PT  - JOURNAL ARTICLE
AU  - C.B. Borg
AU  - N. Braun
AU  - S.A. Heusser
AU  - Y. Bay
AU  - D. Weis
AU  - I. Galleano
AU  - C. Lund
AU  - W. Tian
AU  - L.M. Haugaard-Kedström
AU  - E.P. Bennett
AU  - T. Lynagh
AU  - K. Strømgaard
AU  - J. Andersen
AU  - S.A. Pless
TI  - Mechanism and site of action of big dynorphin on ASIC1a
AID  - 10.1101/816264
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 816264
4099  - http://biorxiv.org/content/early/2019/10/29/816264.short
4100  - http://biorxiv.org/content/early/2019/10/29/816264.full
AB  - 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 acid-mediated 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 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.