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Divergent functional and conformational outcomes in an ion channel-peptide interaction

Christian B. Borg, View ORCID ProfileStephanie A. Heusser, Janne M. Colding, View ORCID ProfileStephan A. Pless
doi: https://doi.org/10.1101/2021.06.21.449215
Christian B. Borg
Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
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Stephanie A. Heusser
Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
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Janne M. Colding
Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
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Stephan A. Pless
Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
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  • ORCID record for Stephan A. Pless
  • For correspondence: stephan.pless@sund.ku.dk
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Abstract

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that contribute to fast synaptic transmission. Pharmacological inhibition of ASIC1a has been shown to reduce neurotoxicity and infarct volumes during stroke. The cysteine knot toxin Psalmotoxin-1 (PcTx1) is one of the most potent and selective inhibitors of ASIC1a. PcTx1 binds at the subunit interface, but both the stoichiometric requirements and the dynamics of the conformational consequences of the ion channel-peptide interaction remain unknown. Here, we use a combination of electrophysiology, voltage-clamp fluorometry and subunit concatenation to decipher the mechanism of PcTx1 inhibition. We observe a long-lived PcTx1-induced conformational change in the ASIC1a extracellular domain that is destabilized by the F350L mutation at the PcTx1 binding site. Concatemeric channel constructs show that two WT ASIC1a subunits are sufficient for WT-like current inhibition, while the presence of a single mutated subunit is enough to destabilize the PcTx1-induced conformation. Our results therefore demonstrate a divergence between the functional effects of PcTx1 on the pore and its conformational consequences in the extracellular domain. It further highlights how engineering of ion channels enables precise control over individual subunits for pharmacological and conformational assessment to determine the mechanism of ion channel–ligand interactions.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted June 22, 2021.
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Divergent functional and conformational outcomes in an ion channel-peptide interaction
Christian B. Borg, Stephanie A. Heusser, Janne M. Colding, Stephan A. Pless
bioRxiv 2021.06.21.449215; doi: https://doi.org/10.1101/2021.06.21.449215
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Divergent functional and conformational outcomes in an ion channel-peptide interaction
Christian B. Borg, Stephanie A. Heusser, Janne M. Colding, Stephan A. Pless
bioRxiv 2021.06.21.449215; doi: https://doi.org/10.1101/2021.06.21.449215

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