Cys-loop receptor binding sites characteristically possess an "aromatic box," where several aromatic amino acid residues surround the bound ligand. A cation-π interaction between one of these residues and the natural agonist is common, although the residue type and location are not conserved. Even in the closely related vertebrate GABA_A and GABA_C receptors, residues in distinct locations perform this role: in GABA_A receptors, a Tyr residue in loop A forms a cation-π interaction with GABA, while in GABA_C receptors it is a loop B residue. GABA-activated Cys-loop receptors also exist in invertebrates, where they have distinct pharmacologies and are the target of a range of pesticides. Here we examine the location of GABA in an insect binding site by incorporating a series of fluorinated Phe derivatives into the receptor binding pocket using unnatural amino acid mutagenesis, and evaluating the resulting receptors when expressed in Xenopus oocytes. A homology model suggests that two aromatic residues (in loops B and C) are positioned such that they could contribute to a cation-π interaction with the primary ammonium of GABA, and the data reveal a clear correlation between the GABA EC_(50) and the cation-π binding ability both at Phe206 (loop B) and Tyr254 (loop C), demonstrating for the first time the contribution of two aromatic residues to a cation-π interaction in a Cys-loop receptor
