Functional Evaluation of Key Interactions Evident in the Structure of the Eukaryotic Cys-Loop Receptor GluCl

[Image: see text] The publication of the first high-resolution crystal structure of a eukaryotic Cys-loop receptor, GluClα, has provided valuable structural information on this important class of ligand-gated ion channels (LGIC). However, limited functional data exist for the GluCl receptors. Before applying the structural insights from GluCl to mammalian Cys-loop receptors such as nicotinic acetylcholine and GABA receptors, it is important to ensure that established functional features of mammalian Cys-loop receptors are present in the more distantly related GluCl receptors. Here, we seek to identify ligand-binding interactions that are generally associated with Cys-loop receptors, including the frequently observed cation−π interaction. Our studies were performed on the highly homologous GluClβ receptor, because GluClα is not activated by glutamate in Xenopus laevis oocytes. Mutagenesis of the signal peptide and pore lining helix was performed to enhance functional expression and sensitivity to applied ligand, respectively. Conventional and unnatural amino acid mutagenesis indicate a strong cation−π interaction between Y206 and the protonated amine of glutamate, as well as other important ionic and hydrogen bond interactions between the ligand and the binding site, consistent with the crystal structure.