-electrode voltage-clamp to −60 mV of a Xenopus oocytes expressing ASIC1a. The extracellular pH was changed as indicated on the top of the Fig. (c) Kinetic scheme of ASIC function. Upon a pH change from 7.4 to 5.0, the channels pass from the closed to the open and then into the desensitized state. (d) pH – current response curve of ASIC1a. The normalized current amplitude is plotted as a function of the pH

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Structural Organization

The ASIC group consists of four ASIC genes resulting in six isoforms in rodents, termed ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4 with a length of 500 to 560 amino acids (Kellenberger and Schild 2015). The members of ENaC/DEG superfamily share the same topology characterized by a large extracellular domain (~370 amino acids), short intracellular amino and carboxy termini (~35 to 90 amino acids), and two transmembrane regions (~20 amino acids). Individual ASIC subunits assemble to form homo- or heterotrimeric channels, except for ASIC2b and ASIC4. ASIC2b is not functional as homomeric channel, but forms functional heteromeric channels together with other ASIC subunits. So far, no role for ASIC4 in homo- or heteromeric channels has been observed. In 2007, Jasti et al. described the first crystal structure of one of the ASIC proteins, the chicken ASIC1a channel (Jasti et al. 2007) which shares 90% sequence identity with human ASIC1a. This structure showed that the channel is formed by three subunits. The shape of the single subunit was compared to that of a hand, and sub-domains were named accordingly (Fig. 1a). Subsequently, structures obtained from chicken ASIC1a representing likely the closed and the toxin-opened conformation were published (Baconguis et al. 2014; Yoder et al. 2018).

Basic Functional Properties

ASICs are transiently activated by a rapid drop in extracellular pH (Fig. 1b). Protons are the main physiological activators of ASICs. As mentioned above, ASICs exist in three different functional states termed closed, open, and desensitized (Fig. 1c). The transient peak current lasts hundreds of milliseconds and is terminated by desensitization. In ASIC3 and some heteromeric ASICs, the desensitization is not complete and allows a small sustained current. Return to physiological pH 7.4 brings the channel into the closed state, allowing subsequent activation by protons. The molecular details of the activation of ASICs are currently not completely understood. Several studies indicate that protonation events close to the thumb (the “acidic pocket”), in the palm, and in the extracellular pore entry contribute to pore opening (see Kellenberger and Schild 2015).

ASIC1a opens at pH 7.0 and reaches its half-maximal activation (pH50) at pH 6.2–6.6 (Fig. 1d). The pH50 values of other ASICs are 5.9–6.3 (ASIC1b), 4.0–4.9 (ASIC2a), and 6.4–6.7 (ASIC3) (Waldmann et al. 1999; Wemmie et al. 2013; Kellenberger and Schild 2015). Many endogenous and exogenous compounds were shown to modulate ASIC function (reviewed in Wemmie et al. 2013; Kellenberger and Schild 2015), as discussed below.

Physiological Roles

ASICs are expressed in all vertebrates; even organisms with rudimentary nervous system have been shown to express at least one of the ASICs. ASICs contribute to acid-evoked currents in many neurons of both the central and peripheral nervous systems (CNS and PNS). ASIC1a, ASIC2a, and ASIC2b are expressed throughout the CNS and PNS (Wemmie et al. 2013; Kellenberger and Schild 2015). In the CNS, ASIC1a, ASIC2a, and ASIC2b show their highest expression in the hippocampus, the cortex, the cerebellum, the olfactory bulb, and the amygdala. ASIC1b and ASIC3 expression is restricted to the PNS (Wemmie et al. 2013). ASIC3 is most abundant in dorsal root ganglia. So far, ASIC4 has not been found in the PNS and shows a more dispersed expression compared to other ASICs in the CNS (rev. in Kellenberger and Schild 2015; Wemmie et al. 2013).

Local pH changes affect the function of almost any protein, including ion channels, and influence therefore many cellular processes. The release of the content of the synaptic vesicles that are acidic leads to a rapid lowering of the pH in the synaptic cleft. Tissue acidification occurs in situations such as inflammation or ischemia (rev. in Boscardin et al. 2016).

The activation of ASICs allows Na+ entry into neurons, inducing depolarization of the neuronal membrane, generating action potentials, and, thus, exciting the neurons. The physiological and pathological roles of ASICs, demonstrated with mouse models deficient of specific ASIC isoforms or with pharmacological approaches using small molecule inhibitors or ASIC-specific toxins, include fear conditioning and anxiety, retinal function, neurodegeneration after ischemic stroke, synaptic plasticity, learning, and memory (Wemmie et al. 2013; Kellenberger and Schild 2015). In addition, studies with mice and rats showed evidence for a role of ASICs in the PNS (mostly ASIC3) and of ASICs of the CNS (mostly ASIC1a) in the sensation of different forms of pain. ASICs also have been proposed to play a role in mechanosensation. While such a role has been demonstrated on the level of the animal, it has so far not been possible to directly show activation of ASICs by mechanical stimuli in cell systems, suggesting that ASICs are part of a mechanotransduction complex that may not be intact in dissociated cells (rev. in Wemmie et al. 2013 and Kellenberger and Schild 2015).

ASIC Regulators

Ions and Polyamines

The activity of ASICs can be modulated by both divalent and trivalent metal ions, including Ni2+, Cu2+, Ca2+, and Zn2+ ions (rev. in Wemmie et al. 2013; Kellenberger and Schild 2015). Ca2+ functions as allosteric modulator of ASIC pH dependence, likely by competing with protons for binding sites, and as a channel blocker (Paukert et al. 2004; Babini et al. 2002).

Depending on the ASIC type of channel, Zn2+ can potentiate or inhibit the channel. Zn2+ has been reported to potentiate proton-induced currents mediated by homomeric ASIC2a and heteromeric ASIC2a-containing channels at micromolar concentrations. In contrast, ASIC1a, ASIC1b, and ASIC3 are inhibited by Zn2+; nanomolar concentrations inhibit homomeric ASIC1a channels, whereas ASIC1b and ASIC3 are only inhibited by micromolar concentrations of Zn2+ (rev. in Wemmie et al. 2013; Kellenberger and Schild 2015; Baron and Lingueglia 2015).

The endogenous polyamine spermine modulates proton-evoked ASIC1- and ASIC1a/ASIC2a-mediated currents (Babini et al. 2002; Baron and Lingueglia 2015).

Neuropeptides

FANaC, a member of the ENaC/DEG ion channel family, is activated by the neuropeptide FMRFamide (Phe-Met-Arg-Phe amide). FMRFamide has also been shown to modulate the function of ASIC1 and ASIC3 by potentiating their responses to acidification. The action on ASICs of FMRFamide and related neuropeptides involves amino acid residues in the palm and a linker between the palm and the thumb (Vick and Askwith 2015; Bargeton et al. 2019). FMRFamide and related neuropeptides increase ASIC activity in several ways, including an acidic shift of the pH dependence of desensitization, a slowing of the current decay, and the generation of a sustained current (Fig. 2).

Fig. 2
figure 2

Sites of drug action. The figure shows the sites of action of toxins and small molecules that inhibit or modulate ASICs. Green lines mark positive modulation, while red lines indicate inhibition. Note that for mit-toxin, the interaction region with ASIC is larger than indicated and stretches over the whole thumb and up to the finger

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Proteases

Channel regulation by different types of proteases (mainly serine proteases) is a topic that has been intensively described for the ENaC. There is also evidence for proteolytic regulation of ASICs. The serine proteases trypsin and matriptase led to an acidic shift of the pH dependence of the peak current of ASIC1a, but not ASIC1b. Furthermore, proteolytic cleavage of ASIC1a, but not ASIC2a, by tissue kallikrein was reported (Su et al. 2011). The relevant protease cleavage sites involved in this process are located in the finger domain of ASIC1a for trypsin and matriptase (Vukicevic et al. 2006; Clark et al. 2010), similar to the cleavage sites in α and γENaC. This points to a regulatory role of the finger domain for ASIC activity.

Protein Kinases

The activation of the tropomyosin-related kinase B (TrkB) has been shown to increase ASIC1a activity and its surface expression via the phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB or Akt) pathway. Moreover, the regulation of ASIC function by other protein kinases has been described in many studies (discussed in Wemmie et al. 2013; Kellenberger and Schild 2015).

Amphiphilic Substances

Bile acids have been shown to activate two members of the ENaC/DEG family of ion channels, the bile acid-sensitive ion channel (BASIC) and ENaC. It is conceivable that bile acids may play a role in ASIC regulation under physiological and pathophysiological conditions as increased ASIC activity in sensory neurons of the gastrointestinal tract may contribute to hyperalgesia and colonic hypersensitivity observed in patients with irritable bowel syndrome with increased bile acid concentrations in their gut. Indeed, it was reported that the function of ASIC1a heterologously expressed in Xenopus laevis oocytes is modulated by bile acids and also other amphiphilic substances like the detergent maltoside (Ilyaskin et al. 2017). In the presence of these bile acids, the whole-cell currents elicited by acidic pH were significantly increased. Molecular docking predicted binding of bile acids to the pore region near the DEG site (G433) in the open conformation of the channel.

Lipids

ASIC function can also be regulated by lipids, e.g., arachidonic acid (AA) (Kellenberger and Schild 2015). It has been proposed that AA acts directly on ASICs to increase channel activity. Recently it has been shown that AA together with the endogenous lipid lysophosphatidylcholine can activate ASIC3 in the absence of any extracellular acidification (Marra et al. 2016).

GMQ and Related Compounds

The synthetic compound 2-guanidine-4-methylquinazoline (GMQ) is an exogenous modulator of ASICs. GMQ has been described as first non-proton activator of ASICs that depends on extracellular Ca2+ and exclusively activates ASIC3 (Yu et al. 2010). Subsequent studies showed that GMQ affects the pH dependence of not only ASIC3 but also ASIC1a, ASIC1b, and ASIC2a, leading to activation of ASIC3, and a decrease of the H+-activated current amplitude of other ASICs (Alijevic and Kellenberger 2012). Based on their structural similarity with GMQ, two natural polyamines, the arginine metabolite agmatine and its analog arcaine, have been proposed to be endogenous non-proton ligands for ASIC3. These two compounds lead to ASIC3 activation similar to GMQ however to a smaller extent. Agmatine also activated heteromeric ASIC3/ASIC1b channels, extending its potential physiological relevance (rev. in Kellenberger and Schild 2015; Baron and Lingueglia 2015).

Drugs

Toxins

Several peptide toxins have been identified that bind to ASICs with nano- or micromolar affinity and act as gating modifiers, inhibitors, or activators. The most important known ASIC toxins comprise psalmotoxin1 (PcTx1) from the venom of the spider Psalmopoeus cambridgei, Hi1a of the Australian funnel-web spider Hadronyche infensa, APETx2 of the sea anemone Anthopleura elegantissima, mambalgin of the black mamba (Dendroaspis polylepis polylepis), and mit-toxin of the Texas coral snake (Micrurus tener tener) (Baron and Lingueglia 2015; Chassagnon et al. 2017). These are peptide toxins composed of ≥40 amino acid residues. PcTx1 and Hi1a are structurally related, with Hi1a containing two tandem PcTx1-like sequences. From co-crystallization and mutagenesis studies, it is known that mit-toxin, PcTx1, and mambalgin bind to the thumb – acidic pocket region of ASIC1a (rev. in Kellenberger and Schild 2015; Baron and Lingueglia 2015). Mit-toxin activates ASIC1a, ASIC1b, and ASIC3 and potentiates ASIC2a opening by protons. PcTx1 inhibits mostly ASIC1a homomers, and mambalgin inhibits ASIC1a and ASIC1b homomers and ASIC1a-containing heteromers, while APETx2 inhibits ASIC3 homomers and ASIC3-containing heteromers. Except for APETx2, which inhibits besides ASICs also voltage-gated Na+ channels (Peigneur et al. 2012; Blanchard et al. 2012), these toxins appear to be specific for ASICs. PcTx1 and mambalgin exert their inhibition by changing the pH dependence of ASICs. PcTx1 shifts the pH dependence of desensitization to more alkaline values, leading to desensitization of ASIC1a at physiological pH and preventing channel opening by subsequent acidification. Mambalgin shifts the pH dependence of activation to more acidic values, thereby leading to a smaller response to acidification. In contrast, the effects of Hi1a and APETx2 appear to be pH-independent. The ASIC toxins have been instrumental in defining some functional roles of ASICs in animal studies (Wemmie et al. 2013).

Small Molecule Inhibitors

Currently there are no ASIC-selective, high affinity small molecule inhibitors available. Amiloride and its derivative benzamil are pore blockers of ENaC/DEG channels. While amiloride inhibits ENaC with an IC50 of ~100 nM, its IC50 of ASIC peak currents is ≥10 μM, and it does not inhibit sustained ASIC currents. At concentrations ≥10 μM, amiloride also inhibits several transporters (Wemmie et al. 2013; Kellenberger and Schild 2015). In spite of these limitations, amiloride has been used in experimental studies in humans. A-317567 inhibits the transient and sustained ASIC current fractions in dorsal root ganglion neurons with an IC50 of 2–10 μM and was shown to be more potent in treating pain than amiloride in animal models (rev. in Kellenberger and Schild 2015; Baron and Lingueglia 2015). Nafamostat mesylate is a synthetic protease inhibitor with potential use as anticoagulant or antitumor agent. It has been shown to inhibit ASIC currents, including the sustained current component of ASIC3, with IC50 values of 2–70 μM (rev. in Kellenberger and Schild 2015; Baron and Lingueglia 2015). The analysis of a number of anti-protozoal diarylamidines identified several compounds that inhibit ASICs with IC50 values of 0.3–40 μM. Among these, diminazene was further characterized in several studies and shown to inhibit ASICs in part by a pore block (Schmidt et al. 2017). Diminazene inhibits the related BASIC channel with similar affinity. Several nonsteroidal anti-inflammatory drugs (NSAIDs) were shown to inhibit ASICs, with IC50 values in the micromolar range, requiring thus much higher concentrations than clinically used. ASIC mRNA levels in sensory neurons are upregulated in inflammation. Interestingly, several NSAIDs, including aspirin, diclofenac, and ibuprofen, prevented this upregulation at therapeutic doses (Voilley et al. 2001). There have been several attempts in developing more specific and potent ASIC inhibitors, resulting in the identification of inhibitors with sub-micromolar affinities (reviewed in Kellenberger and Schild 2015; Baron and Lingueglia 2015; Rash 2017). However, so far, these compounds have not been further developed.