Abstract
Acid-sensing ion channels (ASICs) are thought to be endogenous sensors of acidic pain in inflammatory pathways. It has previously been demonstrated that arachidonic acid (AA), a pain and inflammation promoting molecule, potentiates ASICs. However, a mechanistic understanding of how AA regulates ASICs is lacking. Furthermore, little is known regarding modulation by other polyunsaturated fatty acids (PUFAs). Here we show that PUFAs stabilize the open state of the channel by shifting the pH dependence of activation to more alkaline values, increasing max conductance, and slowing channel desensitization. We examine the effects of 35 PUFAs/PUFA derivatives and show that ASICs can be more strongly potentiated by these lipids than was originally seen for AA. In fact, arachidonoyl glycine (AG) can act as a ligand and activate the channel in the absence of acidic pH. We find that the strength of potentiation is critically dependent upon a negatively charged PUFA head group as well as both the length and the number of doubles bonds in the acyl tail. PUFA-induced shifts in the pH dependence of activation could be eliminated upon mutation of a highly conserved, positively charged arginine in the outer segment of TM1 (R64). Combined our results suggest a hypothesis whereby an electrostatic interaction between the charged PUFA head group and the positively charged arginine side chain potentiates ASIC currents by stabilizing the open state of the channel. This work uncovers a novel putative lipid binding site on ASICs and provides the structural basis for future development of compounds targeting ASICs.
Significance Statement The Acid-sensing ion channels (ASICs) have emerged as potential therapeutic targets for inflammatory pain. Inflammatory mediators like the polyunsaturated fatty acid (PUFA), arachidonic acid (AA), have been shown to potentiate ASICs and partially mediate ASIC-derived pain. However, the mechanistic understanding of AA and other PUFAs interaction with ASICs is lacking. Here, we demonstrate PUFAs stabilize the open state of ASICs, a mechanism that is dependent on both the flexibility of the PUFA tail as well as the charge of the PUFA head group. Further, we uncover a putative PUFA binding site on ASICs governing potentiating. This work provides insights into the molecules regulating ASICs in inflammation, as well as provide a basis for therapeutic targeting of ASIC-derived pain.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Competing Interest Statement: The authors declare no competing interests.