QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

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

This Article Full Text Full Text (PDF) Supplemental Data Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (17) Citing Articles via Google Scholar Google Scholar Articles by Padgett, C. L. Articles by Lummis, S. C. R. Search for Related Content PubMed PubMed Citation Articles by Padgett, C. L. Articles by Lummis, S. C. R.

 Previous Article  |  Next Article 

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

---

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

This article has been cited by other articles:

				S. A. Pless, K. S. Millen, A. P. Hanek, J. W. Lynch, H. A. Lester, S. C. R. Lummis, and D. A. Dougherty A Cation-{pi} Interaction in the Binding Site of the Glycine Receptor Is Mediated by a Phenylalanine Residue J. Neurosci., October 22, 2008; 28(43): 10937 - 10942. [Abstract] [Full Text] [PDF]

				M. Tomizawa, D. Maltby, T. T. Talley, K. A. Durkin, K. F. Medzihradszky, A. L. Burlingame, P. Taylor, and J. E. Casida Atypical nicotinic agonist bound conformations conferring subtype selectivity PNAS, February 5, 2008; 105(5): 1728 - 1732. [Abstract] [Full Text] [PDF]

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help