BPS2026 – A single arginine in ASIC3 is required for maximal channel regulation by DHA

Acid-sensing ion channel 3 (ASIC3) is a pH-gated sodium channel implicated in the sensing of pain due to injury, inflammation, and ischemia. ASIC3 activates upon rapid decreases in extracellular pH and quickly desensitizes following prolonged exposure. Inflammatory mediators including multiple classes of single-chain lipids act on ASICs sensitizing the channels to activate and contribute to an increase in excitability in neurons. We previously demonstrated that polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA), potentiate ASIC3 currents by shifting the pH dependence of activation to more basic conditions and slowing the rate of desensitization. However, the binding site and mechanism of potentiation remains unclear. Using a combination of both molecular dynamics (MD) simulations and patch-clamp electrophysiology, we show that the head group of DHA makes a series of transient interactions with several residues in the outer leaflet on TM1. In the open state, a key arginine (R63) becomes exposed and makes a stable interaction with the head group of the lipid. Mutation of this arginine reduces the impact of DHA on proton sensitivity for channel activation and eliminates the impact on the rate of channel desensitization. Mutation of the more transiently interacting residues near R63 has no impact on the pH sensitivity of the channel but does reduce the effect of DHA on the rate of desensitization. Finally, we show that PUFAs, but not other classes of ASIC3 potentiating lipids, specifically require this critical interaction to slow the rate of desensitization. We hypothesize that PUFAs are loosely coordinated around the channel via transient interactions that allow DHA to make key stable interactions that impact gating. Additionally, the multiple gating changes that occur upon exposure of the channel to lipids likely stem from distinct mechanisms.