Kinetic analysis of ASIC1a delineates conformational signaling from proton-sensing domains to the channel gate

Vullo, Sabrina; Ambrosio, Nicolas; Kučera, Jan; Bignucolo, Olivier; Kellenberger, Stephan

doi:10.7554/elife.66488

Cited by 8 publications

(9 citation statements)

References 57 publications

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“…Figure 2D and E, 3D and E). While non-apparent openings suggested by the linear gating model ( 14) could explain changes in fluorescence at moderate pH, we deem it more likely that these fluorescence changes are a result of protonation events in the ECD that primarily lead to SSD in a model supported by others (19,20). Disruption of the swiveling motion of the β11-12 linker via the N4141K mutation thus limited acute desensitization and SSD, but the dynamic conformational changes of the upper and mid-ECD appeared largely retained (Figure 2E and 3E).…”

Section: Conformational Changes In Ecd and Upper Tmd Are Associated W...mentioning

confidence: 64%

“…To this end, we labeled the channel in two more positions previously reported to yield changes in fluorescence signals, E425C and H72C (Figure 4A, Suppl. Figure S4A) (19, 40, 45).…”

Section: Resultsmentioning

confidence: 99%

“…The degree of SSD is dependent on the proton concentration and subunit composition, but ultimately, channels need to recover from desensitization in high pH to regain the ability to activate fully (14). The different states were proposed to be either connected in a linear gating model, where a desensitized state can only be reached via the open state (14), or a branching model that allows a direct transition into a desensitized state (19, 20).…”

Section: Introductionmentioning

confidence: 99%

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Dynamic conformational changes of acid-sensing ion channels in different desensitizing conditions

Marcher Holm,

Topaktas,

Dannesboe

et al. 2023

Preprint

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Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to fast synaptic transmission and have roles in fear conditioning and nociception. Apart from activation at low pH, ASIC1a also undergoes several types of desensitization, including ‘acute desensitization’ that terminates activation, ‘steady-stated desensitization’ that occurs at sub-activating proton concentrations and limits subsequent activation, and ‘tachyphylaxis’ that results in a progressive decrease in response during a series of activations. Structural insights from a desensitized state of ASIC1 have provided great spatial detail, but dynamic insights into conformational changes in different desensitizing conditions are largely missing. Here, we use electrophysiology and voltage-clamp fluorometry to follow the functional changes of the pore along with conformational changes at several positions in the extracellular and upper transmembrane domain via cysteine-labeled fluorophores. Acute desensitization terminates activation in wild-type but introducing an N414K mutation in the β11-12 linker of mouse ASIC1a interfered with this process. The mutation also affected steady-stated desensitization and led to pronounced tachyphylaxis. Common to all types of desensitization was that the extracellular domain remained sensitive to pH and underwent pH-dependent conformational changes. These conformational changes did, however, not necessarily lead to opening of the pore, which appeared disconnected from the extracellular domain of the protein in mutant channels. N414K-containing channels remained sensitive to a known peptide modulator that increased steady-state desensitization, indicating that the mutation only destabilized but not precluded desensitization. Together, this study contributes to understanding the fundamental properties of ASIC1a desensitization, emphasizing the complex interplay between the conformational changes of the ECD and the pore during channel activation and desensitization.Statement of significanceAcid-sensing ion channels (ASICs) are proton-gated ion channels that contribute to synaptic activity and play roles in acidosis-related diseases. Prolonged acidosis can lead to desensitization in ASIC1a, and modulators that affect this desensitization have shown beneficial effects in pain and stroke. In this study, we investigated the functional and conformational changes during acute desensitization, steady-state desensitization, and tachyphylaxis through a mutation in the β11- β12 linker of ASIC1a. We found that the mutation retained pH-dependent conformational changes of the extracellular domain (ECD) but largely disconnected these movements from the channel pore. Collectively, our work emphasizes the critical role of the β11- β12 linker for the pH-dependent conformational interplay between the ECD and the channel pore.

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Section: Conformational Changes In Ecd and Upper Tmd Are Associated W...mentioning

confidence: 64%

“…To this end, we labeled the channel in two more positions previously reported to yield changes in fluorescence signals, E425C and H72C (Figure 4A, Suppl. Figure S4A) (19, 40, 45).…”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Dynamic conformational changes of acid-sensing ion channels in different desensitizing conditions

Marcher Holm,

Topaktas,

Dannesboe

et al. 2023

Preprint

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“…Functional observations indicate that ASIC1a is activated by H + -induced allosteric changes 40 . Further support for an allosteric activation mechanism came from voltage-clamp fluorometry experiments which showed evidence for conformational changes linked to ASIC1a activation 27, 28, 29, 30 and from the observation that mutation of the two residues involved in ASIC1a pore block by Ca 2+ did not abolish pH-dependent gating 17 . The E425G mutation decreased the Ca 2+ -induced shift of the pH dependence of SSD, while D432C decreased the Ca 2+ -induced shift of the activation pH dependence 17 .…”

Section: Discussionmentioning

confidence: 99%

Identification of the modulatory Ca²⁺binding sites of acid-sensing ion channel 1a

Molton,

Bignucolo,

Kellenberger

2023

Preprint

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Acid-sensing ion channels (ASICs) are neuronal H+-gated, Na+-permeable channels involved in learning, fear sensing, pain sensation and neurodegeneration. An increase in the extracellular Ca2+concentration shifts the pH dependence of ASIC1a to more acidic values. Here, we predicted candidate residues for Ca2+binding on ASIC1a, based on available structural information and molecular dynamics simulations; the function of channels carrying mutations of these residues was then measured. We identify several residues in cavities previously associated with pH-dependent gating, whose mutation decreased the Ca2+-induced shift in ASIC1a pH dependence, likely due to a disruption of Ca2+binding. We show also that Mg2+shares some of the binding sites with Ca2+, and that some of the 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.

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“…A study with toadfish ASIC1 also found a competition between H + and Ca 2+ and concluded, based on a detailed functional analysis, that this channel is activated by an allosteric mechanism and not by a release of Ca 2+ block [32]. A further indication that in ASIC1a, activation involves conformational changes comes from voltage clamp fluorometry studies that detected conformational changes associated with channel opening [33,34].…”

Section: Discussionmentioning

confidence: 99%

Calcium regulates acid-sensing ion channel 3 activation by competing with protons in the channel pore and at an allosteric binding site

et al. 2022

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The extracellular Ca 2+ concentration changes locally under certain physiological and pathological conditions. Such variations affect the function of ion channels of the nervous system and consequently also neuronal signalling. We investigated here the mechanisms by which Ca 2+ controls the activity of acid-sensing ion channel (ASIC) 3. ASICs are neuronal, H + -gated Na + channels involved in several physiological and pathological processes, including the expression of fear, learning, pain sensation and neurodegeneration after ischaemic stroke. It was previously shown that Ca 2+ negatively modulates the ASIC pH dependence. While protons are default activators of ASIC3, this channel can also be activated at pH7.4 by the removal of the extracellular Ca 2+ . Two previous studies concluded that low pH opens ASIC3 by displacing Ca 2+ ions that block the channel pore at physiological pH. We show here that an acidic residue, distant from the pore, together with pore residues, controls the modulation of ASIC3 by Ca 2+ . Our study identifies a new regulatory site in ASIC3 and demonstrates that ASIC3 activation involves an allosteric mechanism together with Ca 2+ unbinding from the channel pore. We provide a molecular analysis of a regulatory mechanism found in many ion channels.

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Kinetic analysis of ASIC1a delineates conformational signaling from proton-sensing domains to the channel gate

Cited by 8 publications

References 57 publications

Dynamic conformational changes of acid-sensing ion channels in different desensitizing conditions

Dynamic conformational changes of acid-sensing ion channels in different desensitizing conditions

Identification of the modulatory Ca²⁺binding sites of acid-sensing ion channel 1a

Calcium regulates acid-sensing ion channel 3 activation by competing with protons in the channel pore and at an allosteric binding site

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Kinetic analysis of ASIC1a delineates conformational signaling from proton-sensing domains to the channel gate

Cited by 8 publications

References 57 publications

Dynamic conformational changes of acid-sensing ion channels in different desensitizing conditions

Dynamic conformational changes of acid-sensing ion channels in different desensitizing conditions

Identification of the modulatory Ca2+binding sites of acid-sensing ion channel 1a

Calcium regulates acid-sensing ion channel 3 activation by competing with protons in the channel pore and at an allosteric binding site

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Identification of the modulatory Ca²⁺binding sites of acid-sensing ion channel 1a