Mechanism and Binding Site of the ASIC1A-Big Dynorphin Interaction

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that are involved in synaptic plasticity, initiation of pain and neuronal death following ischemic stroke. While much of the in vivo regulation and signaling pathways of these channels remain elusive, increasing evidence suggests endogenous neuropeptides to be potentially important physiological modulators of ASIC function and activity. The most potent endogenous ASIC modulator described to date is the opioid neuropeptide big dynorphin (BigDyn). This peptide is upregulated in chronic pain and was found to increase ASIC-mediated neuronal death during acidosis by lowering the pH dependence of steady-state desensitization of ASICs. Defining the molecular details of the ASIC-BigDyn interaction could thus enable the design of compounds targeting a novel site for inhibitors of pain and ischemic stroke. Here, we use a combination of electrophysiology, voltage-clamp fluorometry, synthesis of BigDyn analogs and non-canonical amino acid-mediated photocrosslinking to identify both mechanism and binding site of the ASIC1a-BigDyn interaction. We demonstrate that BigDyn binding induces ASIC1a conformational changes that are distinct from those induced by protonation and likely represent a stabilization of a distinct closed state. Using truncated and alanine-substituted BigDyn analogs, we find that the modulation of ASIC1a by BigDyn is mediated through electrostatic interactions of basic amino acids in the BigDyn N-terminus. Furthermore, mutations that neutralize acidic amino acids in the ASIC1a extracellular domain reduce the effects of BigDyn, suggesting a binding site at the acidic pocket. This notion is confirmed by photocrosslinking using the non-canonical amino acid 4-Azido-phenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a and identify the acidic pocket as the binding site for BigDyn, highlighting this cavity as an important site for the development of ASIC-targeting therapeutics.