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

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Abstract
The binding pockets of Cys-loop receptors are dominated by aromatic amino acids. In the GABAA receptor α1Phe65, β2Tyr97, β2Tyr157, and β2Tyr205 are present at the β2/α1 interface 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 EC50 with 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 EC50 values, 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 GABAA receptor.