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1.
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.  相似文献   

2.
Selective regulation of acid-sensing ion channel 1 by serine proteases   总被引:10,自引:0,他引:10  
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.  相似文献   

3.
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.  相似文献   

4.
Acid-sensing ion channels (ASICs) are modulated by various classes of ligands, including the recently described hydrophobic monoamines, which inhibit and potentiate ASICs in a subunit-specific manner. In particular, memantine inhibits ASIC1a and potentiates ASIC2a homomers. The aim of the present work was to characterize action mechanism of memantine on recombinant ASIC1a expressed in CHO (Chinese hamster ovary) cells. We have demonstrated that effect of memantine on ASIC1a strongly depends on membrane voltage, conditioning pH value and application protocol. When applied simultaneously with activating acidification at hyperpolarized voltages, memantine caused the strongest inhibition. Surprisingly, application of memantine between ASIC1a activations at zero voltage caused significant potentiation. Analysis of the data suggests that memantine produces two separate effects, voltage-dependent open-channel block and shift of steady-state desensitization curve to more acidic values. Putative binding sites are discussed based on the computer docking of memantine to the acidic pocket and the pore region.  相似文献   

5.
Acid-sensing ion channels (ASICs) are neuronal Na+-conducting channels activated by extracellular acidification. ASICs are involved in pain sensation, expression of fear, and neurodegeneration after ischemic stroke. Functional ASICs are composed of three identical or homologous subunits, whose extracellular part has a handlike structure. Currently, it is unclear how protonation of residues in extracellular domains controls ASIC activity. Knowledge of these mechanisms would allow a rational development of drugs acting on ASICs. Protonation may induce conformational changes that control the position of the channel gate. We used voltage-clamp fluorometry with fluorophores attached to residues in different domains of ASIC1a to detect conformational changes. Comparison of the timing of fluorescence and current signals identified residues involved in movements that preceded desensitization and may therefore be associated with channel opening or early steps leading to desensitization. Other residues participated in movements intimately linked to desensitization and recovery from desensitization. Fluorescence signals of all mutants were detected at more alkaline pH than ionic currents. Their midpoint of pH dependence was close to that of steady-state desensitization, whereas the steepness of the pH fluorescence relationship was closer to that of current activation. A sequence of movements was observed upon acidification, and its backward movements during recovery from desensitization occurred in the reverse order, indicating that the individual steps are interdependent. Furthermore, the fluorescence signal of some labeled residues in the finger domain was strongly quenched by a Trp residue in the neighboring β-ball domain. Upon channel activation, their fluorescence intensity increased, indicating that the finger moved away from the β ball. This extensive analysis of activity-dependent conformational changes in ASICs sheds new light on the mechanisms by which protonation controls ASIC activity.  相似文献   

6.
7.
Desensitization is a common feature of ligand-gated ion channels, although the molecular cause varies widely between channel types. Mutations that greatly reduce or nearly abolish desensitization have been described for many ligand-gated ion channels, including glutamate, GABA, glycine, and nicotinic receptors, but not for acid-sensing ion channels (ASICs) until recently. Mutating Gln276 to a glycine (Q276G) in human ASIC1a was reported to mostly abolish desensitization at both the macroscopic and the single channel levels, potentially providing a valuable tool for subsequent studies. However, we find that in both human and chicken ASIC1, the effect of Q276G is modest. In chicken ASIC1, the equivalent Q277G slightly reduces desensitization when using pH 6.5 as a stimulus but desensitizes, essentially like wild-type, when using more acidic pH values. In addition, steady-state desensitization is intact, albeit right-shifted, and recovery from desensitization is accelerated. Molecular dynamics simulations indicate that the Gln277 side chain participates in a hydrogen bond network that might stabilize the desensitized conformation. Consistent with this, destabilizing this network with the Q277N or Q277L mutations largely mimics the Q277G phenotype. In human ASIC1a, the Q276G mutation also reduces desensitization, but not to the extent reported previously. Interestingly, the kinetic consequences of Q276G depend on the human variant used. In the common G212 variant, Q276G slows desensitization, while in the rare D212 variant desensitization accelerates. Our data reveal that while the Q/G mutation does not abolish or substantially impair desensitization as previously reported, it does point to unexpected differences between chicken and human ASICs and the need for careful scrutiny before using this mutation in future studies.  相似文献   

8.
Potentiation of acid-sensing ion channels by sulfhydryl compounds   总被引:2,自引:0,他引:2  
The acid-sensing ion channels (ASICs) are voltage-independent ion channels activated by acidic extracellular pH. ASICs play a role in sensory transduction, behavior, and acidotoxic neuronal death, which occurs during stroke and ischemia. During these conditions, the extracellular concentration of sulfhydryl reducing agents increases. We used perforated patch-clamp technique to analyze the impact of sulfhydryls on H+-gated currents from Chinese hamster ovary (CHO) cells expressing human ASIC1a (hASIC1a). We found that hASIC1a currents activated by pH 6.5 were increased almost twofold by the sulfhydryl-containing reducing agents dithiothreitol (DTT) and glutathione. DTT shifted the pH-dose response of hASIC1a toward a more neutral pH (pH0.5 from 6.54 to 6.69) and slowed channel desensitization. The effect of reducing agents on native mouse hippocampal neurons and transfected mouse ASIC1a was similar. We found that the effect of DTT on hASIC1a was mimicked by the metal chelator TPEN, and mutant hASIC1a channels with reduced TPEN potentiation showed reduced DTT potentiation. Furthermore, the addition of DTT in the presence of TPEN did not result in further increases in current amplitude. These results suggest that the effect of DTT on hASIC1a is due to relief of tonic inhibition by transition metal ions. We found that all ASICs examined remained potentiated following the removal of DTT. This effect was reversed by the oxidizing agent DTNB in hASIC1a, supporting the hypothesis that DTT also impacts ASICs via a redox-sensitive site. Thus sulfhydryl compounds potentiate H+-gated currents via two mechanisms, metal chelation and redox modulation of target amino acids. glutathione; DTT; redox; zinc  相似文献   

9.
Yu Y  Chen Z  Li WG  Cao H  Feng EG  Yu F  Liu H  Jiang H  Xu TL 《Neuron》2010,68(1):61-72
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.  相似文献   

10.
Acid-sensing ion channels (ASICs) are emerging as fundamental players in the regulation of neural plasticity and in pathological conditions. Here we showed that lead (Pb2+), a well known neurotoxic metal ion, reversibly and concentration-dependently inhibited ASIC currents in the acutely dissociated spinal dorsal horn and hippocampal CA1 neurons of rats. In vitro expression of ASIC subunits in combination demonstrated that both ASIC1 and -3 subunits were sensitive to Pb2+. Mechanistically, Pb2+ reduced the pH sensitivity of ASICs independent of membrane voltage change. Moreover, Pb2+ inhibited the ASIC-mediated membrane depolarization and the elevation of intracellular Ca2+ concentration. In addition, we compared the effect of Pb2+ with that of Ca2+ or amiloride to explore the possible interactions of Pb2+ and Ca2+ in regulating ASICs, and we found that Pb2+ inhibited ASIC currents independent of the amiloride/Ca2+ blockade. Because ASIC1b and -3 subunits are mainly expressed in peripheral neurons, our data identified ASIC1a-containing Ca2+-permeable ASIC as a novel central target of Pb2+ action, which may contribute to Pb2+ neurotoxicity.  相似文献   

11.
Cystic fibrosis transmembrane conductance regulator (CFTR) functions as both a chloride channel and an epithelial transport regulator, interacting with Na(+) (epithelial sodium channel), Cl(-), renal outer medullary potassium channel(+), and H(2)O channels and some exchangers (i.e. Na(+)/H(+)) and co-transporters (Na(+)-HCO(3)(minus sign), Na(+)-K(+)-2Cl(-)). Acid-sensitive ion channels (ASICs), members of the epithelial sodium channel/degenerin superfamily, were originally cloned from neuronal tissue, and recently localized in epithelia. Because CFTR has been immunocytochemically and functionally identified in rat, murine, and human brain, the regulation of ASICs by CFTR was tested in oocytes. Our observations show that the proton-gated Na(+) current formed by the heteromultimeric ASIC1a/2a channel was up-regulated by wild type but not by Delta F508-CFTR. In contrast, the acid-gated Na(+) current associated with either the homomultimeric ASIC1a or ASIC2a channel was not influenced by wild type CFTR. The apparent equilibrium dissociation constant for extracellular Na(+) for ASIC1a/2a was increased by CFTR, but CFTR had no effect on the gating behavior or acid sensitivity of ASIC1a/2a. CFTR had no effect on the pH activation of ASIC1a/2a. We conclude that wild type CFTR elevates the acid-gated Na(+) current of ASIC1a/2a in part by altering the kinetics of extracellular Na(+) interaction.  相似文献   

12.
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.  相似文献   

13.
The inhibitory action of non-steroid anti-inflammatory drugs was investigated on acid-sensing ionic channels (ASIC) in isolated hippocampal interneurons and on recombinant ASICs expressed in Chinese hamster ovary (CHO) cells. Diclofenac and ibuprofen inhibited proton-induced currents in hippocampal interneurons (IC50 were 622 ± 34 μM and 3.42 ± 0.50 mM, respectively). This non-competitive effect was fast and fully reversible for both drugs. Aspirin and salicylic acid at 500 μM were ineffective. Diclofenac and ibuprofen decreased the amplitude of proton-evoked currents and slowed the rates of current decay with a good correlation between these effects. Simultaneous application of acid solution and diclofenac was required for its inhibitory effect. Unlike amiloride, the action of diclofenac was voltage-independent and no competition between two drugs was found. Analysis of the action of diclofenac and ibuprofen on activation and desensitization of ASICs showed that diclofenac but not ibuprofen shifted the steady-state desensitization curve to more alkaline pH values. The reason for this shift was slowing down the recovery from desensitization of ASICs. Thus, diclofenac may serve as a neuroprotective agent during pathological conditions associated with acidification.  相似文献   

14.
The exact subunit combinations of functional native acid-sensing ion channels (ASICs) have not been established yet, but both homomeric and heteromeric channels are likely to exist. To determine the ability of different subunits to assemble into heteromeric channels, a number of ASIC1a-, ASIC1b-, ASIC2a-, ASIC2b-, and ASIC3-containing homo- and heteromeric channels were studied by whole-cell patch clamp recordings with respect to pH sensitivity, desensitization kinetics, and level of sustained current normalized to peak current. Analyzing and comparing data for these three features demonstrated unique heteromeric channels in a number of co-expression experiments. Formation of heteromeric ASIC1a+2a and ASIC1b+2a channels was foremost supported by the desensitization characteristics that were independent of proton concentration, a feature none of the respective homomeric channels has. Several lines of evidence supported formation of ASIC1a+3, ASIC1b+3, and ASIC2a+3 heteromeric channels. The most compelling was the desensitization characteristics, which, besides being proton-independent, were faster than those of any of the respective homomeric channels. ASIC2b, which homomerically expressed is not activated by protons per se, did not appear to form unique heteromeric combinations with other subunits and in fact appeared to suppress the function of ASIC1b. Co-expression of three subunits such as ASIC1a+2a+3 and ASIC1b+2a+3 resulted in data that could best be explained by coexistence of multiple channel populations within the same cell. This observation seems to be in good agreement with the fact that ASIC-expressing sensory neurons display a variety of acid-evoked currents.  相似文献   

15.
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.  相似文献   

16.
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.  相似文献   

17.
Acid-sensing ionic channels (ASICs) are involved in such functions of the sensory nervous system as mechanoreception, nociception and perception of acid taste. Phe-Met-Arg-Phe amide-related (FMRFa-related) peptides in micro m concentrations slow down the rate of ASICs desensitization. Here we report that this effect is strongly pH dependent: the lower the pH used to activate ASICs, the larger is the modulatory effect of Arg-Phe amide-related (RFa-related) peptides. Pre-application of the peptides results in a change to the desensitization kinetics of the ASICs-operated current from monoexponential to biexponential: the fast component retains the control kinetics, whereas the slow one is induced by the peptide. The lower the pH, the larger is the slow component, whereas there is practically no modulation at pH 6.6. Phe-Met-Val-Phe amide (FMVFa), which has neutral valine instead of arginine, similarly modulates the kinetics of ASICs, but does not reveal pH dependence of this action. Thus, positively charged arginine regulates the access of the RFa-related peptides to the modulatory site. We suggest that the pH dependence of the modulatory action of RFa-related peptides can be associated with the interaction of a positively charged arginine with histidine residues in the molecule of ASIC.  相似文献   

18.
Stomatin modulates gating of acid-sensing ion channels   总被引:3,自引:0,他引:3  
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.  相似文献   

19.
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.  相似文献   

20.
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.  相似文献   

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