RT Journal Article
SR Electronic
T1 Mechanism and site of action of big dynorphin on ASIC1a
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 816264
DO 10.1101/816264
A1 Borg, C.B.
A1 Braun, N.
A1 Heusser, S.A.
A1 Bay, Y.
A1 Weis, D.
A1 Galleano, I.
A1 Lund, C.
A1 Tian, W.
A1 Haugaard-Kedström, L.M.
A1 Bennett, E.P.
A1 Lynagh, T.
A1 Strømgaard, K.
A1 Andersen, J.
A1 Pless, S.A.
YR 2020
UL http://biorxiv.org/content/early/2020/01/21/816264.abstract
AB 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 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 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 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 azido-phenylalanine. 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.Significance Statement Neuropeptides such as big dynorphin (BigDyn) play important roles in the slow modulation of fast neurotransmission, which is mediated by membrane-embedded receptors. In fact, BigDyn is the most potent known endogenous modulator of one such receptor, the acid-sensing ion channel (ASIC), but the mode of action remains unknown. In this work, we employ a broad array of technologies to unravel the details of where big dynorphin binds to ASIC and how it modulates its activity. As both BigDyn and ASIC are implicated in pain pathways, this work might pave the way towards future analgesics.