Acid-sensing ion channels: trafficking and pathophysiology

Acid-sensing ion channels (ASICs) are proton-gated cation channels that are widely expressed in both the peripheral and central nervous systems. ASICs contribute to a variety of pathophysiological conditions that involve tissue acidosis, such as ischemic stroke, epileptic seizures and multiple sclerosis. Although much progress has been made in researching the structure-function relationship and pharmacology of ASICs, little is known about the trafficking of ASICs and its contribution to ASIC function. The recent identification of the mechanism of membrane insertion and endocytosis of ASIC1a highlights the emerging role of ASIC trafficking in regulating its pathophysiological functions. In this review, we summarize the recent advances and discuss future directions on this topic.     

Selective regulation of acid-sensing ion channel 1 by serine proteases

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that belong to the epithelial Na(+) channel/degenerin family. ASICs are transiently activated by a rapid drop in extracellular pH. Conditions of low extracellular pH, such as ischemia and inflammation in which ASICs are thought to be active, are accompanied by increased protease activity. We show here that serine proteases modulate the function of ASIC1a and ASIC1b but not of ASIC2a and ASIC3. We show that protease exposure shifts the pH dependence of ASIC1a activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in current response if ASIC1a is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of approximately 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provide evidence that this bi-directional regulation of ASIC1a function also occurs in neurons. Thus, we have identified a mechanism that modulates ASIC function and may allow ASIC1a to adapt its gating to situations of persistent extracellular acidification.     

ASIC2 Subunits Facilitate Expression at the Cell Surface and Confer Regulation by PSD-95

Acid-sensing ion channels (ASICs) are Na⁺ channels activated by changes in pH within the peripheral and central nervous systems. Several different isoforms of ASICs combine to form trimeric channels, and their properties are determined by their subunit composition. ASIC2 subunits are widely expressed throughout the brain, where they heteromultimerize with their partnering subunit, ASIC1a. However, ASIC2 contributes little to the pH sensitivity of the channels, and so its function is not well understood. We found that ASIC2 increased cell surface levels of the channel when it is coexpressed with ASIC1a, and genetic deletion of ASIC2 reduced acid-evoked current amplitude in mouse hippocampal neurons. Additionally, ASIC2a interacted with the neuronal synaptic scaffolding protein PSD-95, and PSD-95 reduced cell surface expression and current amplitude in ASICs that contain ASIC2a. Overexpression of PSD-95 also reduced acid-evoked current amplitude in hippocampal neurons. This result was dependent upon ASIC2 since the effect of PSD-95 was abolished in ASIC2−/− neurons. These results lend support to an emerging role of ASIC2 in the targeting of ASICs to surface membranes, and allows for interaction with PSD-95 to regulate these processes.     

Expression and Activity of Acid-Sensing Ion Channels in the Mouse Anterior Pituitary

Acid sensing ion channels (ASICs) are proton-gated cation channels that are expressed in the nervous system and play an important role in fear learning and memory. The function of ASICs in the pituitary, an endocrine gland that contributes to emotions, is unknown. We sought to investigate which ASIC subunits were present in the pituitary and found mRNA expression for all ASIC isoforms, including ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4. We also observed acid-evoked ASIC-like currents in isolated anterior pituitary cells that were absent in mice lacking ASIC1a. The biophysical properties and the responses to PcTx1, amiloride, Ca²⁺ and Zn²⁺ suggested that ASIC currents were mediated predominantly by heteromultimeric channels that contained ASIC1a and ASIC2a or ASIC2b. ASIC currents were also sensitive to FMRFamide (Phe-Met-Arg-Phe amide), suggesting that FMRFamide-like compounds might endogenously regulate pituitary ASICs. To determine whether ASICs might regulate pituitary cell function, we applied low pH and found that it increased the intracellular Ca²⁺ concentration. These data suggest that ASIC channels are present and functionally active in anterior pituitary cells and may therefore influence their function.     

Neurosensory mechanotransduction through acid‐sensing ion channels

Acid‐sensing ion channels (ASICs) are voltage‐insensitive cation channels responding to extracellular acidification. ASIC proteins have two transmembrane domains and a large extracellular domain. The molecular topology of ASICs is similar to that of the mechanosensory abnormality 4‐ or 10‐proteins expressed in touch receptor neurons and involved in neurosensory mechanotransduction in nematodes. The ASIC proteins are involved in neurosensory mechanotransduction in mammals. The ASIC isoforms are expressed in Merkel cell–neurite complexes, periodontal Ruffini endings and specialized nerve terminals of skin and muscle spindles, so they might participate in mechanosensation. In knockout mouse models, lacking an ASIC isoform produces defects in neurosensory mechanotransduction of tissue such as skin, stomach, colon, aortic arch, venoatrial junction and cochlea. The ASICs are thus implicated in touch, pain, digestive function, baroreception, blood volume control and hearing. However, the role of ASICs in mechanotransduction is still controversial, because we lack evidence that the channels are mechanically sensitive when expressed in heterologous cells. Thus, ASIC channels alone are not sufficient to reconstruct the path of transducing molecules of mechanically activated channels. The mechanotransducers associated with ASICs need further elucidation. In this review, we discuss the expression of ASICs in sensory afferents of mechanoreceptors, findings of knockout studies, technical issues concerning studies of neurosensory mechanotransduction and possible missing links. Also we propose a molecular model and a new approach to disclose the molecular mechanism underlying the neurosensory mechanotransduction.     

Subtype-specific Modulation of Acid-sensing Ion Channel (ASIC) Function by 2-Guanidine-4-methylquinazoline

Acid-sensing ion channels (ASICs) are neuronal Na⁺-selective channels that are transiently activated by extracellular acidification. ASICs are involved in fear and anxiety, learning, neurodegeneration after ischemic stroke, and pain sensation. The small molecule 2-guanidine-4-methylquinazoline (GMQ) was recently shown to open ASIC3 at physiological pH. We have investigated the mechanisms underlying this effect and the possibility that GMQ may alter the function of other ASICs besides ASIC3. GMQ shifts the pH dependence of activation to more acidic pH in ASIC1a and ASIC1b, whereas in ASIC3 this shift goes in the opposite direction and is accompanied by a decrease in its steepness. GMQ also induces an acidic shift of the pH dependence of inactivation of ASIC1a, -1b, -2a, and -3. As a consequence, the activation and inactivation curves of ASIC3 but not other ASICs overlap in the presence of GMQ at pH 7.4, thereby creating a window current. At concentrations >1 mm, GMQ decreases maximal peak currents by reducing the unitary current amplitude. Mutation of residue Glu-79 in the palm domain of ASIC3, previously shown to be critical for channel opening by GMQ, disrupted the GMQ effects on inactivation but not activation. This suggests that this residue is involved in the consequences of GMQ binding rather than in the binding interaction itself. This study describes the mechanisms underlying the effects of a novel class of ligands that modulate the function of all ASICs as well as activate ASIC3 at physiological pH.     

Complex action of tyramine,tryptamine and histamine on native and recombinant ASICs

Proton-gated channels of the ASIC family are widely distributed in the mammalian brain, and, according to the recent data, participate in synaptic transmission. However, ASIC-mediated currents are small, and special efforts are required to detect them. This prompts the search for endogenous ASIC ligands, which can activate or potentiate these channels. A recent finding of the potentiating action of histamine on recombinant homomeric ASIC1a has directed attention to amine-containing compounds. In the present study, we have analyzed the action of histamine, tyramine, and tryptamine on native and recombinant ASICs. None of the compounds caused potentiation of native ASICs in hippocampal interneurons. Furthermore, when applied simultaneously with channel activation, they produced voltage-dependent inhibition. Experiments on recombinant ASIC1a and ASIC2a allowed for an interpretation of these findings. Histamine and tyramine were found to be inactive on the ASIC2a, while tryptamine demonstrated weak inhibition. However, they induce both voltage-dependent inhibition of open channels and voltage-independent potentiation of closed/desensitized channels on the ASIC1a. We suggest that the presence of an ASIC2a subunit in heteromeric native ASICs prevents potentiation but not inhibition. As a result, the inhibitory action of histamine, which is masked by a strong potentiating effect on the ASIC1a homomers, becomes pronounced in experiments with native ASICs.     

Interaction of acid-sensing ion channel (ASIC) 1 with the tarantula toxin psalmotoxin 1 is state dependent

Acid-sensing ion channels (ASICs) are Na(+) channels gated by extracellular H(+). Six ASIC subunits that are expressed in neurons have been characterized. The tarantula toxin psalmotoxin 1 has been reported to potently and specifically inhibit homomeric ASIC1a and has been useful to characterize ASICs in neurons. Recently we have shown that psalmotoxin 1 inhibits ASIC1a by increasing its apparent affinity for H(+). However, the mechanism by which PcTx1 increases the apparent H(+) affinity remained unclear. Here we show that PcTx1 also interacts with ASIC1b, a splice variant of ASIC1a. However, PcTx1 does not inhibit ASIC1b but promotes its opening; under slightly acidic conditions, PcTx1 behaves like an agonist for ASIC1b. Our results are most easily explained by binding of PcTx1 with different affinities to different states (closed, open, and desensitized) of the channel. For ASIC1b, PcTx1 binds most tightly to the open state, promoting opening, whereas for ASIC1a, it binds most tightly to the open and the desensitized state, promoting desensitization.     

Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.     

Stomatin modulates gating of acid-sensing ion channels

Acid-sensing ion channels (ASICs) are H(+)-gated members of the degenerin/epithelial Na(+) channel (DEG/ENaC) family in vertebrate neurons. Several ASICs are expressed in sensory neurons, where they play a role in responses to nociceptive, taste, and mechanical stimuli; others are expressed in central neurons, where they participate in synaptic plasticity and some forms of learning. Stomatin is an integral membrane protein found in lipid/protein-rich microdomains, and it is believed to regulate the function of ion channels and transporters. In Caenorhabditis elegans, stomatin homologs interact with DEG/ENaC channels, which together are necessary for normal mechanosensation in the worm. Therefore, we asked whether stomatin interacts with and modulates the function of ASICs. We found that stomatin co-immunoprecipitated and co-localized with ASIC proteins in heterologous cells. Moreover, stomatin altered the function of ASIC channels. Stomatin potently reduced acid-evoked currents generated by ASIC3 without changing steady state protein levels or the amount of ASIC3 expressed at the cell surface. In contrast, stomatin accelerated the desensitization rate of ASIC2 and heteromeric ASICs, whereas current amplitude was unaffected. These data suggest that stomatin binds to and alters the gating of ASICs. Our findings indicate that modulation of DEG/ENaC channels by stomatin-like proteins is evolutionarily conserved and may have important implications for mammalian nociception and mechanosensation.     

Nonproton ligand sensing domain is required for paradoxical stimulation of acid-sensing ion channel 3 (ASIC3) channels by amiloride

Acid-sensing ion channels (ASICs), which belong to the epithelial sodium channel/degenerin family, are activated by extracellular protons and are inhibited by amiloride (AMI), an important pharmacological tool for studying all known members of epithelial sodium channel/degenerin. In this study, we reported that AMI paradoxically opened homomeric ASIC3 and heteromeric ASIC3 plus ASIC1b channels at neutral pH and synergistically enhanced channel activation induced by mild acidosis (pH 7.2 to 6.8). The characteristic profile of AMI stimulation of ASIC3 channels was reminiscent of the channel activation by the newly identified nonproton ligand, 2-guanidine-4-methylquinazoline. Using site-directed mutagenesis, we showed that ASIC3 activation by AMI, but not its inhibitory effect, was dependent on the integrity of the nonproton ligand sensing domain in ASIC3 channels. Moreover, the structure-activity relationship study demonstrated the differential requirement of the 5-amino group in AMI for the stimulation or inhibition effect, strengthening the different interactions within ASIC3 channels that confer the paradoxical actions of AMI. Furthermore, using covalent modification analyses, we provided strong evidence supporting the nonproton ligand sensing domain is required for the stimulation of ASIC3 channels by AMI. Finally, we showed that AMI causes pain-related behaviors in an ASIC3-dependent manner. These data reinforce the idea that ASICs can sense nonproton ligands in addition to protons. The results also indicate caution in the use of AMI for studying ASIC physiology and in the development of AMI-derived ASIC inhibitors for treating pain syndromes.     

Acidosis,Acid-Sensing Ion Channels,and Neuronal Cell Death

Acidosis is a common feature of many neuronal diseases and often accompanied with adverse consequences such as pain and neuronal injury. Before the discovery of acid-sensing ion channels (ASICs), protons were usually considered as a modulator of other ion channels, such as voltage-gated calcium channels, N-methyl-d-aspartate, and γ-amino butyric acid(A) receptor channels. Accordingly, the functional effects of acidosis were considered as consequences of modulations of these channels. Since the first cloning of ASICs in 1997, the conventional view on acidosis-mediated pain and cell injury has been dramatically changed. To date, ASICs, which are directly activated by extracellular protons, are shown to mediate most of the acidosis-associated physiological and pathological functions. For example, ASIC1a channels are reported to mediate acidosis-induced ischemic neuronal death. In this article, we will review the possible mechanisms that underlie ASIC1a channel-mediated neuronal death and discuss ASIC1a channel modulators involved in this process.     

Acid-sensing ion channels in mouse olfactory bulb M/T neurons

The olfactory bulb contains the first synaptic relay in the olfactory pathway, the sensory system in which odorants are detected enabling these chemical stimuli to be transformed into electrical signals and, ultimately, the perception of odor. Acid-sensing ion channels (ASICs), a family of proton-gated cation channels, are widely expressed in neurons of the central nervous system. However, no direct electrophysiological and pharmacological characterizations of ASICs in olfactory bulb neurons have been described. Using a combination of whole-cell patch-clamp recordings and biochemical and molecular biological analyses, we demonstrated that functional ASICs exist in mouse olfactory bulb mitral/tufted (M/T) neurons and mainly consist of homomeric ASIC1a and heteromeric ASIC1a/2a channels. ASIC activation depolarized cultured M/T neurons and increased their intracellular calcium concentration. Thus, ASIC activation may play an important role in normal olfactory function.     

Acid sensing ion channels regulate neuronal excitability by inhibiting BK potassium channels

Acid sensing ion channels (ASICs), Ca²⁺ and voltage-activated potassium channels (BK) are widely present throughout the central nervous system. Previous studies have shown that when expressed together in heterologous cells, ASICs inhibit BK channels, and this inhibition is relieved by acidic extracellular pH. We hypothesized that ASIC and BK channels might interact in neurons, and that ASICs may regulate BK channel activity. We found that ASICs inhibited BK currents in cultured wild-type cortical neurons, but not in ASIC1a/2/3 triple knockout neurons. The inhibition in the wild-type was partially relieved by a drop in extracellular pH to 6. To test the consequences of ASIC-BK interaction for neuronal excitability, we compared action potential firing in cultured cortical neurons from wild-type and ASIC1a/2/3 null mice. We found that in the knockout, action potentials were narrow and exhibited increased after-hyperpolarization. Moreover, the excitability of these neurons was significantly increased. These findings are consistent with increased BK channel activity in the neurons from ASIC1a/2/3 null mice. Our data suggest that ASICs can act as endogenous pH-dependent inhibitors of BK channels, and thereby can reduce neuronal excitability.     

The tarantula toxin psalmotoxin 1 inhibits acid-sensing ion channel (ASIC) 1a by increasing its apparent H+ affinity

Acid-sensing ion channels (ASICs) are ion channels activated by extracellular protons. They are involved in higher brain functions and perception of pain, taste, and mechanical stimuli. Homomeric ASIC1a is potently inhibited by the tarantula toxin psalmotoxin 1. The mechanism of this inhibition is unknown. Here we show that psalmotoxin 1 inhibits ASIC1a by a unique mechanism: the toxin increases the apparent affinity for H(+) of ASIC1a. Since ASIC1a is activated by H(+) concentrations that are only slightly larger than the resting H(+) concentration, this increase in H(+) affinity is sufficient to shift ASIC1a channels into the desensitized state. As activation of ASIC1a has recently been linked to neurodegeneration associated with stroke, our results suggest chronic desensitization of ASIC1a by a slight increase of its H(+) affinity as a possible way of therapeutic intervention in stroke.     

Zebrafish acid-sensing ion channel (ASIC) 4, characterization of homo- and heteromeric channels, and identification of regions important for activation by H+

There are four genes for acid-sensing ion channels (ASICs) in the genome of mammalian species. Whereas ASIC1 to ASIC3 form functional H+-gated Na+ channels, ASIC4 is not gated by H+, and its function is unknown. Zebrafish has two ASIC4 paralogs: zASIC4.1 and zASIC4.2. Whereas zASIC4.1 is gated by extracellular H+, zASIC4.2 is not. This differential response to H+ makes zASIC4 paralogs a good model to study the properties of this ion channel. In this study, we found that surface expression of homomeric zASIC4.2 is higher than that of zASIC4.1. Surface expression of zASIC4.1 was much increased by formation of heteromeric channels, suggesting that zASIC4.1 contributes to heteromeric ASICs in zebrafish neurons. Robust surface expression of H+-insensitive zASIC4.2 suggests that zASIC4.2 functions as a homomer and is gated by an as yet unknown stimulus, different from H+. Moreover, we identified a small region just distal to the first transmembrane domain that is crucial for the differential H+ response of the two paralogs. This post-TM1 domain may have a general role in gating of members of this gene family.     

Protein kinase C stimulates the acid-sensing ion channel ASIC2a via the PDZ domain-containing protein PICK1

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. They are expressed in central and sensory neurons where they are involved in neuromodulation and in pain perception. Recently, the PDZ domain-containing protein PICK1 (protein interacting with C-kinase) has been shown to interact with ASIC1a and ASIC2a, raising the possibility that protein kinase C (PKC) could regulate ASICs. We now show that the amplitude of the ASIC2a current, which was only modestly increased ( approximately +30%) by the PKC activator 1-oleyl-2-acetyl-sn-glycerol (OAG, 50 microm) in the absence of PICK1, was strongly potentiated ( approximately +300%) in the presence of PICK1. This PICK1-dependent regulatory effect was inhibited in the presence of a PKC inhibitory peptide and required the PDZ domain of PICK1 as well as the PDZ-binding domain of ASIC2a. We have also shown the direct PICK1-dependent phosphorylation of ASIC2a by [(32)P]phosphate labeling and immunoprecipitation and identified a major phosphorylation site, (39)TIR, on the N terminus part of ASIC2a. The OAG-induced increase in ASIC2a current amplitude did not involve any change in the unitary conductance of the ASIC2a channel, whether co-expressed with PICK1 or not. These data provide the first demonstration of a regulation of ASICs by protein kinase phosphorylation and its potentiation by the partner protein PICK1.     

Inhibition of Neuronal Degenerin/Epithelial Na+ Channels by the Multiple Sclerosis Drug 4-Aminopyridine

The voltage-gated K⁺ (Kv) channel blocker 4-aminopyridine (4-AP) is used to target symptoms of the neuroinflammatory disease multiple sclerosis (MS). By blocking Kv channels, 4-AP facilitates action potential conduction and neurotransmitter release in presynaptic neurons, lessening the effects of demyelination. Because they conduct inward Na⁺ and Ca²⁺ currents that contribute to axonal degeneration in response to inflammatory conditions, acid-sensing ion channels (ASICs) contribute to the pathology of MS. Consequently, ASICs are emerging as disease-modifying targets in MS. Surprisingly, as first demonstrated here, 4-AP inhibits neuronal degenerin/epithelial Na⁺ (Deg/ENaC) channels, including ASIC and BLINaC. This effect is specific for 4-AP compared with its heterocyclic base, pyridine, and the related derivative, 4-methylpyridine; and akin to the actions of 4-AP on the structurally unrelated Kv channels, dose- and voltage-dependent. 4-AP has differential actions on distinct ASICs, strongly inhibiting ASIC1a channels expressed in central neurons but being without effect on ASIC3, which is enriched in peripheral sensory neurons. The voltage dependence of the 4-AP block and the single binding site for this inhibitor are consistent with 4-AP binding in the pore of Deg/ENaC channels as it does Kv channels, suggesting a similar mechanism of inhibition in these two classes of channels. These findings argue that effects on both Kv and Deg/ENaC channels should be considered when evaluating the actions of 4-AP. Importantly, the current results are consistent with 4-AP influencing the symptoms of MS as well as the course of the disease because of inhibitory actions on Kv and ASIC channels, respectively.     

Proton-binding sites of acid-sensing ion channel 1

Acid-sensing ion channels (ASICs) are proton-gated cation channels that exist throughout the mammalian central and peripheral nervous systems. ASIC1 is the most abundant of all the ASICs and is likely to modulate synaptic transmission. Identifying the proton-binding sites of ASCI1 is required to elucidate its pH-sensing mechanism. By using the crystal structure of ASIC1, the protonation states of each titratable site of ASIC1 were calculated by solving the Poisson-Boltzmann equation under conditions wherein the protonation states of all these sites are simultaneously in equilibrium. Four acidic-acidic residue pairs--Asp238-Asp350, Glu220-Asp408, Glu239-Asp346, and Glu80-Glu417--were found to be highly protonated. In particular, the Glu80-Glu417 pair in the inner pore was completely protonated and possessed 2 H(+), implying its possible importance as a proton-binding site. The pK(a) of Glu239, which forms a pair with a possible pH-sensing site Asp346, differs among each homo-trimer subunit due to the different H-bond pattern of Thr237 in the different protein conformations of the subunits. His74 possessed a pK(a) of ≈6-7. Conservation of His74 in the proton-sensitive ASIC3 that lacks a residue corresponding to Asp346 may suggest its possible pH-sensing role in proton-sensitive ASICs.