Publications by authors named "Ariele P Hanek"

6 Publications

  • Page 1 of 1

A cation-π interaction at a phenylalanine residue in the glycine receptor binding site is conserved for different agonists.

Mol Pharmacol 2011 Apr 25;79(4):742-8. Epub 2011 Jan 25.

School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia.

Cation-π interactions have been demonstrated to play a major role in agonist-binding in Cys-loop receptors. However, neither the aromatic amino acid contributing to this interaction nor its location is conserved among Cys-loop receptors. Likewise, it is not clear how many different agonists of a given receptor form a cation-π interaction or, if they do, whether it is with the same aromatic amino acid as the major physiological agonist. We demonstrated previously that Phe159 in the glycine receptor (GlyR) α1 subunit forms a strong cation-π interaction with the principal agonist, glycine. In the current study, we investigated whether the lower efficacy agonists of the human GlyR β-alanine and taurine also form cation-π interactions with Phe159. By incorporating a series of unnatural amino acids, we found cation-π interactions between Phe159 and the amino groups of β-alanine and taurine. The strengths of these interactions were significantly weaker than for glycine. Modeling studies suggest that β-alanine and taurine are orientated subtly differently in the binding pocket, with their amino groups further from Phe159 than that of glycine. These data therefore show that similar agonists can have similar but not identical orientations and interactions in the binding pocket and provide a possible explanation for the lower potencies of β-alanine and taurine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/mol.110.069583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063724PMC
April 2011

Photochemical proteolysis of an unstructured linker of the GABAAR extracellular domain prevents GABA but not pentobarbital activation.

Mol Pharmacol 2010 Jul 2;78(1):29-35. Epub 2010 Apr 2.

Division of Chemistry and Chemical Engineering, Division of Biology, California Institute of Technology, Pasadena, California, USA.

The GABA type A receptor (GABA(A)R) is the major inhibitory receptor in the mammalian central nervous system and the target of numerous pharmaceuticals. The alpha-subunit of these pentameric Cys-loop neurotransmitter-gated ion channels contributes to the binding of both GABA and allosteric modulators such as the benzodiazepines, suggesting a role for this subunit in the conformational changes associated with activation of the receptor. Herein we use the nonsense suppression methodology to incorporate a photoactivatable unnatural amino acid and photochemically cleave the backbone of the alpha subunit of the alpha(1)beta(2) GABA(A)R in a linker region that is believed to span the subunit. Proteolytic cleavage impairs GABA but not pentobarbital activation, strongly suggesting that conformational changes involving this linker region are critical to the GABA activation pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/mol.109.059832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2912059PMC
July 2010

A cation-pi interaction in the binding site of the glycine receptor is mediated by a phenylalanine residue.

J Neurosci 2008 Oct;28(43):10937-42

School of Biomedical Sciences and Queensland Brain Institute, University of Queensland, Brisbane, Queensland 4072, Australia.

Cys-loop receptor binding sites characteristically contain many aromatic amino acids. In nicotinic ACh and 5-HT3 receptors, a Trp residue forms a cation-pi interaction with the agonist, whereas in GABA(A) receptors, a Tyr performs this role. The glycine receptor binding site, however, contains predominantly Phe residues. Homology models suggest that two of these Phe side chains, Phe159 and Phe207, and possibly a third, Phe63, are positioned such that they could contribute to a cation-pi interaction with the primary amine of glycine. Here, we test this hypothesis by incorporation of a series of fluorinated Phe derivatives using unnatural amino acid mutagenesis. The data reveal a clear correlation between the glycine EC(50) value and the cation-pi binding ability of the fluorinated Phe derivatives at position 159, but not at positions 207 or 63, indicating a single cation-pi interaction between glycine and Phe159. The data thus provide an anchor point for locating glycine in its binding site, and demonstrate for the first time a cation-pi interaction between Phe and a neurotransmitter.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.2540-08.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2649377PMC
October 2008

A stereochemical test of a proposed structural feature of the nicotinic acetylcholine receptor.

J Am Chem Soc 2008 Oct 10;130(40):13216-8. Epub 2008 Sep 10.

Division of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.

Understanding the gating mechanism of the nicotinic acetylcholine receptor (nAChR) and similar channels constitutes a significant challenge in chemical neurobiology. In the present work, we use a stereochemical probe to evaluate a proposed pin-into-hydrophobic socket mechanism for the alphaVal46 side chain of the nAChR. Utilizing nonsense suppression methodology we incorporated isoleucine (Ile), O-methyl threonine (Omt) and threonine (Thr) as well as their side chain epimers (the allo counterparts). Surprisingly, our results indicate that only the pro-S methyl group of the alphaVal46 side chain is sensitive to changes in hydrophobicity, consistent with the precise geometrical requirements of the pin-into-socket mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja8015457DOI Listing
October 2008

Unnatural amino acid mutagenesis of the GABA(A) receptor binding site residues reveals a novel cation-pi interaction between GABA and beta 2Tyr97.

J Neurosci 2007 Jan;27(4):886-92

Department of Biochemistry, University of Cambridge, Cambridge CB2 1AG, United Kingdom.

The binding pockets of Cys-loop receptors are dominated by aromatic amino acids. In the GABA(A) receptor alpha1Phe65, beta2Tyr97, beta2Tyr157, and beta2Tyr205 are present at the beta2/alpha1 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 beta(2)97 revealed a approximately 20-fold increase in EC50 with the addition of each fluorine atom to a phenylalanine, indicating a cation-pi interaction between GABA and this residue. This is the first example of a cation-pi interaction in loop A of a Cys-loop receptor. Along with previous studies that identified cation-pi 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 alpha(1)65 (in loop D) is tolerant to subtle changes. Conversely, mutating either beta2Tyr157 (in loop B) or beta2Tyr205 (in loop C) to Phe substantially disrupts receptor function. Substitution of 4-F-Phe, however, at either position, or 4-MeO-Phe at beta2Tyr157, 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.4791-06.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2649369PMC
January 2007

A unified view of the role of electrostatic interactions in modulating the gating of Cys loop receptors.

J Biol Chem 2005 Dec 10;280(50):41655-66. Epub 2005 Oct 10.

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.

In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on gamma-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M508635200DOI Listing
December 2005