Slowing of the Time Course of Acidification Decreases the Acid-Sensing Ion Channel 1a Current Amplitude and Modulates Action Potential Firing in Neurons

Acid-sensing ion channels (ASICs) are H+-activated neuronal Na+ channels. They are involved in fear behavior, learning, neurodegeneration after ischemic stroke and in pain sensation. ASIC activation has so far been studied only with fast pH changes, although the pH changes associated with many roles of ASICs are slow. It is currently not known whether slow pH changes can open ASICs at all. Here, we investigated to which extent slow pH changes can activate ASIC1a channels and induce action potential signaling. To this end, ASIC1a current amplitudes and charge transport in transfected Chinese hamster ovary cells, and ASIC-mediated action potential signaling in cultured cortical neurons were measured in response to defined pH ramps of 1-40 s duration from pH7.4 to pH6.6 or 6.0. A kinetic model of the ASIC1a current was developed and integrated into the Hodgkin-Huxley action potential model. Interestingly, whereas the ASIC1a current amplitude decreased with slower pH ramps, action potential firing was higher upon intermediate than fast acidification in cortical neurons. Indeed, fast pH changes (<4 s) induced short action potential bursts, while pH changes of intermediate speed (4-10 s) induced longer bursts. Slower pH changes (>10 s) did in many experiments not generate action potentials. Computer simulations corroborated these observations. We provide here the first description of ASIC function in response to defined slow pH changes. Our study shows that ASIC1a currents, and neuronal activity induced by ASIC1a currents, strongly depend on the speed of pH changes. Importantly, with pH changes that take >10 s to complete, ASIC1a activation is inefficient. Therefore, it is likely that currently unknown modulatory mechanisms allow ASIC activity in situations such as ischemia and inflammation

Accelerated Current Decay Kinetics of a Rare Human Acid-Sensing ion Channel 1a Variant That Is Used in Many Studies as Wild Type

Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels that are activated by extracellular acidification and are involved in fear sensing, learning, neurodegeneration after ischemia, and in pain sensation. We have recently found that the human ASIC1a (hASIC1a) wild type (WT) clone which has been used by many laboratories in recombinant expression studies contains a point mutation that occurs with a very low frequency in humans. Here, we compared the function and expression of ASIC1a WT and of this rare variant, in which the highly conserved residue Gly212 is substituted by Asp. Residue 212 is located at a subunit interface that undergoes changes during channel activity. We show that the modulation of channel function by commonly used ASIC inhibitors and modulators, and the pH dependence, are the same or only slightly different between hASIC1a-G212 and -D212. hASIC1a-G212 has however a higher current amplitude per surface-expressed channel and considerably slower current decay kinetics than hASIC1a-D212, and its current decay kinetics display a higher dependency on the type of anion present in the extracellular solution. We demonstrate for a number of channel mutants previously characterized in the hASIC1a-D212 background that they have very similar effects in the hASIC1a-G212 background. Taken together, we show that the variant hASIC1a-D212 that has been used as WT in many studies is, in fact, a mutant and that the properties of hASIC1a-D212 and hASIC1a-G212 are sufficiently close that the conclusions made in previous pharmacology and structure-function studies remain valid

Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures

The epithelial sodium channel (ENaC) is responsible for Na+ and fluid absorption across colon, kidney, and airway epithelia. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its NH2-terminal region lacks structure. Here we found that an 18 amino acid peptide, S18, which corresponded to residues G22-A39 of the SPLUNC1 NH2 terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold), while SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the βENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid (ASL) height was abnormally low (4.2 ± 0.6 μm), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 ± 0.6 μm, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease

Analysis of gating of Acid-Sensing Ion Channels (ASICs)

Les cellules neuronales, qui sont les unités composant le système nerveux, peuvent « sentir » tous les changements du milieu qui se passe soit à l'interne ou aussi à l'externe de la cellule. Un neurone se sert d'un riche répertoire de « senseurs », ou récepteurs, pour percevoir tous ces changements. De plus ce qui est incroyable, c'est que chaqu'un de ces récepteurs est spécialisé à détecter un type de changement seulement. Par exemple, un neurone aura des récepteurs spécifiques pour détecter des changements du taux de CO2 dans le sang, d'autres pour détecter le niveau de sucre ou la quantité de neurotransmetteurs et d'autres, appelé ASICs, pour sentir des acidifications extracellulaires. Pourquoi les neurones ont ces ASICs pour sentir ces acidifications ? Pendant un accident vasculaire cérébral (AVC) ischémique, notre cerveau devient comme un citron. Des acidifications lentes s'établent et ces ASICs sont donc très sollicités sous ces conditions et peuvent causer la mort des neurones. Mais si bien contrôlé, une acidité peut aussi être bénéfique et agir comme un neurotransmetteur et donc promouvoir la communication neuronale via les ASICs. La majorité des scientifiques a étudié ces ASICs suite à des acidifications rapides. C'est donc indispensable de connaitre comment ces ASICs se comportent suite à des acidifications de nature plus lentes associés aux genres d'acidifications qu'on a dans nos cerveaux. J'ai donc d'abord établit un modèle informatique pour simuler le comportement de ces ASICs suite aux acidifications lentes. Par les expériences, j'ai confirmé les prédictions des simulations en observant que les neurones sont beaucoup plus sollicités lors des acidifications lentes plutôt que des acidifications rapides. De plus, lorsque les changements de pH étaient accompagnées de changements de milieu qui simulaient les conditions de milieu lors de fortes communications neuronales, ces acidifications lentes stimulaient encore plus les neurones. Le comportement des ASICs s'avéré être prédit par le modèle informatique, qui pourra donc constituer un outil pour prédire la réponse d'ASICs en conditions plus physiologiques. Dans un autre projet, plutôt pharmacologique, j'ai améliorée certains modulateurs d'ASICs en découvrant des composés plus puissants. J'ai découvert des composés qui bloquent la réponse d'ASICs. Ils pourront donc être bénéfiques pour réduire les conséquences d'un AVC. J'ai découvert aussi des composés particuliers qui potentialisent l'activité d'ASICs. Ils pourront alors promouvoir la communication neuronale et améliorer nos performances cognitives. -- L'acidité est généralement associée à la douleur, spécialement dans le système nerveux périphérique (SNP). Dans le système nerveux central (SNC), au contraire, elle peut promouvoir la communication neuronale. Les neurones ont plusieurs types de senseurs d'acidité, entre autres les « acid-sensing ion channels » (ASICs). Ils sont des canaux ioniques qui s'ouvrent de façon transitoire en réponse à une acidification extracellulaire pour permettre au sodium d'entrer dans le neurone et induire des potentiels d'actions. Le comportement des ASICs en réponse au pH peut être décrit avec trois états de conformation différents : l'état fermé, ouvert et désensibilisé. Les transitions cinétiques reliant ces trois états composent le modèle cinétique des ASICs, qui au début de ma thèse n'avait pas été établit. En utilisant des techniques d'électrophysiologie, j'ai décrit le modèle cinétique d'ASICla et -3, qui sont respectivement des sous-unités exprimé dans le SNC et SNP. Avec les données récoltées j'ai construit un modèle computationnel pour prédire la réponse d'ASIC au changement de pH et aux vitesses de ces changements. J'ai modelé et confirmé expérimentalement avec succès le comportement des ASICs suite à un changement rapide ou lent de pH, qui sont des événements pouvant se dérouler dans notre cerveau en condition normale ou lors d'une ischémie cérébrale, et au niveau du SNP pendant une inflammation. Il est attendu qu'une acidification rapide ouvre les ASICs, tandis qu'une acidification lente les désensibilise. Cependant, j'ai observé que, une acidification lente peut ouvrir les ASICs et générer des petits courants. J'ai modelé la réponse neuronale à ces petits courants et montré expérimentalement qu'elles peuvent engendrer plus de potentiels d'actions et accroitre l'excitabilité neuronale par rapport aux grands courants issus des acidifications rapides. Finalement j'ai montré que ASICla est nécessaire pour moduler l'excitation neuronale en réponse à des changements ioniques, outre que le pH, qui ont heu pendant une haute activité de transmission nerveuse. Le deuxième projet de ma thèse s'est basé sur la découverte d'une molécule appelé 2- guanidine-4-methylquinazoline (GMQ), qui pouvait moduler ASICla et -3. Malgré des propriétés intéressantes, GMQ ne montre qu'une faible spécificité et puissance. Nous avons alors instauré une collaboration avec un chimiste afin de faire un screening sur ASICla et -3 avec la technique d'électrophysiologie pour tester différents composés dérivant du GMQ. J'ai donc trouvé des toutes nouvelles molécules actives sur ASICla et -3, montrant une puissance plus élevée et une meilleure spécificité entre les deux sous-unités. J'ai finalement identifié la première molécule chimique qui a le pouvoir de potentialiser le courant d'ASICla. La potentialisation pharmacologique d'ASICla pourrait aboutir à des performances accrues de la communication neuronale et améliorer les fonctions cognitives. -- Acidity is often associated with pain, especially when we consider the peripheral nervous system (PNS). On the other hand, protons participate in neuronal communication in the central nervous system (CNS). Neurons have différent types of acid sensors. Among these sensors are the acid-sensing ion channels (ASICs). These channels open transiently in response to extracellular acidifications and allow sodium entering the cell. This entry of sodium causes depolarizations in neurons and eventually induces action potentials. The ASIC behavior in response to pH can be described by the transitions between three main conformational states of these channels: the closed, the open and the desensitized state. The gating transitions describe how the ASICs switch from one state to another as a fonction of the pH and time. The gating transitions connect the three conformational states and compose the so-called ASIC kinetic scheme, which at the beginning of my thesis was still poorly defined. Using patch-clamp electrophysiology I measured the transition rates of the kinetic scheme of ASIC la and -3 isoforms that are highly expressed in the CNS and PNS respectively. With the collected data I built a computational model that I used to predict the behavior of ASICs in response to any extent of pH changes and also to any speed by how these pH changes occur. I modeled and successfully confirmed by mean of experiments the ASIC behavior in response to rapid and slow acidification. Slow acidifications can occur in vivo in our brain under normal condition, during an ischemic stroke and neuronal inflammation. Rapid acidifications promote the opening of ASICs, while slow acidifications are thought to rather promote desensitization. However, I showed that a slow pH change can resuit in ASIC opening and generate small currents. I modeled the neuronal response to these small currents and showed then experimentally that they can induce higher neuronal excitation than the higher currents of rapid acidifications. Finally, I showed that ASIC la is necessary to mediate neuronal excitation in response to ion changes, other than pH, normally occurring with high neurotransmission activity. The second project of my thesis was based on the discovery of a small molecule called 2-guanidine-4-methylquinazoline (GMQ) that has been identified to modulate ASIC la and ASIC3. Despite interesting properties on ASICs gating, GMQ présents low specificity and low potency. Therefore, in collaboration with a chemist, we started a screening of GMQ-derivatives by patch-clamp electrophysiology and identified new compounds active on ASIC la and -3 that show higher specificity among the two isoforms, and higher potency. I also identified the first known chemical that enhances ASIC la currents. The pharmacological ASIC la potentiation may enhance neuronal communication and improve cognitive function

Changes in H+, K+, and Ca2+ Concentrations, as Observed in Seizures, Induce Action Potential Signaling in Cortical Neurons by a Mechanism That Depends Partially on Acid-Sensing 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 Ca2+ concentration decreases. We hypothesized that the concomitant changes in H+, K+ and Ca2+ 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 Ca2+ 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 Ca2+ together with the increase in the K+ concentration led to a hyperpolarizing shift of the activation voltage dependence of voltage-gated Na+ channels and to an increase in their current amplitude. 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

Molecular determinants of desensitization in an ENaC/degenerin channel

Epithelial Na(+) channel (ENaC)/degenerin family members are involved in mechanosensation, blood pressure control, pain sensation, and the expression of fear. Several of these channel types display a form of desensitization that allows the channel to limit Na(+) influx during prolonged stimulation. We used site-directed mutagenesis and chemical modification, functional analysis, and molecular dynamics simulations to investigate the role of the lower palm domain of the acid-sensing ion channel 1, a member of the ENaC/degenerin family. The lower palm domains of this trimeric channel are arranged around a central vestibule, at ∼20 Å above the plasma membrane and are covalently linked to the transmembrane channel parts. We show that the lower palm domains approach one another during desensitization. Residues in the palm co-determine the pH dependence of desensitization, its kinetics, and the stability of the desensitized state. Mutations of palm residues impair desensitization by preventing the closing movement of the palm. Overexpression of desensitization-impaired channel mutants in central neurons allowed-in contrast to overexpression of wild type-a sustained signaling response to rapid pH fluctuations. We identify and describe here the function of an important regulatory domain that most likely has a conserved role in ENaC/degenerin channels.-Roy, S., Boiteux, C., Alijevic, O., Liang, C., Bernèche, S., Kellenberger, S. Molecular determinants of desensitization in an ENaC/degenerin channel

Heteroarylguanidines as Allosteric Modulators of ASIC1a and ASIC3 Channels

Acid-sensing ion channels (ASICs) are neuronal Na⁺-selective ion channels that open in response to extracellular acidification. They are involved in pain, fear, learning, and neurodegeneration after ischemic stroke. 2-Guanidine-4-methylquinazoline (GMQ) was recently discovered as the first nonproton activator of ASIC3. GMQ is of interest as a gating modifier and pore blocker of ASICs. It has however a low potency, and exerts opposite effects on ASIC1a and ASIC3. To further explore the molecular mechanisms of GMQ action, we have used the guanidinium moiety of GMQ as a scaffold and tested the effects of different GMQ derivatives on the ASIC pH dependence and maximal current. We report that GMQ derivatives containing quinazoline and quinoline induced, as GMQ, an alkaline shift of the pH dependence of activation in ASIC3 and an acidic shift in ASIC1a. Another group of 2-guanidinopyridines shifted the pH dependence of both ASIC1a and ASIC3 to more acidic values. Several compounds induced an alkaline shift of the pH dependence of ASIC1a/2a and ASIC2a/3 heteromers. Compared to GMQ, guanidinopyridines showed a 20-fold decrease in the IC₅₀ for ASIC1a and ASIC3 current inhibition at pH 5. Strikingly, 2-guanidino-quinolines and -pyridines showed a concentration-dependent biphasic effect that resulted at higher concentrations in ASIC1a and ASIC3 inhibition (IC₅₀ > 100 μM), while causing at lower concentration a potentiation of ASIC1a, but not ASIC3 currents (EC₅₀ ≈ 10 μM). In conclusion, we describe a new family of small molecules as ASIC ligands and identify an ASIC subtype-specific potentiation by a subgroup of these compounds

Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures.

Item does not contain fulltextThe epithelial sodium channel (ENaC) is responsible for Na(+) and fluid absorption across colon, kidney, and airway epithelia. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its NH2-terminal region lacks structure. Here we found that an 18 amino acid peptide, S18, which corresponded to residues G22-A39 of the SPLUNC1 NH2 terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 ( approximately 2.5 fold), while SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the betaENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid (ASL) height was abnormally low (4.2 +/- 0.6 mum), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 +/- 0.6 mum, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease.1 december 201