The epithelial Na(+) channel (ENaC) and the acid-sensing ion channels (ASICs) form subfamilies within the ENaC/degenerin family of Na(+) channels. ENaC mediates transepithelial Na(+) transport, ...thereby contributing to Na(+) homeostasis and the maintenance of blood pressure and the airway surface liquid level. ASICs are H(+)-activated channels found in central and peripheral neurons, where their activation induces neuronal depolarization. ASICs are involved in pain sensation, the expression of fear, and neurodegeneration after ischemia, making them potentially interesting drug targets. This review summarizes the biophysical properties, cellular functions, and physiologic and pathologic roles of the ASIC and ENaC subfamilies. The analysis of the homologies between ENaC and ASICs and the relation between functional and structural information shows many parallels between these channels, suggesting that some mechanisms that control channel activity are shared between ASICs and ENaC. The available crystal structures and the discovery of animal toxins acting on ASICs provide a unique opportunity to address the molecular mechanisms of ENaC and ASIC function to identify novel strategies for the modulation of these channels by pharmacologic ligands.
Acid-sensing ion channels (ASICs) are neuronal, proton-gated, Na.sup.+ -selective ion channels. They are involved in various physiological and pathological processes such as neurodegeneration after ...stroke, pain sensation, fear behavior and learning. To obtain information on the activation mechanism of ASIC1a, we attempted in this study to impose distance constraints between paired residues in different channel domains by using cross-linkers reacting with engineered Cys residues, and we measured how this affected channel function. First, the optical tweezer 4'-Bis(maleimido)azobenzene (BMA) was used, whose conformation changes depending on the wavelength of applied light. After exposure of channel mutants to BMA, an activation of the channel by light was only observed with a mutant containing a Cys mutation in the extracellular pore entry, I428C. Western blot analysis indicated that BMA did not cross-link Cys428 residues. Extracellular application of methanethiosulfonate (MTS) cross-linkers of different lengths changed the properties of several Cys mutants, in many cases likely without cross-linking two Cys residues. Our observations suggest that intersubunit cross-linking occurred in the wrist mutant A425C and intrasubunit cross-linking in the acidic pocket mutant D237C/I312C. In these mutants, exposure to cross-linkers favored a non-conducting channel conformation and induced an acidic shift of the pH dependence and a decrease of the maximal current amplitude. Overall, the cross-linking approaches appeared to be inefficient, possibly due to the geometrical requirements for successful reactions of the two ends of the cross-linking compound.
Acid‐sensing ion channels (ASICs) and the epithelial Na+ channel (ENaC) are both members of the ENaC/degenerin family of amiloride‐sensitive Na+ channels. ASICs act as proton sensors in the nervous ...system where they contribute, besides other roles, to fear behaviour, learning and pain sensation. ENaC mediates Na+ reabsorption across epithelia of the distal kidney and colon and of the airways. ENaC is a clinically used drug target in the context of hypertension and cystic fibrosis, while ASIC is an interesting potential target. Following a brief introduction, here we will review selected aspects of ASIC and ENaC function. We discuss the origin and nature of pH changes in the brain and the involvement of ASICs in synaptic signalling. We expose how in the peripheral nervous system, ASICs cover together with other ion channels a wide pH range as proton sensors. We introduce the mechanisms of aldosterone‐dependent ENaC regulation and the evidence for an aldosterone‐independent control of ENaC activity, such as regulation by dietary K+. We then provide an overview of the regulation of ENaC by proteases, a topic of increasing interest over the past few years. In spite of the profound differences in the physiological and pathological roles of ASICs and ENaC, these channels share many basic functional and structural properties. It is likely that further research will identify physiological contexts in which ASICs and ENaC have similar or overlapping roles.
Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate chemical communication between neurons at synapses. A variant iGluR subfamily, the Ionotropic Receptors (IRs), was ...recently proposed to detect environmental volatile chemicals in olfactory cilia. Here, we elucidate how these peripheral chemosensors have evolved mechanistically from their iGluR ancestors. Using a
Drosophila model, we demonstrate that IRs act in combinations of up to three subunits, comprising individual odor-specific receptors and one or two broadly expressed coreceptors. Heteromeric IR complex formation is necessary and sufficient for trafficking to cilia and mediating odor-evoked electrophysiological responses in vivo and in vitro. IRs display heterogeneous ion conduction specificities related to their variable pore sequences, and divergent ligand-binding domains function in odor recognition and cilia localization. Our results provide insights into the conserved and distinct architecture of these olfactory and synaptic ion channels and offer perspectives into the use of IRs as genetically encoded chemical sensors.
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► IRs are variant olfactory-expressed iGluRs essential for odor detection ► IRs function in combinations of up to three different common and variable subunits ► IR complex assembly is necessary and sufficient for activity in heterologous cells ► IR ligand-binding domains function in odor recognition and cilia localization
Institut de Pharmacologie et de Toxicologie,
Université de Lausanne, Lausanne,
Switzerland
Kellenberger, Stephan and
Laurent Schild.
Epithelial Sodium Channel/Degenerin Family of Ion Channels: A
...Variety of Functions for a Shared Structure. Physiol. Rev. 82: 735-767, 2002. The recently discovered
epithelial sodium channel (ENaC)/degenerin (DEG) gene family encodes
sodium channels involved in various cell functions in metazoans.
Subfamilies found in invertebrates or mammals are functionally
distinct. The degenerins in Caenorhabditis elegans
participate in mechanotransduction in neuronal cells, FaNaC in snails
is a ligand-gated channel activated by neuropeptides, and the
Drosophila subfamily is expressed in gonads and neurons. In
mammals, ENaC mediates Na + transport in epithelia and is
essential for sodium homeostasis. The ASIC genes encode
proton-gated cation channels in both the central and peripheral
nervous system that could be involved in pain transduction. This review
summarizes the physiological roles of the different channels belonging
to this family, their biophysical and pharmacological characteristics,
and the emerging knowledge of their molecular structure. Although
functionally different, the ENaC/DEG family members share functional
domains that are involved in the control of channel activity and in the
formation of the pore. The functional heterogeneity among the members
of the ENaC/DEG channel family provides a unique opportunity to address the molecular basis of basic channel functions such as activation by
ligands, mechanotransduction, ionic selectivity, or block by pharmacological ligands.
Acid-sensing ion channels (ASICs) are proton-activated Na⁺ channels expressed in the nervous system, where they are involved in learning, fear behaviors, neurodegeneration, and pain sensation. In ...this work, we study the role in pH sensing of two regions of the ectodomain enriched in acidic residues: the acidic pocket, which faces the outside of the protein and is the binding site of several animal toxins, and the palm, a central channel domain. Using voltage clamp fluorometry, we find that the acidic pocket undergoes conformational changes during both activation and desensitization. Concurrently, we find that, although proton sensing in the acidic pocket is not required for channel function, it does contribute to both activation and desensitization. Furthermore, protonation-mimicking mutations of acidic residues in the palm induce a dramatic acceleration of desensitization followed by the appearance of a sustained current. In summary, this work describes the roles of potential pH sensors in two extracellular domains, and it proposes a model of acidification-induced conformational changes occurring in the acidic pocket of ASIC1a.
Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels activated by extracellular acidification. ASICs are involved in learning, fear sensing, pain sensation and neurodegeneration. ...Increasing the extracellular Ca2+ concentration decreases the H+ sensitivity of ASIC1a, suggesting a competition for binding sites between H+ and Ca2+ ions. Here, we predicted candidate residues for Ca2+ binding on ASIC1a, based on available structural information and our molecular dynamics simulations. With functional measurements, we identified several residues in cavities previously associated with pH-dependent gating, whose mutation reduced the modulation by extracellular Ca2+ of the ASIC1a pH dependence of activation and desensitization. This occurred likely owing to a disruption of Ca2+ binding. Our results link one of the two predicted Ca2+-binding sites in each ASIC1a acidic pocket to the modulation of channel activation. Mg2+ regulates ASICs in a similar way as does Ca2+. We show that Mg2+ shares some of the binding sites with Ca2+. Finally, we provide evidence that some of the ASIC1a Ca2+-binding sites are functionally conserved in the splice variant ASIC1b. Our identification of divalent cation-binding sites in ASIC1a shows how Ca2+ affects ASIC1a gating, elucidating a regulatory mechanism present in many ion channels.Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels activated by extracellular acidification. ASICs are involved in learning, fear sensing, pain sensation and neurodegeneration. Increasing the extracellular Ca2+ concentration decreases the H+ sensitivity of ASIC1a, suggesting a competition for binding sites between H+ and Ca2+ ions. Here, we predicted candidate residues for Ca2+ binding on ASIC1a, based on available structural information and our molecular dynamics simulations. With functional measurements, we identified several residues in cavities previously associated with pH-dependent gating, whose mutation reduced the modulation by extracellular Ca2+ of the ASIC1a pH dependence of activation and desensitization. This occurred likely owing to a disruption of Ca2+ binding. Our results link one of the two predicted Ca2+-binding sites in each ASIC1a acidic pocket to the modulation of channel activation. Mg2+ regulates ASICs in a similar way as does Ca2+. We show that Mg2+ shares some of the binding sites with Ca2+. Finally, we provide evidence that some of the ASIC1a Ca2+-binding sites are functionally conserved in the splice variant ASIC1b. Our identification of divalent cation-binding sites in ASIC1a shows how Ca2+ affects ASIC1a gating, elucidating a regulatory mechanism present in many ion channels.
Acid-sensing ion channels (ASICs) are activated by extracellular acidification. Because ASIC currents are transient, these channels appear to be ideal sensors for detecting the onset of rapid pH ...changes. ASICs are involved in neuronal death after ischemic stroke, and in the sensation of inflammatory pain. Ischemia and inflammation are associated with a slowly developing, long-lasting acidification. Recent studies indicate however that ASICs are unable to induce an electrical signaling activity under standard experimental conditions if pH changes are slow. In situations associated with slow and sustained pH drops such as high neuronal signaling activity and ischemia, the extracellular K
+
concentration increases, and the Ca
2+
concentration decreases. We hypothesized that the concomitant changes in H
+
, K
+
, and Ca
2+
concentrations may allow a long-lasting ASIC-dependent induction of action potential (AP) signaling. We show that for acidification from pH7.4 to pH7.0 or 6.8 on cultured cortical neurons, the number of action potentials and the firing time increased strongly if the acidification was accompanied by a change to higher K
+
and lower Ca
2+
concentrations. Under these conditions, APs were also induced in neurons from ASIC1a
–/–
mice, in which a pH of ≤ 5.0 would be required to activate ASICs, indicating that ASIC activation was not required for the AP induction. Comparison between neurons of different ASIC genotypes indicated that the ASICs modulate the AP induction under such changed ionic conditions. Voltage-clamp measurements of the Na
+
and K
+
currents in cultured cortical neurons showed that the lowering of the pH inhibited Na
+
and K
+
currents. In contrast, the lowering of the Ca
2+
together with the increase in the K
+
concentration led to a hyperpolarizing shift of the activation voltage dependence of voltage-gated Na
+
channels. We conclude that the ionic changes observed during high neuronal activity mediate a sustained AP induction caused by the potentiation of Na
+
currents, a membrane depolarization due to the changed K
+
reversal potential, the activation of ASICs, and possibly effects on other ion channels. Our study describes therefore conditions under which slow pH changes induce neuronal signaling by a mechanism involving ASICs.
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The pH in the different tissues and organs of our body is kept within tight limits. Local pH changes occur, however, temporarily under physiological conditions, as for example in ...synapses during neuronal activity. In pathological situations, such as in ischemia, inflammation, and tumor growth, long-lasting acidification develops. Acid-sensing ion channels (ASICs) are low pH-activated Na+-permeable ion channels that are widely expressed in the central and peripheral nervous systems. ASICs act as pH sensors, leading to neuronal excitation when the pH drops. Animal studies have shown that ASICs are involved in several physiological and pathological processes, such as pain sensation, learning, fear sensing, and neurodegeneration after ischemic stroke. ASIC inhibitors could be used as analgesic and anxiolytic drugs, and as drugs for the treatment of ischemic stroke. For these reasons, ASICs have recently attracted increasing attention. Currently, no drugs are clinically used as ASIC modulators. ASICs are however targets of several peptide toxins from animals. Much effort is invested in research studying the function of these channels. We review here the available pharmacological agents acting on ASICs, which include small molecules and animal toxins. We then discuss the current understanding of the molecular mechanisms by which pH controls ASIC activity. Knowledge of the function of ASICs at the molecular level should allow the development of new pharmacological strategies for targeting these promising ion channels.
BACKGROUND AND PURPOSE APETx2, a toxin from the sea anemone Anthropleura elegantissima, inhibits acid‐sensing ion channel 3 (ASIC3)‐containing homo‐ and heterotrimeric channels with IC50 values < 100 ...nM and 0.1–2 µM respectively. ASIC3 channels mediate acute acid‐induced and inflammatory pain response and APETx2 has been used as a selective pharmacological tool in animal studies. Toxins from sea anemones also modulate voltage‐gated Na+ channel (Nav) function. Here we tested the effects of APETx2 on Nav function in sensory neurones.
EXPERIMENTAL APPROACH Effects of APETx2 on Nav function were studied in rat dorsal root ganglion (DRG) neurones by whole‐cell patch clamp.
KEY RESULTS APETx2 inhibited the tetrodotoxin (TTX)‐resistant Nav 1.8 currents of DRG neurones (IC50, 2.6 µM). TTX‐sensitive currents were less inhibited. The inhibition of Nav 1.8 currents was due to a rightward shift in the voltage dependence of activation and a reduction of the maximal macroscopic conductance. The inhibition of Nav 1.8 currents by APETx2 was confirmed with cloned channels expressed in Xenopus oocytes. In current‐clamp experiments in DRG neurones, the number of action potentials induced by injection of a current ramp was reduced by APETx2.
CONCLUSIONS AND IMPLICATIONS APETx2 inhibited Nav 1.8 channels, in addition to ASIC3 channels, at concentrations used in in vivo studies. The limited specificity of this toxin should be taken into account when using APETx2 as a pharmacological tool. Its dual action will be an advantage for the use of APETx2 or its derivatives as analgesic drugs.
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