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.     

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.     

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.     

Neuroprotective Effects of Paeoniflorin on 6-OHDA-Lesioned Rat Model of Parkinson’s Disease

Paeoniflorin (PF) is the main active component extracted from the roots of Paeonialactiflora, a traditional Chinese medicine used for the treatment of neurodegenerative disorders, especially Parkinson’s disease (PD). The degeneration of dopaminergic (DA-) neurons in PD may be caused by pathological activation of acid-sensing ion channels (ASICs). Thus, we designed a series of experiments to evaluate the therapeutic effects of PF and to test whether its effects are related to its inhibitory effect on ASIC1a. We found that systemic administration of PF or ASICs blockers (psalmotoxin-1 and amiloride) improved behavioral symptoms, delayed DA-neuronal loss and attenuated the reduction of dopamine (DA) and its metabolites in a rat model of 6-hydroxydopamine (6-OHDA)-induced PD. In addition, our data showed that PF, like ASICs blockers, regulated the expression of ASIC1a, decreased the level of α-synuclein (α-SYN), and improved autophagic dysfunction. Further experiments showed that ASIC1a knockdown down-regulated the α-SYN level and alleviated the autophagic injury in the 6-OHDA-treated ASIC1a-silenced PC12 cells. In summary, these findings indicate that PF enhanced the autophagic degradation of α-SYN and, thus, protected DA-neurons against the neurotoxicity caused by 6-OHDA. These findings also provide experimental evidence that PF may be a neuroprotectant for PD by acting on ASIC1a and that ASIC1a may be involved in the pathogenesis of PD.     

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.     

ASIC3, a sensor of acidic and primary inflammatory pain

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular acidosis that are expressed in both central and peripheral nervous systems. Although peripheral ASICs seem to be natural sensors of acidic pain (e.g., in inflammation, ischaemia, lesions or tumours), a direct demonstration is still lacking. We show that approximately 60% of rat cutaneous sensory neurons express ASIC3-like currents. Native as well as recombinant ASIC3 respond synergistically to three different inflammatory signals that are slight acidifications (approximately pH 7.0), hypertonicity and arachidonic acid (AA). Moderate pH, alone or in combination with hypertonicity and AA, increases nociceptors excitability and produces pain suppressed by the toxin APETx2, a specific blocker of ASIC3. Both APETx2 and the in vivo knockdown of ASIC3 with a specific siRNA also have potent analgesic effects against primary inflammation-induced hyperalgesia in rat. Peripheral ASIC3 channels are thus essential sensors of acidic pain and integrators of molecular signals produced during inflammation where they contribute to primary hyperalgesia.     

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.     

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.     

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.     

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.     

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.     

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.     

Acid-sensing ion channels contribute to the effect of acidosis on the function of dendritic cells

As an H(+)-gated subgroup of the degenerin/epithelial Na(+) channel family, acid-sensing ion channels (ASICs) were reported to be involved in various physiological and pathological processes in neurons. However, little is known about the role of ASICs in the function of dendritic cells (DCs). In this study, we investigated the expression of ASICs in mouse bone marrow-derived DCs and their possible role in the function of DCs. We found that ASIC1, ASIC2, and ASIC3 are expressed in DCs at the mRNA and protein levels, and extracellular acid can evoke ASIC-like currents in DCs. We also demonstrated that acidosis upregulated the expression of CD11c, MHC class II, CD80, and CD86 and enhanced the Ag-presenting ability of DCs via ASICs. Moreover, the effect of acidosis on DCs can be abolished by the nonsteroidal anti-inflammatory drugs ibuprofen and diclofenac. These results suggest that ASICs are involved in the acidosis-mediated effect on DC function.     

A Rapid Facilitation of Acid-Sensing Ion Channels Current by Corticosterone in Cultured Hippocampal Neurons

Acid-sensing ion channels (ASIC) play an important role in the central neuronal system and excessive activation of ASICs induces neuronal damage. Recent studies show that ASIC1a, a subunit of ASIC, is involved in stress processes but the mechanisms by which ASIC1a is regulated by corticosterone (CORT), a stress-induced hormone, are as yet unelucidated. In the present study, to explore the effects of CORT on ASIC1a in cultured hippocampal neurons, the whole-cell patch clamp technique was used. We present data showing that extracellular application of 1 and 10 μM CORT increase the inward current when solution of pH 6.0 is applied to the exterior of the cell. Moreover, extracellular application of membrane-impermeable CORT-BSA (1 μM) maintains current elevation induced by the action of ASIC1a. However, intracellular application of CORT (1 μM) did not increase ASIC1a current. Subsequent extracellular application of CORT enhanced the amplitude of ASIC1a current. Also, RU38486 (10 μM), an antagonist of nuclear glucocorticoids receptor, did not block an increase of ASIC1a current induced by CORT. In addition, CORT application further resulted in a significant enhancement of ASIC1a current in the presence of phorbol 12-myristate 13-acetate (0.5 μM) or bryostatin1 (1 μM), which are both protein kinase C (PKC) agonists. On the contrary, after pretreatment with GF109203X (3 μM), an antagonist of PKC, CORT did not elevate ASIC1a current. These data indicate that the rapid increase of ASIC1a current induced by CORT may be caused by the activation of corticosteroid receptors found on the cell membranes of hippocampal neurons and it may involve a PKC-dependent mechanism.     

Modulation of acid-sensing ion channel currents, acid-induced increase of intracellular Ca2+, and acidosis-mediated neuronal injury by intracellular pH

Acid-sensing ion channels (ASICs), activated by lowering extracellular pH (pH(o)), play an important role in normal synaptic transmission in brain and in the pathology of brain ischemia. Like pH(o), intracellular pH (pH(i)) changes dramatically in both physiological and pathological conditions. Although it is known that a drop in pH(o) activates the ASICs, it is not clear whether alterations of pH(i) have an effect on these channels. Here we demonstrate that the overall activities of ASICs, including channel activation, inactivation, and recovery from desensitization, are tightly regulated by pH(i). In cultured mouse cortical neurons, bath perfusion of the intracellular alkalizing agent quinine increased the amplitude of the ASIC current by approximately 50%. In contrast, intracellular acidification by withdrawal of NH(4)Cl or perfusion of propionate inhibited the current. Increasing pH buffering capacity in the pipette solution with 40 mm HEPES attenuated the effects of quinine and NH(4)Cl. The effects of intracellular alkalizing/acidifying agents were mimicked by using intracellular solutions with pH directly buffered at high/low values. Increasing pH(i) induced a shift in H(+) dose-response curve toward less acidic pH but a shift in the steady state inactivation curve toward more acidic pH. In addition, alkalizing pH(i) induced an increase in the recovery rate of ASICs from desensitization. Consistent with its effect on the ASIC current, changing pH(i) has a significant influence on the acid-induced increase of intracellular Ca(2+), membrane depolarization, and acidosis-mediated neuronal injury. Our findings suggest that changes in pH(i) may play an important role in determining the overall function of ASICs in both physiological and pathological conditions.     

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.     

Regulating Factors in Acid-Sensing Ion Channel 1a Function

In recent years, research of acid sensing ion channels (ASICs) has increased tremendously, especially studies focusing on ASIC1a, which plays a critical role in many important physiologic and pathological functions. This review will discuss factors regulating ASIC1a expression and activity in various conditions and will provide a theoretical basis for clinical development and application of ASIC1a modifiers.     

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.