Subunit-dependent cadmium and nickel inhibition of acid-sensing ion channels

Acid-sensing ion channels (ASIC) are ligand-gated cation channels that are highly expressed in peripheral sensory and central neurons. ASIC are transiently activated by decreases in extracellular pH and are thought to play important roles in sensory perception, neuronal transmission, and excitability, and in the pathology of neurological conditions, such as brain ischemia. We demonstrate here that the heavy metals Ni(2+) and Cd(2+) dose-dependently inhibit ASIC currents in hippocampus CA1 neurons and in Chinese hamster ovary (CHO) cells heterologously expressing these channels. The effects of both Ni(2+) and Cd(2+) were voltage-independent, fast, and reversible. Neither metal affected activation and desensitization kinetics but rather decreased pH-sensitivity. Moreover, distinct ASIC isoforms were differentially inhibited by Ni(2+) and Cd(2+). External application of 1 mM Ni(2+) rapidly inhibited homomeric ASIC1a and heteromeric ASIC1a/2a channels without affecting ASIC1b, 2a, and ASIC3 homomeric channels and ASIC1a/3 and 2a/3 heteromeric channels. In contrast, external Cd(+) (1 mM) inhibited ASIC2a and ASIC3 homomeric channels and ASIC1a/2a, 1a/3, and 2a/3 heteromeric channels but not ASIC1a homomeric channels. The acid-sensing current in isolated rat hippocampus CA1 neurons, thought to be carried primarily by ASIC1a and 1a/2a, was inhibited by 1 mM Ni(2+). The current study identifies ASIC as a novel target for the neurotoxic heavy metals Cd(2+) and Ni(2+).     

Inhibition of G-protein-coupled inward rectifying K+ channels by intracellular acidosis

G-protein-coupled inward rectification K(+) (GIRK) channels play an important role in modulation of synaptic transmission and cellular excitability. The GIRK channels are regulated by diverse intra- and extracellular signaling molecules. Previously, we have shown that GIRK1/GIRK4 channels are activated by extracellular protons. The channel activation depends on a histidine residue in the M1-H5 linker and may play a role in neurotransmission. Here, we show evidence that the heteromeric GIRK1/GIRK4 channels are inhibited by intracellular acidification. This inhibition was produced by selective decrease in the channel open probability with a modest drop in the single-channel conductance. The inhibition does not seem to require G-proteins as it was seen in two G-protein coupling-defective GIRK mutants and in excised patches in the absence of exogenous G-proteins. Three histidine residues in intracellular domains were critical for the inhibition. Individual mutation of His-64, His-228, or His-352 in GIRK4 abolished or greatly diminished the inhibition in homomeric GIRK4. Mutations of any of these histidine residues in GIRK4 or their counterparts in GIRK1 were sufficient to eliminate the pH(i) sensitivity of the heteromeric GIRK1/GIRK4 channels. Thus, the molecular and biophysical bases for the inhibition of GIRK channels by intracellular protons are illustrated. Because of the inequality of the pH(i) and pH(o) in most cells and their relatively independent controls by cellular versus systemic mechanisms, such pH(i) sensitivity may allow these channels to regulate cellular excitability in certain physiological and pathophysiological conditions when intracellular acidosis occurs.     

Single channel properties of rat acid-sensitive ion channel-1alpha, -2a,and -3 expressed in Xenopus oocytes

The mammalian nervous system expresses proton-gated ion channels known as acid-sensing ion channels (ASICs). Depending on their location and specialization some neurons express more than one type of ASIC where they may form homo- or heteromeric channels. Macroscopic characteristics of the ASIC currents have been described, but little is known at the single channel level. Here, we have examined the properties of unitary currents of homomeric rat ASIC1alpha, ASIC2a, and ASIC3 expressed in Xenopus oocytes with the patch clamp technique. We describe and characterize properties unique to each of these channels that can be used to distinguish the various types of ASIC channels expressed in mammalian neurons. The amplitudes of the unitary currents in symmetrical Na(+) are similar for the three types of channels (23-18 pS) and are not voltage dependent. However, ASIC1alpha exhibits three subconductance states, ASIC2a exhibits only one, and ASIC3 none. The kinetics of the three types of channels are different: ASIC1alpha and ASIC2a shift between modes of activity, each mode has different open probability and kinetics. In contrast, the kinetics of ASIC3 are uniform throughout the burst of activity. ASIC1alpha, ASIC2a, and ASIC3 are activated by external protons with apparent pH(50) of 5.9, 5.0, and 5.4, respectively. Desensitization in the continual presence of protons is fast and complete in ASIC1alpha and ASIC3 (2.0 and 4.5 s(-1), respectively) but slow and only partial in ASIC2a (0.045 s(-1)). The response to external Ca(2+) also differs: micro M concentrations of extracellular Ca(2+) are necessary for proton gating of ASIC3 (EC(50) = 0.28 micro M), whereas ASIC1alpha and ASIC2a do not require Ca(2+). In addition, Ca(2+) inhibits ASIC1alpha (K(D) = 9.2 +/- 2 mM) by several mechanisms: decrease in the amplitude of unitary currents, shortening of the burst of activity, and decrease in the number of activated channels. Contrary to previous reports, our results indicate that the Ca(2+) permeability of ASIC1alpha is very small.     

pH Dependency and desensitization kinetics of heterologously expressed combinations of acid-sensing ion channel subunits

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.     

Identification of the Ca2+ blocking site of acid-sensing ion channel (ASIC) 1: implications for channel gating

Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by H+ in the physiological range of pH. The apparent affinity for H+ of ASIC1a and 1b is modulated by extracellular Ca2+ through a competition between Ca2+ and H+. Here we show that, in addition to modulating the apparent H+ affinity, Ca2+ blocks ASIC1a in the open state (IC50 approximately 3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (IC50 > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by Ca2+. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial Na+ channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by Ca2+ of ASIC1a, showing that these two amino acids (E425 and D432) are crucial for Ca2+ block. It has previously been suggested that relief of Ca2+ block opens ASIC3 channels. However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by H+ and modulation of H+ affinity by Ca2+. These results show that channel block by Ca2+ and H+ gating are not intrinsically linked.     

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.     

Zn2+ inhibits glycine transport by glycine transporter subtype 1b

In the central nervous system, glycine is a co-agonist with glutamate at the N-methyl-D-aspartate subtype of glutamate receptors and also an agonist at inhibitory, strychnine-sensitive glycine receptors. The GLYT1 subtypes of glycine transporters (GLYTs) are responsible for regulation of glycine at excitatory synapses, whereas a combination of GLYT1 and GLYT2 subtypes of glycine transporters are used at inhibitory glycinergic synapses. Zn2+ is stored in synaptic vesicles with glutamate in a number of regions of the brain and is believed to play a role in modulation of excitatory neurotransmission. In this study we have investigated the actions of Zn2+ on the glycine transporters, GLYT1b and GLYT2a, expressed in Xenopus laevis oocytes and we demonstrate that Zn2+ is a noncompetitive inhibitor of GLYT1 but has no effect on GLYT2. We have also investigated the molecular basis for these differences and the relationship between the Zn2+ and proton binding sites on GLYT1. Using site-directed mutagenesis, we identified 2 histidine residues, His-243 in the large second extracellular loop (ECL2) and His-410 in the fourth extracellular loop (ECL4), as two coordinates in the Zn2+ binding site of GLYT1b. In addition, our study suggests that the molecular determinants of proton regulation of GLYT1b are localized to the 2 histidine residues (His-410 and His-421) of ECL4. The ability of Zn2+ and protons to regulate the rate of glycine transport by interacting with residues situated in ECL4 of GLYT1b suggests that this region may influence the substrate translocation mechanism.     

Isolation of a tarantula toxin specific for a class of proton-gated Na+ channels

Acid sensing is associated with nociception, taste transduction, and perception of extracellular pH fluctuations in the brain. Acid sensing is carried out by the simplest class of ligand-gated channels, the family of H(+)-gated Na(+) channels. These channels have recently been cloned and belong to the acid-sensitive ion channel (ASIC) family. Toxins from animal venoms have been essential for studies of voltage-sensitive and ligand-gated ion channels. This paper describes a novel 40-amino acid toxin from tarantula venom, which potently blocks (IC(50) = 0.9 nm) a particular subclass of ASIC channels that are highly expressed in both central nervous system neurons and sensory neurons from dorsal root ganglia. This channel type has properties identical to those described for the homomultimeric assembly of ASIC1a. Homomultimeric assemblies of other members of the ASIC family and heteromultimeric assemblies of ASIC1a with other ASIC subunits are insensitive to the toxin. The new toxin is the first high affinity and highly selective pharmacological agent for this novel class of ionic channels. It will be important for future studies of their physiological and physio-pathological roles.     

Protons open acid-sensing ion channels by catalyzing relief of Ca2+ blockade

Acid-sensing ion channels (ASICs) open when extracellular pH drops and they are enhanced by lactate, making them specialized for detecting lactic acidosis. Highly expressed on cardiac nociceptors and some other sensory neurons, ASICs may help trigger pain caused by tissue ischemia. We report that H(+) opens ASIC3 by speeding release of Ca(2+) from a high-affinity binding site (K(Ca) = 150 nM) on the extracellular side of the pore. The bound Ca(2+) blocks permeation and the channel conducts when multiple H(+) ions relieve this block. Activation through Ca(2+) explains sensitivity to lactate, which decreases extracellular [Ca(2+)], and it may prove relevant in CNS pathologies (stroke, seizure) that simultaneously drop pH and Ca(2+).     

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.     

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.     

Independent Contribution of Extracellular Proton Binding Sites to ASIC1a Activation

Acid-sensing ion channels (ASICs) are a group of trimeric cation permeable channels gated by extracellular protons that are mainly expressed in the nervous system. Despite the structural information available for ASIC1, there is limited understanding of the molecular mechanism that allows these channels to sense and respond to drops in extracellular pH. In this report, we employed the substituted cysteine accessibility method and site-directed mutagenesis to examine the mechanism of activation of ASIC1a by extracellular protons. We found that the modification of E238C and D345C channels by MTSET reduced proton apparent affinity for activation. Furthermore, the introduction of positively charged residues at position 345 rendered shifted biphasic proton activation curves. Likewise, channels bearing mutations at positions 79 and 416 in the palm domain of the channel showed reduced proton apparent affinity and biphasic proton activation curves. Of significance, the effect of the mutations at positions 79 and 345 on channel activation was additive. E79K-D345K required a change to a pH lower than 2 for maximal activation. In summary, this study provides direct evidence for the presence of two distinct proton coordination sites in the extracellular region of ASIC1a, which jointly facilitate pore opening in response to extracellular acidification.     

The multivalent PDZ domain-containing protein CIPP is a partner of acid-sensing ion channel 3 in sensory neurons

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular pH. They are present in the brain, where they are thought to participate in signal transduction associated with local pH variations, and in sensory neurons, where they have been involved in pain perception associated with tissue acidosis and in mechanoperception. The ASIC3 subunit is mainly expressed in dorsal root ganglion neurons. Its expression is associated with a rapidly inactivating current followed by a slowly activating sustained current thought to be required for the tonic sensation of pain caused by acids. We report here the interaction of this channel subunit with the multivalent PDZ (PSD-95 Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein CIPP. This interaction requires the C-terminal region of ASIC3 and the fourth PDZ domain of CIPP. Co-expression of CIPP and ASIC3 in COS cells increases the maximal ASIC3 peak current density by a factor of 5 and slightly shifts the pH(0.5) for activation from pH 6.2 to pH 6.4. CIPP mRNA is found at a significant level in the same dorsal root ganglion neuronal cell population that expresses the ASIC3 subunit, i.e. mainly in the small nociceptive neurons. CIPP is thus a scaffolding protein that could both enhance the surface expression of ASIC3 and bring together ASIC3 and functionally related proteins in the membrane of sensory neurons.     

DEG/ENaC/ASIC channels vary in their sensitivity to anti-hypertensive and non-steroidal anti-inflammatory drugs

The degenerin channels, epithelial sodium channels, and acid-sensing ion channels (DEG/ENaC/ASICs) play important roles in sensing mechanical stimuli, regulating salt homeostasis, and responding to acidification in the nervous system. They have two transmembrane domains separated by a large extracellular domain and are believed to assemble as homomeric or heteromeric trimers. Based on studies of selected family members, these channels are assumed to form nonvoltage-gated and sodium-selective channels sensitive to the anti-hypertensive drug amiloride. They are also emerging as a target of nonsteroidal anti-inflammatory drugs (NSAIDs). Caenorhabditis elegans has more than two dozen genes encoding DEG/ENaC/ASIC subunits, providing an excellent opportunity to examine variations in drug sensitivity. Here, we analyze a subset of the C. elegans DEG/ENaC/ASIC proteins to test the hypothesis that individual family members vary not only in their ability to form homomeric channels but also in their drug sensitivity. We selected a panel of C. elegans DEG/ENaC/ASICs that are coexpressed in mechanosensory neurons and expressed gain-of-function or d mutants in Xenopus laevis oocytes. We found that only DEGT‑1d, UNC‑8d, and MEC‑4d formed homomeric channels and that, unlike MEC‑4d and UNC‑8d, DEGT‑1d channels were insensitive to amiloride and its analogues. As reported for rat ASIC1a, NSAIDs inhibit DEGT‑1d and UNC‑8d channels. Unexpectedly, MEC‑4d was strongly potentiated by NSAIDs, an effect that was decreased by mutations in the putative NSAID-binding site in the extracellular domain. Collectively, these findings reveal that not all DEG/ENaC/ASIC channels are amiloride-sensitive and that NSAIDs can both inhibit and potentiate these channels.     

Influence of extracellular pH on the modulatory effect of zinc ions on Kv1.3 potassium channels.

In the present study we applied the whole-cell patch-clamp technique to study the influence of extracellular pH (pH(o)) on the modulatory effect exerted by zinc ions (Zn(2+)) on voltage-gated potassium channels Kv1.3 expressed in human lymphocytes. Obtained data provide evidence that lowering of pH(o) from the 7.35 to 6.4 slowed significantly the current activation rate, shifted the activation midpoint by about 16 mV towards positive membrane potentials and reduced the current amplitude to about 0.55 of the control value. In contrast, raising the pH(o) from 7.35 to 8.4 did not affect significantly the activation midpoint and current amplitude. Application of Zn(2+) in the concentration range from 100 microM to 1 mM at pH(o)=6.4 slowed additionally the activation rate, shifted the activation midpoint by about 20 mV towards positive membrane potentials and reduced the current amplitude in a concentration-dependent manner. The total effect exerted by Zn(2+) and protons at pH(o) = 6.4 was more significant than the effect exerted by Zn alone. Both the magnitude of the shift and the degree of current inhibition by Zn(2+) were independent on pH(o) in the range from 6.4 to 8.4. The data might suggest that the effects exerted by protons and zinc ions occur independently on each other and probably involve different mechanisms. Changing the holding potential from -90 mV to -60 mV at pH(o)=7.35 abolished the Zn(2+)-induced inhibition of the current amplitudes at concentrations below 300 microM. At pH(o)=6.4 the total inhibition caused by Zn(2+) and protons was also diminished, however, a significant reduction was observed at 100 microM concentration. In contrast, changing the holding potential did not change the Zn(2+)- and proton-induced shift of the activation midpoint. Altogether, obtained data suggest that extracellular protons exert the modulatory effects that are additive to the effects exerted by Zn(2+) on the channels. Possible physiological significance of these additive effects is discussed.     

Molecular determinants for activation of G-protein-coupled inward rectifier K+ (GIRK) channels by extracellular acidosis

Synaptic cleft acidification occurs following vesicle release. Such a pH change may affect synaptic transmissions in which G-protein-coupled inward rectifier K(+) (GIRK) channels play a role. To elucidate the effect of extracellular pH (pH(o)) on GIRK channels, we performed experiments on heteromeric GIRK1/GIRK4 channels expressed in Xenopus oocytes. A decrease in pH(o) to 6.2 augmented GIRK1/GIRK4 currents by approximately 30%. The channel activation was reversible and dependent on pH(o) levels. This effect was produced by selective augmentation of single channel conductance without change in the open-state probability. To determine which subunit was involved, we took advantage of homomeric expression of GIRK1 and GIRK4 by introducing a single mutation. We found that homomeric GIRK1-F137S and GIRK4-S143T channels were activated at pH(o) 6.2 by approximately 20 and approximately 70%, respectively. Such activation was eliminated when a histidine residue in the M1-H5 linker was mutated to a non-titratable glutamine, i.e. H116Q in GIRK1 and H120Q in GIRK4. Both of these histidines were required for pH sensing of the heteromeric channels, because the mutation of one of them diminished but not abolished the pH(o) sensitivity. The pH(o) sensitivity of the heteromeric channels was completely lost when both were mutated. Thus, these results suggest that the GIRK-mediated synaptic transmission is determined by both neurotransmitter and protons with the transmitter accounting for only 70% of the effect on postsynaptic cell and protons released together with the transmitter contributing to the other 30%.     

ASIC2b-dependent regulation of ASIC3, an essential acid-sensing ion channel subunit in sensory neurons via the partner protein PICK-1

ASIC3, an acid-sensing ion channel subunit expressed essentially in sensory neurons, has been proposed to be involved in pain. We show here for the first time that native ASIC3-like currents were increased in cultured dorsal root ganglion (DRG) neurons following protein kinase C (PKC) stimulation. This increase was induced by the phorbol ester PDBu and by pain mediators, such as serotonin, which are known to activate the PKC pathway through their binding to G protein-coupled receptors. We demonstrate that this regulation involves the silent ASIC2b subunit, an ASIC subunit also expressed in sensory neurons. Indeed, heteromultimeric ASIC3/ASIC2b channels, but not homomeric ASIC3 channels, are positively regulated by PKC. The increase of ASIC3/ASIC2b current is accompanied by a shift in its pH dependence toward more physiological pH values and may lead to an increase of sensory neuron excitability. This regulation by PKC requires PICK-1 (protein interacting with C kinase), a PDZ domain-containing protein, which interacts with the ASIC2b C terminus.     

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.