Unnatural Amino Acid Mutagenesis of the GABA_A Receptor 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_A 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 GABA_A receptor.
Additional Information
© 2007 Society for Neuroscience. Received July 28, 2006; revised Dec. 14, 2006; accepted Dec. 14, 2006. This work was supported by the Wellcome Trust (S.C.R.L. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Science), Merck Sharpe and Dohme, the Biotechnology and Biological Sciences Research Council (a CASE studentship to C.L.P.), and the National Institutes of Health (NIH) (NS11756; NS34407). A.P.H. is a recipient of NIH Predoctoral Trainee Grant 5T32GMO7616.Attached Files
Published - 886.full.pdf
Supplemental Material - Fig1S.pdf
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Additional details
- PMCID
- PMC2649369
- Eprint ID
- 102589
- Resolver ID
- CaltechAUTHORS:20200416-150305764
- Wellcome Trust
- Merck Sharp & Dohme Research Laboratories
- Dohme
- Biotechnology and Biological Sciences Research Council (BBSRC)
- NIH
- NS11756
- NIH
- NS34407
- NIH Predoctoral Fellowship
- 5T32GMO7616
- Created
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2020-04-16Created from EPrint's datestamp field
- Updated
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2021-11-16Created from EPrint's last_modified field