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The Journal of Neuroscience, January 24, 2007, 27(4):886-892; doi:10.1523/JNEUROSCI.4791-06.2007

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Cellular/Molecular
Unnatural Amino Acid Mutagenesis of the GABA_A Receptor Binding Site Residues Reveals a Novel Cation– Interaction between GABA and ß₂Tyr97

Claire L. Padgett,¹ Ariele P. Hanek,² Henry A. Lester,² Dennis A. Dougherty,² and Sarah C. R. Lummis¹

¹Department of Biochemistry, University of Cambridge, Cambridge CB2 1AG, United Kingdom, and ²California Institute of Technology, Pasadena, California 91125

Correspondence should be addressed to Sarah C. R. Lummis, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1AG, UK. Email: sl120{at}cam.ac.uk

The binding pockets of Cys-loop receptors are dominated by aromatic amino acids. In the GABA_A receptor ₁Phe65, ß₂Tyr97, ß₂Tyr157, and ß₂Tyr205 are present at the ß₂/₁ 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 ß₂97 revealed a 20-fold increase in EC₅₀ with the addition of each fluorine atom to a phenylalanine, indicating a cation– interaction between GABA and this residue. This is the first example of a cation– interaction in loop A of a Cys-loop receptor. Along with previous studies that identified cation– 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 ₁65 (in loop D) is tolerant to subtle changes. Conversely, mutating either ß₂Tyr157 (in loop B) or ß₂Tyr205 (in loop C) to Phe substantially disrupts receptor function. Substitution of 4-F-Phe, however, at either position, or 4-MeO-Phe at ß₂Tyr157, 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 GABA_A receptor.

Key words: ligand-gated ion channel; Cys-loop receptor; cation– interaction; GABA_A receptor binding site; unnatural amino acids; homology model

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Received July 28, 2006; revised Dec. 14, 2006; accepted Dec. 14, 2006.

Correspondence should be addressed to Sarah C. R. Lummis, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1AG, UK. Email: sl120{at}cam.ac.uk

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