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Unnatural Amino Acid Mutagenesis of the GABA_A Receptor Binding Site Residues Reveals a Novel Cation-π Interaction between GABA and β₂Tyr97

Padgett, Claire L. and Hanek, Ariele P. and Lester, Henry A. and Dougherty, Dennis A. and Lummis, Sarah C. R. (2007) Unnatural Amino Acid Mutagenesis of the GABA_A Receptor Binding Site Residues Reveals a Novel Cation-π Interaction between GABA and β₂Tyr97. Journal of Neuroscience, 27 (4). pp. 886-892. ISSN 0270-6474. PMCID PMC2649369. doi:10.1523/jneurosci.4791-06.2007. https://resolver.caltech.edu/CaltechAUTHORS:20200416-150305764

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1523/jneurosci.4791-06.2007DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2649369PubMed CentralArticle
ORCID:
AuthorORCID
Lester, Henry A.0000-0002-5470-5255
Dougherty, Dennis A.0000-0003-1464-2461
Lummis, Sarah C. R.0000-0001-9410-9805
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.
Funders:
Funding AgencyGrant Number
Wellcome TrustUNSPECIFIED
Merck Sharp & Dohme Research LaboratoriesUNSPECIFIED
DohmeUNSPECIFIED
Biotechnology and Biological Sciences Research Council (BBSRC)UNSPECIFIED
NIHNS11756
NIHNS34407
NIH Predoctoral Fellowship5T32GMO7616
Subject Keywords:ligand-gated ion channel; Cys-loop receptor; cation–π interaction; GABA_A receptor binding site; unnatural amino acids; homology model
Issue or Number:4
PubMed Central ID:PMC2649369
DOI:10.1523/jneurosci.4791-06.2007
Record Number:CaltechAUTHORS:20200416-150305764
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200416-150305764
Official Citation:Unnatural Amino Acid Mutagenesis of the GABAA Receptor Binding Site Residues Reveals a Novel Cation–π Interaction between GABA and β2Tyr97. Claire L. Padgett, Ariele P. Hanek, Henry A. Lester, Dennis A. Dougherty, Sarah C. R. Lummis. Journal of Neuroscience 24 January 2007, 27 (4) 886-892; DOI: 10.1523/JNEUROSCI.4791-06.2007
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:102589
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:16 Apr 2020 22:18
Last Modified:16 Nov 2021 18:13

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