Fatty Acids Inhibit a Pentameric Ligand-Gated Ion Channel through One of Two Binding Sites

Abstract

Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are inhibited by fatty acids. To investigate the structural determinants of fatty acid inhibition of pLGICs, we developed a new photolabeling reagent, KK-242, for the identification of fatty acid binding sites. by giant liposome patch-clamping, fatty acids inhibit the model pLGIC, Erwinia ligand-gated ion channel (ELIC), decreasing peak response to agonist and increasing the rate and extent of current decay. Polyunsaturated fatty acids (PUFAs) are more efficacious than saturated fatty acids. This modulatory effect is similar to previous reports of fatty acid inhibition of the GABA(A) receptor and nicotinic acetylcholine receptor, and is consistent with a model where fatty acids stabilize a pre-activated state. KK-242 also inhibits ELIC. KK-242 photolabels two fatty acid binding sites per subunit, which are also binding sites for docosahexaenoic acid and palmitic acid. To probe the functional significance of these sites, a cysteine was introduced to each site and modified with hexadecyl-methanethiosulfonate (MTS). Modification of one site with hexadecyl-MTS recapitulates the inhibitory effect of fatty acids on ELIC function. The results show that fatty acids bind to two sites, and inhibit ELIC by binding to just one of these sites. KK-242 may be a useful tool for identification of fatty acid binding sites in other membrane proteins.