Mechanism and site of action of big dynorphin on ASIC1a

Abstract

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to synaptic plasticity, 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 upregulated 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 non-canonical amino acidmediated photocrosslinking. We demonstrate that BigDyn binding induces ASIC1a conformational changes that are different from those induced by protonation and likely represent a distinct closed state. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is 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 non-canonical 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highlight this cavity as an important site for the development of ASIC-targeting therapeutics.Note: This manuscript has not been peer-reviewed 3

High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

“…Cell culture and transfection. HEK 293T cells (ATCC ® ), in which endogenous hASIC1a was removed by CRISPR/Cas9 (Borg et al, 2020), were grown in monolayer in T75 or T175 flasks (Orange Scientific, Belgium) in DMEM (Gibco, Denmark) supplemented with 10 % FBS (Thermo Fisher Scientific, Denmark) and 1 % penicillin-streptomycin (Thermo Fisher Scientific) and incubated at 37 ˚C in a humidified 5 % CO2 atmosphere. For APC experiments, cells were seeded into six-well plates (Orange Scientific) at a density of 300.000 cells/well and transfected the next day with Trans-IT LT1 (Mirus, WI, USA) and 1:1:1:1 µg DNA encoding hASIC1a TAG variants, ncAA-RS, tRNA and eGFP Y40TAG or Y151TAG, respectively.…”

“…Here, we focused on AzF-containing variants for which we had previously detected crosslinking to BigDyn on Western blots to evaluate if the observed peptide-channel interaction also results in functional modulation (Borg et al, 2020). Cells were exposed to SSD-inducing pH conditions in the presence or absence of 3 µM BigDyn and the resulting currents upon pH 5.6 activation were normalized to control currents after incubation at pH 7.6 ( Figure 4A+B).…”

“…BigDyn is likely to be highly flexible without a strong propensity to adopt a secondary fold (O'Connor et al, 2015;Ferre et al, 2019) and therefore samples a greater conformational space and is thus more likely to undergo covalent crosslinking at multiple sites (9/9 sites tested at the acidic pocket, (Borg et al, 2020)). By contrast, PcTx1 folds into a compact and highly stable conformation and will consequently undergo covalent crosslinking at relatively fewer sites (4/9 sites tested at the acidic pocket, Figure 5).…”

The current chemical biology tool box for studying ion channels

“…Apart from trapping intra-or inter-subunit interactions in ion channels, ncAA photocrosslinkers can also covalently trap ligand-receptor complexes in G protein-coupled receptors and transporters (Coin et al 2013;Rannversson et al 2016). This has recently been extended to ion channels, when we used AzF in the extracellular domain of ASICs to show that the neuropeptide big dynorphin binds to the acidic pocket of ASIC1a (Borg et al 2020). The ribosome is shown in khaki, while the clover leaf-shaped structures indicate endogenous (green) or orthogonal (red) tRNAs carrying naturally occurring amino acids and ncAAs, respectively.…”

“…This has recently been extended to ion channels, when we used AzF in the extracellular domain of ASICs to show that the neuropeptide big dynorphin binds to the acidic pocket of ASIC1a (Borg et al . 2020).…”