Polyunsaturated fatty acids inhibit a pentameric ligand‐gated ion channel through one of two specific binding sites

Abstract

Polyunsaturated fatty acids (PUFAs) inhibit pentameric ligand-gated ion channels (pLGICs), including the GABAAreceptor (GABAAR) and nicotinic acetylcholine receptor (nAchR). PUFA inhibition of pLGICs is thought to occur through an allosteric mechanism mediated by direct binding to specific sites. However, the sites of PUFA binding, and those sites that mediate channel inhibition are not fully elucidated. We investigated fatty acid binding sites in the prototypic pLGIC, Erwinia ligand-gated ion channel (ELIC), using a novel fatty acid photolabeling reagent, KK-242. The PUFA, docosahexaenoic acid (DHA), inhibited ELIC channel responses, similar to the effect reported in the GABAAR and nAchR. KK-242 also produced an inhibitory effect in ELIC, and photolabeled two binding sites in the outer transmembrane domain (TMD). In contrast, the commercially-available fatty acid photolabeling reagent, pacFA, did not photolabel ELIC. Fatty acid binding to these photolabeled sites was specific for DHA over palmitic acid (PA), and DHA preferentially bound to these sites in the agonist-bound state. Coarse-grained simulations corroborated the selectivity of polyunsaturated fatty acid binding to these sites. Cysteines introduced in the fatty acid binding sites of the outer TMD were efficiently modified by hexadecyl-methanethiosulfonate (hMTS), while inner TMD sites were not accessible to hMTS, possibly due to the presence of tightly bound phospholipids. hMTS modification of only one of these binding sites recapitulated the inhibitory effect of fatty acids on ELIC function. The results demonstrate that PUFAs occupy multiple sites in the outer TMD of ELIC, but that a single intrasubunit binding site mediates PUFA inhibition of ELIC. Additionally, KK-242 is a useful photolabeling reagent for the identification of fatty acid binding sites in other membrane proteins with advantages over currently available reagents.