Protons at the gate: DEG/ENaC ion channels help us feel and remember

The DEG/ENaC ion channel family contributes to channels of striking functional diversity. Neuronally expressed family members include the C. elegans degenerins that mediate touch and are thought to be mechanically gated, and the mammalian ASICs, which are gated by protons. ASICs affect a range of sensory functions that includes perception of gentle touch, harsh touch, heat, sour taste, and pain. Family member ASIC1 is now implicated in long-term potentiation, suggesting that minute fluxes in synaptic pH may activate ASICs to enhance learning.     

Endogenous arginine-phenylalanine-amide-related peptides alter steady-state desensitization of ASIC1a

The acid-sensing ion channels (ASICs) are proton-gated, voltage-insensitive cation channels expressed throughout the nervous system. ASIC1a plays a role in learning, pain, and fear-related behaviors. In addition, activation of ASIC1a during prolonged acidosis following cerebral ischemia induces neuronal death. ASICs undergo steady-state desensitization, a characteristic that limits ASIC1a activity and may play a prominent role in the prevention of ASIC1a-evoked neuronal death. In this study, we found exogenous and endogenous arginine-phenylalanine-amide (RF-amide)-related peptides decreased the pH sensitivity of ASIC1a steady-state desensitization. During conditions that normally induced steady-state desensitization, these peptides profoundly enhanced ASIC1a activity. We also determined that human ASIC1a required more acidic pH to undergo steady-state desensitization compared with mouse ASIC1a. Surprisingly, steady-state desensitization of human ASIC1a was also affected by a greater number of peptides compared with mouse ASIC1a. Mutation of five amino acids in a region of the extracellular domain changed the characteristics of human ASIC1a to those of mouse ASIC1a, suggesting that this region plays a pivotal role in neuropeptide and pH sensitivity of steady-state desensitization. Overall, these experiments lend vital insight into steady-state desensitization of ASIC1a and expand our understanding of the structural determinants of RF-amide-related peptide modulation. Furthermore, our finding that endogenous peptides shift steady-state desensitization suggests that RF-amides could impact the role of ASIC1a in both pain and neuronal damage following stroke and ischemia.     

A nonproton ligand sensor in the acid-sensing ion channel

Acid-sensing ion channels (ASICs) have long been considered as extracellular proton (H(+))-gated cation channels, and peripheral ASIC3 channels seem to be a natural sensor of acidic pain. Here, we report the identification of a nonproton sensor on ASIC3. We show first that 2-guanidine-4-methylquinazoline (GMQ) causes persistent ASIC3 channel activation at the normal pH. Using GMQ as a probe and combining mutagenesis and covalent modification analysis, we then uncovered a ligand sensor lined by residues around E423 and E79 of the extracellular "palm" domain of the ASIC3 channel that is crucial for activation by nonproton activators. Furthermore, we show that GMQ activates sensory neurons and causes pain-related behaviors in an ASIC3-dependent manner, indicating the functional significance of ASIC activation by nonproton ligands. Thus, natural ligands beyond protons may activate ASICs under physiological and pathological conditions through the nonproton ligand sensor, serving for channel activation independent of abrupt and marked acidosis.     

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.     

Acid-Sensing Ion Channel 1a Contributes to Airway Hyperreactivity in Mice

Neurons innervating the airways contribute to airway hyperreactivity (AHR), a hallmark feature of asthma. Several observations suggested that acid-sensing ion channels (ASICs), neuronal cation channels activated by protons, might contribute to AHR. For example, ASICs are found in vagal sensory neurons that innervate airways, and asthmatic airways can become acidic. Moreover, airway acidification activates ASIC currents and depolarizes neurons innervating airways. We found ASIC1a protein in vagal ganglia neurons, but not airway epithelium or smooth muscle. We induced AHR by sensitizing mice to ovalbumin and found that ASIC1a^-/- mice failed to exhibit AHR despite a robust inflammatory response. Loss of ASIC1a also decreased bronchoalveolar lavage fluid levels of substance P, a sensory neuropeptide secreted from vagal sensory neurons that contributes to AHR. These findings suggest that ASIC1a is an important mediator of AHR and raise the possibility that inhibiting ASIC channels might be beneficial in asthma.     

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.     

Ca2+-Permeable Acid-sensing Ion Channels and Ischemic Brain Injury

Acidosis is a common feature of brain in acute neurological injury, particularly in ischemia where low pH has been assumed to play an important role in the pathological process. However, the cellular and molecular mechanisms underlying acidosis-induced injury remain unclear. Recent studies have demonstrated that activation of Ca²⁺-permeable acid-sensing ion channels (ASIC1a) is largely responsible for acidosis-mediated, glutamate receptor-independent, neuronal injury. In cultured mouse cortical neurons, lowering extracellular pH to the level commonly seen in ischemic brain activates amiloride-sensitive ASIC currents. In the majority of these neurons, ASICs are permeable to Ca²⁺, and an activation of these channels induces increases in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i). Activation of ASICs with resultant [Ca²⁺]_i loading induces time-dependent neuronal injury occurring in the presence of the blockers for voltage-gated Ca²⁺ channels and the glutamate receptors. This acid-induced injury is, however, inhibited by the blockers of ASICs, and by reducing [Ca²⁺]_o. In focal ischemia, intracerebroventricular administration of ASIC1a blockers, or knockout of the ASIC1a gene protects brain from injury and does so more potently than glutamate antagonism. Furthermore, pharmacological blockade of ASICs has up to a 5 h therapeutic time window, far beyond that of glutamate antagonists. Thus, targeting the Ca²⁺-permeable acid-sensing ion channels may prove to be a novel neuroprotective strategy for stroke patients.     

Protons and Psalmotoxin-1 reveal nonproton ligand stimulatory sites in chicken acid-sensing ion channel: Implication for simultaneous modulation in ASICs

Acid-sensing ion channels (ASICs) are proton-sensitive, sodium-selective channels expressed in the nervous system that sense changes in extracellular pH. These ion channels are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds. In the case of chicken ASIC1, the spider toxin Psalmotoxin-1 (PcTx1) activates the channel, resulting in an inward current. Furthermore, a growing class of ligands containing a guanidine group has been identified that stimulate peripheral ASICs (ASIC3), but exert subtle influence on other ASIC subtypes. The effects of the guanidine compounds on cASIC1 have not been the focus of previous study. Here, we investigated the interaction of the guanidine compound 2-guanidine-4-methylquinazoline (GMQ) on cASIC1 proton activation and PcTx1 stimulation. Exposure of expressed cASIC1 to PcTx1 resulted in biphasic currents consisting of a transient peak followed by an irreversible cASIC1 PcTx1 persistent current. This cASIC1 PcTx1 persistent current may be the result of locking the cASIC1 protein into a desensitized transition state. The guanidine compound GMQ increased the apparent affinity of protons on cASIC1 and decreased the half-maximal constant of the cASIC1 steady-state desensitization profile. Furthermore, GMQ stimulated the cASIC1 PcTx1 persistent current in a concentration-dependent manner, which resulted in a non-desensitizing inward current. Our data suggests that GMQ may have multiple sites within cASIC1 and may act as a “molecular wedge” that forces the PcTx1-desensitized ASIC into an open state. Our findings indicate that guanidine compounds, such as GMQ, may alter acid-sensing ion channel activity in combination with other stimuli, and that additional ASIC subtypes (along with ASIC3) may serve to sense and mediate signals from multiple stimuli.     

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.     

Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death

Acid-sensing ion channels (ASICs) composed of ASIC1a subunit exhibit a high Ca(2+) permeability and play important roles in synaptic plasticity and acid-induced cell death. Here, we show that ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity, as a result of activation of NR2B-containing N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) during ischemia. Furthermore, NR2B-specific antagonist, CaMKII inhibitor, or overexpression of mutated form of ASIC1a with Ser478 or Ser479 replaced by alanine (ASIC1a-S478A, ASIC1a-S479A) in cultured hippocampal neurons prevented ischemia-induced enhancement of ASIC currents, cytoplasmic Ca(2+) elevation, as well as neuronal death. Thus, NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/CaMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.     

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.     

Protons and Psalmotoxin-1 reveal nonproton ligand stimulatory sites in chicken acid-sensing ion channel

Acid-sensing ion channels (ASICs) are proton-sensitive, sodium-selective channels expressed in the nervous system that sense changes in extracellular pH. These ion channels are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds. In the case of chicken ASIC1, the spider toxin Psalmotoxin-1 (PcTx1) activates the channel, resulting in an inward current. Furthermore, a growing class of ligands containing a guanidine group has been identified that stimulate peripheral ASICs (ASIC3), but exert subtle influence on other ASIC subtypes. The effects of the guanidine compounds on cASIC1 have not been the focus of previous study. Here, we investigated the interaction of the guanidine compound 2-guanidine-4-methylquinazoline (GMQ) on cASIC1 proton activation and PcTx1 stimulation. Exposure of expressed cASIC1 to PcTx1 resulted in biphasic currents consisting of a transient peak followed by an irreversible cASIC1 PcTx1 persistent current. This cASIC1 PcTx1 persistent current may be the result of locking the cASIC1 protein into a desensitized transition state. The guanidine compound GMQ increased the apparent affinity of protons on cASIC1 and decreased the half-maximal constant of the cASIC1 steady-state desensitization profile. Furthermore, GMQ stimulated the cASIC1 PcTx1 persistent current in a concentration-dependent manner, which resulted in a non-desensitizing inward current. Our data suggests that GMQ may have multiple sites within cASIC1 and may act as a “molecular wedge” that forces the PcTx1-desensitized ASIC into an open state. Our findings indicate that guanidine compounds, such as GMQ, may alter acid-sensing ion channel activity in combination with other stimuli, and that additional ASIC subtypes (along with ASIC3) may serve to sense and mediate signals from multiple stimuli.     

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.     

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.     

Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels

Ca2+ toxicity remains the central focus of ischemic brain injury. The mechanism by which toxic Ca2+ loading of cells occurs in the ischemic brain has become less clear as multiple human trials of glutamate antagonists have failed to show effective neuroprotection in stroke. Acidosis is a common feature of ischemia and is assumed to play a critical role in brain injury; however, the mechanism(s) remain ill defined. Here, we show that acidosis activates Ca2+ -permeable acid-sensing ion channels (ASICs), inducing glutamate receptor-independent, Ca2+ -dependent, neuronal injury inhibited by ASIC blockers. Cells lacking endogenous ASICs are resistant to acid injury, while transfection of Ca2+ -permeable ASIC1a establishes sensitivity. In focal ischemia, intracerebroventricular injection of ASIC1a blockers or knockout of the ASIC1a gene protects the brain from ischemic injury and does so more potently than glutamate antagonism. Thus, acidosis injures the brain via membrane receptor-based mechanisms with resultant toxicity of [Ca2+]i, disclosing new potential therapeutic targets for stroke.     

Extracellular Chloride Modulates the Desensitization Kinetics of Acid-sensing Ion Channel 1a (ASIC1a)

Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. The recent crystallization of ASIC1a identified potential binding sites for Cl⁻ in the extracellular domain that are highly conserved between ASIC isoforms. However, the significance of Cl⁻ binding is unknown. We investigated the effect of Cl⁻ substitution on heterologously expressed ASIC1a current and H⁺-gated currents from hippocampal neurons recorded by whole-cell patch clamp. Replacement of extracellular Cl⁻ with the impermeable and inert anion methanesulfonate (MeSO₃⁻) caused ASIC1a currents to desensitize at a faster rate and attenuated tachyphylaxis. However, peak current amplitude, pH sensitivity, and selectivity were unchanged. Other anions, including Br⁻, I⁻, and thiocyanate, also altered the kinetics of desensitization and tachyphylaxis. Mutation of the residues that form the Cl⁻-binding site in ASIC1a abolished the modulatory effects of anions. The results of anion substitution on native ASIC channels in hippocampal neurons mirrored those in heterologously expressed ASIC1a and altered acid-induced neuronal death. Anion modulation of ASICs provides new insight into channel gating and may prove important in pathological brain conditions associated with changes in pH and Cl⁻.     

Acid-sensing ion channels (ASICs): therapeutic targets for neurological diseases and their regulation

Extracellular acidification occurs not only in pathological conditions such as inflammation and brain ischemia, but also in normal physiological conditions such as synaptic transmission. Acid-sensing ion channels (ASICs) can detect a broad range of physiological pH changes during pathological and synaptic cellular activities. ASICs are voltage-independent, proton-gated cation channels widely expressed throughout the central and peripheral nervous system. Activation of ASICs is involved in pain perception, synaptic plasticity, learning and memory, fear, ischemic neuronal injury, seizure termination, neuronal degeneration, and mechanosensation. Therefore, ASICs emerge as potential therapeutic targets for manipulating pain and neurological diseases. The activity of these channels can be regulated by many factors such as lactate, Zn²⁺, and Phe-Met-Arg-Phe amide (FMRFamide)-like neuropeptides by interacting with the channel’s large extracellular loop. ASICs are also modulated by G protein-coupled receptors such as CB₁ cannabinoid receptors and 5-HT₂. This review focuses on the physiological roles of ASICs and the molecular mechanisms by which these channels are regulated. [BMB Reports 2013; 46(6): 295-304]     

细胞外调控分子对酸敏感离子通道的功能调控及其机制

酸敏感离子通道(acid-Sensing ion channels,ASlCs)是一类由细胞外质子(H )激活的配体门控阳离子通道.迄今为止,人们在哺乳动物体内已经发现了6种ASICs亚基蛋白,它们分布在多种组织器官中.越来越多的研究表明:ASICs参与了机体的生理、病理过程,如:学习、记忆、痛觉、脑中风和肿瘤.在过去的10年中,人们发现多种内源性或外源性分子可以调控ASICs通道活性.由于这些细胞外调控分子与多种生理和病理功能有关,因此研究细胞外调控分子对ASICs的调控及其分子机制,可以帮助我们更多地了解ASICs功能以及结构信息,也为人们设计ASICs靶点特异性药物提供了理论依据.文章将系统地介绍细胞外调控分子对ASICs的功能调控及其作用机制,特别是该研究领域的最新进展.