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Proc Natl Acad Sci U S A. 2020 Mar 31;117(13):7447-7454. doi: 10.1073/pnas.1919323117. Epub 2020 Mar 12.

Mechanism and site of action of big dynorphin on ASIC1a.

Author information

1
Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark.
2
Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark.
3
Copenhagen Center for Glycomics, Department of Odontology, University of Copenhagen, 2200 Copenhagen, Denmark.
4
Department of Molecular Biology, Vipergen ApS, 1610 Copenhagen, Denmark.
5
Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark; stephan.pless@sund.ku.dk.

Abstract

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to neurotransmission, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is up-regulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs, and noncanonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding results in an ASIC1a closed resting conformation that is distinct from open and desensitized states induced by protons. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is primarily mediated through electrostatic interactions of basic amino acids in the BigDyn N terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the noncanonical amino acid azidophenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn, and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics.

KEYWORDS:

acid-sensing ion channel; ligand–receptor interaction; neuropeptide; noncanonical amino acids; voltage-clamp fluorometry

PMID:
32165542
DOI:
10.1073/pnas.1919323117

Conflict of interest statement

The authors declare no competing interest.

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