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.