ASIC1a Channel Research Articles

Mutagenesis of the Peptide Inhibitor of ASIC3 Channel Introduces Binding to Thumb Domain of ASIC1a but Reduces Analgesic Activity.

Acid-sensing ion channels (ASICs), which act as proton-gating sodium channels, have garnered attention as pharmacological targets. ASIC1a isoform, notably prevalent in the central nervous system, plays an important role in synaptic plasticity, anxiety, neurodegeneration, etc. In the peripheral nervous system, ASIC1a shares prominence with ASIC3, the latter well established for its involvement in pain signaling, mechanical sensitivity, and inflammatory hyperalgesia. However, the precise contributions of ASIC1a in peripheral functions necessitate thorough investigation. To dissect the specific roles of ASICs, peptide ligands capable of modulating these channels serve as indispensable tools. Employing molecular modeling, we designed the peptide targeting ASIC1a channel from the sea anemone peptide Ugr9-1, originally targeting ASIC3. This peptide (A23K) retained an inhibitory effect on ASIC3 (IC50 9.39 µM) and exhibited an additional inhibitory effect on ASIC1a (IC50 6.72 µM) in electrophysiological experiments. A crucial interaction between the Lys23 residue of the A23K peptide and the Asp355 residue in the thumb domain of the ASIC1a channel predicted by molecular modeling was confirmed by site-directed mutagenesis of the channel. However, A23K peptide revealed a significant decrease in or loss of analgesic properties when compared to the wild-type Ugr9-1. In summary, using A23K, we show that negative modulation of the ASIC1a channel in the peripheral nervous system can compromise the efficacy of an analgesic drug. These results provide a compelling illustration of the complex balance required when developing peripheral pain treatments targeting ASICs.

Redistribution of ASIC1a channels triggered by IL-6: Potential role of ASIC1a in neuroinflammation

Cytokines, particularly IL-6, play a crucial role in modulating immune responses in the central nervous system (CNS). Elevated IL-6 levels have been observed in neuroinflammatory conditions, as well as in the sera and brains of patients with neurodegenerative diseases such as Parkinson's, Huntington's, Multiple Sclerosis, and Alzheimer's. Additionally, alterations in regional brain pH have been noted in these conditions. Acid-sensing ion channels (ASICs), including ASIC1a, activated by low pH levels, are highly abundant in the CNS and have recently been associated with various neurological disorders. Our study examined the impact of IL-6 on ASIC1a channels in cell cultures, demonstrating IL-6-induced the redistribution of cytosolic ASIC1a channels to the cell membrane. This redistribution was accompanied by increased ASIC1a current amplitude upon activation, as well as elevated levels of phosphorylated CaMKII and ERK kinases. Additionally, we observed posttranslational modifications on the ASIC1a channel itself. These findings provide insight into a potential link between inflammatory processes and neurodegenerative mechanisms, highlighting ASIC1a channels as promising therapeutic targets in these conditions.

Molecular Basis for Mambalgin-2 Interaction with Heterotrimeric α-ENaC/ASIC1a/γ-ENaC Channels in Cancer Cells.

Cancer progression is characterized by microenvironmental acidification. Tumor cells adapt to low environmental pH by activating acid-sensing trimeric ion channels of the DEG/ENaC family. The α-ENaC/ASIC1a/γ-ENaC heterotrimeric channel is a tumor-specific acid-sensing channel, and its targeting can be considered a new strategy for cancer therapy. Mambalgin-2 from the Dendroaspis polylepis venom inhibits the α-ENaC/ASIC1a/γ-ENaC heterotrimer more effectively than the homotrimeric ASIC1a channel, initially proposed as the target of mambalgin-2. Although the molecular basis of such mambalgin selectivity remained unclear. Here, we built the models of the complexes of mambalgin-2 with the α-ENaC/ASIC1a/γ-ENaC and ASIC1a channels, performed MD and predicted the difference in the binding modes. The importance of the 'head' loop region of mambalgin-2 for the interaction with the hetero-, but not with the homotrimeric channel was confirmed by site-directed mutagenesis and electrophysiology. A new mode of allosteric regulation of the ENaC channels by linking the thumb domain of the ASIC1a subunit with the palm domain of the γ-ENaC subunit was proposed. The data obtained provide new insights into the regulation of various types of acid-sensing ion channels and the development of new strategies for cancer treatment.

Dual contribution of ASIC1a channels in the spinal processing of pain information by deep projection neurons revealed by computational modeling

Dorsal horn of the spinal cord is an important crossroad of pain neuraxis, especially for the neuronal plasticity mechanisms that can lead to chronic pain states. Windup is a well-known spinal pain facilitation process initially described several decades ago, but its exact mechanism is still not fully understood. Here, we combine both ex vivo and in vivo electrophysiological recordings of rat spinal neurons with computational modeling to demonstrate a role for ASIC1a-containing channels in the windup process. Spinal application of the ASIC1a inhibitory venom peptides mambalgin-1 and psalmotoxin-1 (PcTx1) significantly reduces the ability of deep wide dynamic range (WDR) neurons to develop windup in vivo. All deep WDR-like neurons recorded from spinal slices exhibit an ASIC current with biophysical and pharmacological characteristics consistent with functional expression of ASIC1a homomeric channels. A computational model of WDR neuron supplemented with different ASIC1a channel parameters accurately reproduces the experimental data, further supporting a positive contribution of these channels to windup. It also predicts a calcium-dependent windup decrease for elevated ASIC conductances, a phenomenon that was experimentally validated using the Texas coral snake ASIC-activating toxin (MitTx) and calcium-activated potassium channel inhibitory peptides (apamin and iberiotoxin). This study supports a dual contribution to windup of calcium permeable ASIC1a channels in deep laminae projecting neurons, promoting it upon moderate channel activity, but ultimately leading to calcium-dependent windup inhibition associated to potassium channels when activity increases.

Structure and analysis of nanobody binding to the human ASIC1a ion channel.

ASIC1a is a proton-gated sodium channel involved in modulation of pain, fear, addiction, and ischemia-induced neuronal injury. We report isolation and characterization of alpaca-derived nanobodies (Nbs) that specifically target human ASIC1a. Cryo-electron microscopy of the human ASIC1a channel at pH 7.4 in complex with one of these, Nb.C1, yielded a structure at 2.9 Å resolution. It is revealed that Nb.C1 binds to a site overlapping with that of the Texas coral snake toxin (MitTx1) and the black mamba venom Mambalgin-1; however, the Nb.C1-binding site does not overlap with that of the inhibitory tarantula toxin psalmotoxin-1 (PcTx1). Fusion of Nb.C1 with PcTx1 in a single polypeptide markedly enhances the potency of PcTx1, whereas competition of Nb.C1 and MitTx1 for binding reduces channel activation by the toxin. Thus, Nb.C1 is a molecular tool for biochemical and structural studies of hASIC1a; a potential antidote to the pain-inducing component of coral snake bite; and a candidate to potentiate PcTx1-mediated inhibition of hASIC1a in vivo for therapeutic applications.

Validation of an ASIC1a Ligand-Gated Assay on an Automated Patch Clamp Platform and its Use for Novel Ligand Screening

There is growing interest in automated patch clamp assays for ligand-gated targets, many of which are expressed throughout the nervous system and underlie complex neurological diseases. The Acid-Sensing Ion Channel (ASIC) family comprises combinations of ASIC1-4 proteins that form cation-selective channels in the central and peripheral nervous system. ASIC1a proteins are involved in synaptic plasticity, and implicated in stroke, ischemia and pain. Small molecules and toxins modulate ASIC1a channels, and fragments can bind to ASIC3 channels (Wolkenberg et al., 2010), so we screened a diverse low molecular weight library for novel ASIC1a modulators. All data was generated on the gigaseal QPatch48 system (Sophion) using coated metal pipetting tips and a CHO cell line stably expressing human ASIC1a. Currents were elicited from a holding potential of −60 mV by rapid application of saline or compound-containing solutions of varied pH. EC50 of pH activation and IC50 of test compound inhibition were determined by cumulative applications to the same cell. A proprietary low MW library was tested at 200 μM (N=2) and active hits were confirmed by cumulative IC50 testing (N=3). We validated the pharmacology of the hASIC1a cell line using toxins (e.g. IC50 of 120 nM for Mambalgin-3), small molecules (Benzamil IC50 of 4.0 μM), and clinical development compounds, which paved the way to screen a proprietary library of low molecular weight compounds in an effort to find chemical building blocks for the design of novel and efficacious ASIC1a modulators. A hit rate of 3.0% was achieved which included several novel scaffolds with low μM potency, which can be used in biophysical binding assays and structure-based drug design to develop ligand-efficient modulators of the human ASIC1a channel.

Mambalgin-1 pain-relieving peptide locks the hinge between α4 and α5 helices to inhibit rat acid-sensing ion channel 1a

Acid-sensing ion channels (ASICs) are proton-gated cationic channels involved in pain and other processes, underscoring the potential therapeutic value of specific inhibitors such as the three-finger toxin mambalgin-1 (Mamb-1) from snake venom. A low-resolution structure of the human-ASIC1a/Mamb-1 complex obtained by cryo-electron microscopy has been recently reported, implementing the structure of the chicken-ASIC1/Mamb-1 complex previously published. Here we combine structure-activity relationship of both the rat ASIC1a channel and the Mamb-1 toxin with a molecular dynamics simulation to obtain a detailed picture at the level of side-chain interactions of the binding of Mamb-1 on rat ASIC1a channels and of its inhibition mechanism. Fingers I and II of Mamb-1 but not the core of the toxin are required for interaction with the thumb domain of ASIC1a, and Lys-8 of finger I potentially interacts with Tyr-358 in the thumb domain. Mamb-1 does not interfere directly with the pH sensor as previously suggested, but locks by several contacts a key hinge between α4 and α5 helices in the thumb domain of ASIC1a to prevent channel opening. Our results provide an improved model of inhibition of mammalian ASIC1a channels by Mamb-1 and clues for further development of optimized ASIC blockers.

Sevanol and Its Analogues: Chemical Synthesis, Biological Effects and Molecular Docking.

Among acid-sensing ion channels (ASICs), ASIC1a and ASIC3 subunits are the most widespread and prevalent in physiological and pathophysiological conditions. They participate in synaptic plasticity, learning and memory, as well as the perception of inflammatory and neurological pain, making these channels attractive pharmacological targets. Sevanol, a natural lignan isolated from Thymus armeniacus, inhibits the activity of ASIC1a and ASIC3 isoforms, and has a significant analgesic and anti-inflammatory effect. In this work, we described the efficient chemical synthesis scheme of sevanol and its analogues, which allows us to analyze the structure–activity relationships of the different parts of this molecule. We found that the inhibitory activity of sevanol and its analogues on ASIC1a and ASIC3 channels depends on the number and availability of the carboxyl groups of the molecule. At the structural level, we predicted the presence of a sevanol binding site based on the presence of molecular docking in the central vestibule of the ASIC1a channel. We predicted that this site could also be occupied in part by the FRRF-amide peptide, and the competition assay of sevanol with this peptide confirmed this prediction. The intravenous (i.v.), intranasal (i.n.) and, especially, oral (p.o.) administration of synthetic sevanol in animal models produced significant analgesic and anti-inflammatory effects. Both non-invasive methods of sevanol administration (i.n. and p.o.) showed greater efficacy than the invasive (i.v.) method, thus opening new horizons for medicinal uses of sevanol.

Structural and Functional Analysis of Gly212 Mutants Reveals the Importance of Intersubunit Interactions in ASIC1a Channel Function.

Acid-sensing ion channels (ASICs) act as pH sensors in neurons. ASICs contribute to pain sensation, learning, fear behavior and to neuronal death after ischemic stroke. Extracellular acidification induces a transient activation and subsequent desensitization of these Na+-selective channels. ASICs are trimeric channels made of identical or homologous subunits. We have previously shown that mutation of the highly conserved Gly212 residue of human ASIC1a to Asp affects the channel function. Gly212 is located in the proximity of a predicted Cl– binding site at a subunit interface. Here, we have measured the function of a series of Gly212 mutants. We show that substitution of Gly212 affects the ASIC1a pH dependence and current decay kinetics. Intriguingly, the mutations to the acidic residues Asp and Glu have opposing effects on the pH dependence and the current decay kinetics. Analysis of molecular dynamics simulation trajectories started with the coordinates of the closed conformation indicates that the immediate environment of residue 212 in G212E, which shifts the pH dependence to more alkaline values, adopts a conformation closer to the open state. The G212D and G212E mutants have a different pattern of intersubunit salt bridges, that, in the case of G212E, leads to an approaching of neighboring subunits. Based on the comparison of crystal structures, the conformational changes in this zone appear to be smaller during the open-desensitized transition. Nevertheless, MD simulations highlight differences between mutants, suggesting that the changed function upon substitution of residue 212 is due to differences in intra- and intersubunit interactions in its proximity.

Alkaloid Lindoldhamine Inhibits Acid-Sensing Ion Channel 1a and Reveals Anti-Inflammatory Properties.

Acid-sensing ion channels (ASICs), which are present in almost all types of neurons, play an important role in physiological and pathological processes. The ASIC1a subtype is the most sensitive channel to the medium’s acidification, and it plays an important role in the excitation of neurons in the central nervous system. Ligands of the ASIC1a channel are of great interest, both fundamentally and pharmaceutically. Using a two-electrode voltage-clamp electrophysiological approach, we characterized lindoldhamine (a bisbenzylisoquinoline alkaloid extracted from the leaves of Laurus nobilis L.) as a novel inhibitor of the ASIC1a channel. Lindoldhamine significantly inhibited the ASIC1a channel’s response to physiologically-relevant stimuli of pH 6.5–6.85 with IC50 range 150–9 μM, but produced only partial inhibition of that response to more acidic stimuli. In mice, the intravenous administration of lindoldhamine at a dose of 1 mg/kg significantly reversed complete Freund’s adjuvant-induced thermal hyperalgesia and inflammation; however, this administration did not affect the pain response to an intraperitoneal injection of acetic acid (which correlated well with the function of ASIC1a in the peripheral nervous system). Thus, we describe lindoldhamine as a novel antagonist of the ASIC1a channel that could provide new approaches to drug design and structural studies regarding the determinants of ASIC1a activation.

Fear extinction requires ASIC1a-dependent regulation of hippocampal-prefrontal correlates

Extinction of conditioned fear necessitates the dynamic involvement of hippocampus, medial prefrontal cortex (mPFC), and basolateral amygdala (BLA), but key molecular players that regulate these circuits to achieve fear extinction remain largely unknown. Here, we report that acid-sensing ion channel 1a (ASIC1a) is a crucial molecular regulator of fear extinction, and that this function requires ASIC1a in ventral hippocampus (vHPC), but not dorsal hippocampus, mPFC, or BLA. While genetic disruption or pharmacological inhibition of ASIC1a in vHPC attenuated the extinction of conditioned fear, overexpression of the channel in this area promoted fear extinction. Channelrhodopsin-2-assisted circuit mapping revealed that fear extinction involved an ASIC1a-dependent modification of the long-range hippocampal-prefrontal correlates in a projection-specific manner. Gene expression profiling analysis and validating experiments identified several neuronal activity-regulated and memory-related genes, including Fos, Npas4, and Bdnf, as the potential mediators of ASIC1a regulation of fear extinction. Mechanistically, genetic overexpression of brain-derived neurotrophic factor (BDNF) in vHPC or supplement of BDNF protein in mPFC both rescued the deficiency in fear extinction and the deficits on extinction-driven adaptations of hippocampal-prefrontal correlates caused by the Asic1a gene inactivation in vHPC. Together, these results establish ASIC1a as a critical constituent in fear extinction circuits and thus a promising target for managing adaptive behaviors.

Bile acids potentiate proton-activated currents in Xenopus laevis oocytes expressing human acid-sensing ion channel (ASIC1a).

Acid‐sensing ion channels (ASICs) are nonvoltage‐gated sodium channels transiently activated by extracellular protons and belong to the epithelial sodium channel (ENaC)/Degenerin (DEG) family of ion channels. Bile acids have been shown to activate two members of this family, the bile acid‐sensitive ion channel (BASIC) and ENaC. To investigate whether bile acids also modulate ASIC function, human ASIC1a was heterologously expressed in Xenopus laevis oocytes. Exposing oocytes to tauro‐conjugated cholic (t‐CA), deoxycholic (t‐DCA), and chenodeoxycholic (t‐CDCA) acid at pH 7.4 did not activate ASIC1a‐mediated whole‐cell currents. However, in ASIC1a expressing oocytes the whole‐cell currents elicited by pH 5.5 were significantly increased in the presence of these bile acids. Single‐channel recordings in outside‐out patches confirmed that t‐DCA enhanced the stimulatory effect of pH 5.5 on ASIC1a channel activity. Interestingly, t‐DCA reduced single‐channel current amplitude by ~15% which suggests an interaction of t‐DCA with a region close to the channel pore. Molecular docking predicted binding of bile acids to the pore region near the degenerin site (G433) in the open conformation of the channel. Site‐directed mutagenesis demonstrated that the amino acid residue G433 is critically involved in the potentiating effect of bile acids on ASIC1a activation by protons.

Identifying key modulators of panic attacks in panic disorder: A translational model

Panic disorder (PD) has a prevalence of 4% in the general population, but its pathophysiology remains elusive. This research focuses on identifying molecular and genetic modulators of unpredictable, recurrent panic attacks which characterize PD. Prior studies revealed that the serotonin transporter, 5-HTT, is involved in fear behavior and that genetic variations might affect panic-related behavior. In order to test these genetic variations on a molecular level a translational model consisting of humans and rodents was used. As follow-up of a human study, 40 male mice (background C57BL/6), wild-type and heterozygous 5-HTT knock-out, were exposed to either 9% CO2 or room air to experimentally provoke fearrelated behavior. Furthermore, the acid-sensing ion channel ASIC1a is hypothesized to be a linking factor between fear detection and fear response. It was tested whether this ASIC1a is expressed in serotonergic neurons in the dorsal raphe nucleus, main source of serotonin, the pivotal neurotransmitter in panic responses. Localization of the ASIC1a channel was determined by use of immunofluorescence. Analysis of the behavioral tests showed no significant difference between genotypes in both air and carbon dioxide condition. Carbon dioxide exposure however increased freezing time in both genotypes. ASIC1a has been indicated in the dorsal raphe and hippocampus in perfusion-fixated rat tissue, but could not yet be detected in the dorsal raphe of the fresh-frozen tissue of tested mice.

The Human Acid-Sensing Ion Channel ASIC1a: Evidence for a Homotetrameric Assembly State at the Cell Surface.

The chicken acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. We address here the oligomeric state of the functional ASIC1 in situ at the cell surface. The oligomeric states of functional ASIC1a and mutants with additional cysteines introduced in the extracellular pore vestibule were resolved on SDS-PAGE. The functional ASIC1 complexes were stabilized at the cell surface of Xenopus laevis oocytes or CHO cells either using the sulfhydryl crosslinker BMOE, or sodium tetrathionate (NaTT). Under these different crosslinking conditions ASIC1a migrates as four distinct oligomeric states that correspond by mass to multiples of a single ASIC1a subunit. The relative importance of each of the four ASIC1a oligomers was critically dependent on the availability of cysteines in the transmembrane domain for crosslinking, consistent with the presence of ASIC1a homo-oligomers. The expression of ASIC1a monomers, trimeric or tetrameric concatemeric cDNA constructs resulted in functional channels. The resulting ASIC1a complexes are resolved as a predominant tetramer over the other oligomeric forms, after stabilization with BMOE or NaTT and SDS-PAGE/western blot analysis. Our data identify a major ASIC1a homotetramer at the surface membrane of the cell expressing functional ASIC1a channel.

The role of ASICs in cerebral ischemia

Cerebral ischemia is a leading cause of death and long-term disabilities worldwide. Excessive intracellular Ca(2+) accumulation in neurons has been considered essential for neuronal injury associated with cerebral ischemia. Although the involvement of glutamate receptors in neuronal Ca(2+) accumulation and toxicity has been the subject of intensive investigation, inhibitors for these receptors showed little effect in clinical trials. Thus, additional Ca(2+) toxicity pathway(s) must be involved. Acidosis is a common feature in cerebral ischemia and was known to cause brain injury. The mechanisms were, however, unclear. The finding that ASIC1a channels are highly enriched in brain neurons, their activation by ischemic acidosis, and their demonstrated Ca(2+) permeability suggested a role for these channels in Ca(2+) accumulation and neuronal injury associated with cerebral ischemia. Indeed, a number of studies have now provided solid evidence supporting the involvement of ASIC1a channel activation in ischemic brain injury.

Bile Salts Activate BLINaC - An Epithelial Na+ Channel in the Liver

Brain Liver Intestine Na+ Channel (BLINaC) is an ion channel of the DEG/ENaC gene family of unknown function. Expression of rat BLINaC (rBLINaC) in Xenopus oocytes leads to small unselective currents that are only weakly sensitive to amiloride. rBLINaC is potently inhibited by micromolar concentrations of extracellular Ca2+ and removal of Ca2+ leads to robust currents and increases Na+ selectivity. Recently we identified the fenamate flufenamic acid (FFA) and related compounds as agonists of rBLINaC. Application of millimolar concentrations of FFA to rBLINaC expressing oocytes induces a robust, Na+-selective current, which is partially blocked by amiloride. The identification of FFA as an artificial activator for BLINaC suggests the presence of an endogenous ligand.In rodents, the blinac mRNA is expressed mainly in brain, liver and intestine; in humans, it is mainly expressed in the small intestine. Immunhistochemical stainings of mouse liver revealed prominent expression of the BLINaC protein in the apical membrane of cholangiocytes lining the bile duct of mice, potentially implicating BLINaC in the generation of secondary bile. Guided by this localisation we tested whether the channel is affected by bile. The application of diluted bile to Xenopus oocytes expressing rBLINaC indeed induced a strong and reversible amiloride-sensitive unselective current. We identified bile salts as the BLINaC-activating molecules in bile and are currently trying to unravel the mechanistic basis of this activation. Bile salts are amphiphatic molecules that affect the structure, curvature and fluidity of membranes and might thus be activating BLINaC indirectly via a membrane dependent mechanism. Using a chimeric approach with ASIC1a, a related but bile salt-insensitive channel, we are currently defining the domains of BLINaC that are crucial for sensing membrane modulation induced by bile salts.

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

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

Subunit Oligomerization of Human ASIC1a in Xenopus Laevis Oocytes

The crystal structure of the chicken Acid Sensing Ion Channel 1 (ASIC1) shows a homotrimeric channel complex. Because of conflicting results regarding the subunit stoichiometry of other members of the ASIC ion channel family, we have determined the mass of the functional human ASIC1a channel complex expressed in Xenopus oocytes. We have used sulfhydryl crosslinkers or oxidizing agents to stabilize the native oligomeric ASIC1a complex. In the cut-open oocyte recording system, the intracellular perfusion of 2-20 mM sodium tetrathionate (NaTT) did not affect hASIC1a activity. Western blot analysis shows that NaTT induces a shift in the molecular weight of ASIC1a from the monomeric 70 kDa, to higher molecular weights of 140 and 280 kDa. ASIC1a purification after surface biotinylation and crosslinking shows that the major form of ASIC1a at the plasma membrane corresponds to a 280 kDa channel complex. The use of different sulfhydryl crosslinkers allowed to stabilize on Western blot oligomeric ASIC1a complexes of 280, 210 and 140 kDa corresponding to tetramers, trimers and dimers. This subunit crosslinking of the ASIC1a complex depends on cysteines in the C-terminus of the protein. We confirmed by size exclusion chromatography the presence of a hASIC1a channel complex of a mass corresponding approximatively to 280 kDa. In Xenopus oocytes sulfhydryl crosslinkers or oxidizing reagents stabilized a hASIC1a complex of 280 kDa without affecting channel activity; the mass of this channel complex cannot simply account for a trimer. The C-terminus of ASIC1a subunits is involved in subunit-subunit interactions and may be important for the oligomerization of the functional channel complex. This work was supported by a grant from the SNF.

High-density expression of Ca2+-permeable ASIC1a channels in NG2 glia of rat hippocampus.

NG2 cells, a fourth type of glial cell in the mammalian CNS, undergo reactive changes in response to a wide variety of brain insults. Recent studies have demonstrated that neuronally expressed acid-sensing ion channels (ASICs) are implicated in various neurological disorders including brain ischemia and seizures. Acidosis is a common feature of acute neurological conditions. It is postulated that a drop in pH may be the link between the pathological process and activation of NG2 cells. Such postulate immediately prompts the following questions: Do NG2 cells express ASICs? If so, what are their functional properties and subunit composition? Here, using a combination of electrophysiology, Ca2+ imaging and immunocytochemistry, we present evidence to demonstrate that NG2 cells of the rat hippocampus express high density of Ca2+-permeable ASIC1a channels compared with several types of hippocampal neurons. First, nucleated patch recordings from NG2 cells revealed high density of proton-activated currents. The magnitude of proton-activated current was pH dependent, with a pH for half-maximal activation of 6.3. Second, the current-voltage relationship showed a reversal close to the equilibrium potential for Na+. Third, psalmotoxin 1, a blocker specific for the ASIC1a channel, largely inhibited proton-activated currents. Fourth, Ca2+ imaging showed that activation of proton-activated channels led to an increase of [Ca2+]i. Finally, immunocytochemistry showed co-localization of ASIC1a and NG2 proteins in the hippocampus. Thus the acid chemosensor, the ASIC1a channel, may serve for inducing membrane depolarization and Ca2+ influx, thereby playing a crucial role in the NG2 cell response to injury following ischemia.

A Combined Computational and Functional Approach Identifies New Residues Involved in pH-dependent Gating of ASIC1a

Acid-sensing ion channels (ASICs) are key receptors for extracellular protons. These neuronal nonvoltage-gated Na(+) channels are involved in learning, the expression of fear, neurodegeneration after ischemia, and pain sensation. We have applied a systematic approach to identify potential pH sensors in ASIC1a and to elucidate the mechanisms by which pH variations govern ASIC gating. We first calculated the pK(a) value of all extracellular His, Glu, and Asp residues using a Poisson-Boltzmann continuum approach, based on the ASIC three-dimensional structure, to identify candidate pH-sensing residues. The role of these residues was then assessed by site-directed mutagenesis and chemical modification, combined with functional analysis. The localization of putative pH-sensing residues suggests that pH changes control ASIC gating by protonation/deprotonation of many residues per subunit in different channel domains. Analysis of the function of residues in the palm domain close to the central vertical axis of the channel allowed for prediction of conformational changes of this region during gating. Our study provides a basis for the intrinsic ASIC pH dependence and describes an approach that can also be applied to the investigation of the mechanisms of the pH dependence of other proteins.