Unnatural Amino Acid Mutagenesis of the GABAAReceptor Binding Site Residues Reveals a Novel Cation–π Interaction between GABA and β2Tyr97

Abstract

The binding pockets of Cys-loop receptors are dominated by aromatic amino acids. In the GABA_Areceptor α₁Phe65, β₂Tyr97, β₂Tyr157, and β₂Tyr205 are present at the β₂/α₁interface and have been implicated in forming an important part of the GABA binding site. Here, we have probed interactions of these residues using subtle chemical changes: unnatural amino acid mutagenesis was used to introduce a range of Phe analogs, and mutant receptors expressed in oocytes were studied using voltage-clamp electrophysiology. Serial mutations at β₂97 revealed a ∼20-fold increase in EC₅₀with the addition of each fluorine atom to a phenylalanine, indicating a cation–π interaction between GABA and this residue. This is the first example of a cation–π interaction in loop A of a Cys-loop receptor. Along with previous studies that identified cation–π interactions in loop B and loop C, the result emphasizes that the location of this interaction is not conserved in the Cys-loop family. The data further show that α₁65 (in loop D) is tolerant to subtle changes. Conversely, mutating either β₂Tyr157 (in loop B) or β₂Tyr205 (in loop C) to Phe substantially disrupts receptor function. Substitution of 4-F-Phe, however, at either position, or 4-MeO-Phe at β₂Tyr157, resulted in receptors with wild-type EC₅₀values, suggesting a possible hydrogen bond. The molecular scale insights provided by these data allow the construction of a model for GABA docking to the agonist binding site of the GABA_Areceptor.