PUFA modulation of ASIC3 involves both specific and lipid solvent-like interactions.

Inflammatory mediators including polyunsaturated fatty acids (PUFAs) are known to potentiate ASIC3 by inducing changes in multiple gating features, yet the molecular basis for their interactions remains poorly defined. Using all-atom MD simulations and electrophysiology, we show that DHA accumulates around ASIC3 through loosely coordinated interactions with a membrane-facing electropositive region along the outer leaflet of TM1. In the open state, the carboxylate head group strongly binds to a critical arginine (R63) along with nearby polar residues that are necessary to slow the rate in channel desensitization rate but not to increase the pH sensitivity for channel activation. Moreover, mutating R63 disrupted effects on ASIC3 desensitization induced by PUFAs but not N-acyl amino acids (NAAAs) or lysophosphatidylcholines (LPCs). Our results provide the first detailed description of a functional PUFA binding site on ASICs, offering new insights into lipid modulation and potential strategies for developing novel therapeutics for pain and inflammation.