Unnatural Amino Acid Mutagenesis of the GABA<sub>A</sub>Receptor Binding Site Residues Reveals a Novel Cation–π Interaction between GABA and β<sub>2</sub>Tyr97

Padgett, Claire L.; Hanek, Ariele P.; Lester, Henry A.; Dougherty, Dennis A.; Lummis, Sarah C. R.

doi:10.1523/jneurosci.4791-06.2007

Cited by 106 publications

(131 citation statements)

References 30 publications

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“…The data include not only fluorinated analogs, but also a Phe derivative with a cyano group (CN) in the 4 position. 4-CN-Phe is slightly larger in size than the fluorinated Phe derivatives but is well tolerated in Cys-loop receptor binding sites (Padgett et al, 2007;Pless et al, 2008). This substitution yielded a ␤-alanine EC 50 between those for 3,4-F 2 -Phe and 3,4,5-F 3 -Phe, consistent with the cation-binding ability of 4-CN-Phe.…”

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confidence: 63%

“…Like all Cys-loop receptors, the GlyR binding site is lined with aromatic residues, and we have shown previously that a cation-interaction between the positively charged amine of glycine and Phe159 in loop B makes a substantial contribution to agonist binding (Pless et al, 2008). Likewise, other studies have shown that aromatic side chains form cationinteractions with the principal agonists of other Cys-loop receptors, such as nACh, 5-HT 3 , MOD-1 (modulation of locomotion defective 1), and GABA A receptors (Zhong et al, 1998;Beene et al, 2002;Mu et al, 2003;Lummis et al, 2005;Padgett et al, 2007;Dougherty, 2008). However, the nature and location of the aromatic residue that interacts with the cation is not conserved among Cys-loop receptors or among different agonists for a given receptor (Beene et al, 2002;Mu et al, 2003;Dougherty, 2008;Xiu et al, 2009).…”

Section: Introductionmentioning

confidence: 74%

“…(Tyr97 in the ␣1␤2 GABA A receptor) and loop C (Trp226 on the MOD-1 receptor) have also been reported (Zhong et al, 1998;Beene et al, 2002;Mu et al, 2003;Lummis et al, 2005;Padgett et al, 2007;Pless et al, 2008). …”

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confidence: 85%

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A Cation-π Interaction at a Phenylalanine Residue in the Glycine Receptor Binding Site Is Conserved for Different Agonists

Pless

Hanek²,

Price³

et al. 2011

Mol Pharmacol

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Cation-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-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) ␣1 subunit forms a strong cation-interaction with the principal agonist, glycine. In the current study, we investigated whether the lower efficacy agonists of the human GlyR ␤-alanine and taurine also form cation-interactions with Phe159. By incorporating a series of unnatural amino acids, we found cation-interactions between Phe159 and the amino groups of ␤-alanine and taurine. The strengths of these interactions were significantly weaker than for glycine. Modeling studies suggest that ␤-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 ␤-alanine and taurine.

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Section: Downloaded Frommentioning

confidence: 63%

Section: Introductionmentioning

confidence: 74%

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A Cation-π Interaction at a Phenylalanine Residue in the Glycine Receptor Binding Site Is Conserved for Different Agonists

Pless

Hanek²,

Price³

et al. 2011

Mol Pharmacol

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“…Mutation of the homologous W55 residue in the GABA A -R ␥2-subunit, F77C, abolished binding by (28). In the GABA A -R ␣1-subunit, it was shown that for the homologous Trp-55 subtle changes were caused by unnatural amino acid mutations (29). In the muscle nAChR it was shown that mutation W57F in the ␦-subunit and W55F in the ⑀-subunit have only minor effects on acetylcholine-sensitivity but mainly affect channel gating by reducing the channel opening rate (30).…”

Section: Discussionmentioning

confidence: 99%

Crystal Structures of a Cysteine-modified Mutant in Loop D of Acetylcholine-binding Protein

Brams

Gay

Saez

et al. 2011

Journal of Biological Chemistry

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Cys-loop receptors (CLRs) 2 belong to a class of ligandgated ion channels that are involved in fast synaptic transmission in the central nervous system and the neuromuscular junction. This transmission can either be excitatory or inhibitory depending on the charge of ions that pass through the ion conduction pathway of the channel. Excitatory transmission is mediated by nicotinic acetylcholine receptors (nAChR) and 5-HT 3 serotonin receptors, which selectively pass cations. On the other hand, inhibitory transmission is mediated by glycine receptors (GlyR) and ␥-aminobutyric acid (GABA A and GABA C ) receptors, which selectively conduct anions. In both types of receptors, the ion conduction pathway lies along the central axis formed by five channel subunits, which can either be identical for homomeric CLRs or non-identical for heteromeric CLRs. Opening and closing of the ion conduction pathway is controlled by a gate that is allosterically coupled to the extracellular ligand-binding domain (for a recent review see Ref. 1).Detailed structural data are still lacking for an intact eukaryotic CLR, but structural information has been obtained from 4 Å resolution electron microscopic images of the Torpedo nAChR (2) and higher resolution x-ray crystal structures of AChBP, a molluscan homolog of the extracellular domain of nAChRs (3, 4), the monomeric ␣1 nAChR subunit (5), and two prokaryotic homologs ELIC (6) and GLIC (7,8), which presumably represent the closed and open state of a CLR. Despite their different pharmacological properties, these CLRs share a common architectural arrangement of aromatic residues in their ligand-binding site. The ligand-binding site for CLRs is found at the interface between two subunits and ligands interact with amino acids from both the principal face (containing loops A, B, and C) and complementary face (containing loops D, E, and F). We have focused our attention on an aromatic residue that lies on the complementary face of the binding pocket and is highly conserved among eukaryotic and prokaryotic CLRs.Recently, we found that the tryptophan residue at position 55 of the rat ␣7 nAChR (Trp-55) was the site where synthetic * This research was supported, in whole or in part, by the Intramural Research Program of the National Institutes of Health, NIEHS (to E. A. G., J. C. S., and J. L. Y.), by G.0257.08), and the Belgian Federal Science Policy Office, Interuniversity Attraction Poles program (P6/31) (to J. T.), EU FP7 2020288 NeuroCypres (to C. U., A. B. S., and R. C. vd. S.), and KULeuven Onderzoekstoelage OT/08/048 (to S. V. S. and C. U.). □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. The atomic coordinates and structure factors (codes 2XZ6 and 2XZ5)

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“…Finally, in nicotinoids, one fundamental bound conformation is conserved for all AChBP and nAChR subtypes (7)(8)(9)(10)23). The same agonist molecule can also adopt different binding orientations at other Cys-loop receptors, depending on the nature and position of aromatic amino acid side chains, that is, serotonin (5-HT) at 5-HT 3 versus MOD-1 receptors and ␥-aminobutyric acid (GABA) at GABA A versus GABA C receptors (24,25).…”

Section: Discussionmentioning

confidence: 99%

Atypical nicotinic agonist bound conformations conferring subtype selectivity

Tomizawa

Maltby

Talley

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

116

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The nicotinic acetylcholine (ACh) receptor (nAChR) plays a crucial role in excitatory neurotransmission and is an important target for drugs and insecticides. Diverse nAChR subtypes with various subunit combinations confer differential selectivity for nicotinic drugs. We investigated the subtype selectivity of nAChR agonists by comparing two ACh-binding proteins (AChBPs) as structural surrogates with distinct pharmacological profiles [i.e., Lymnaea stagnalis (Ls) AChBP of low neonicotinoid and high nicotinoid sensitivities and Aplysia californica (Ac) AChBP of high neonicotinoid sensitivity] mimicking vertebrate and insect nAChR subtypes, respectively. The structural basis of subtype selectivity was examined here by photoaffinity labeling. Two azidoneonicotinoid probes in the Ls-AChBP surprisingly modified two distinct and distant subunit interface sites: loop F Y164 of the complementary or (؊)-face subunit and loop C Y192 of the principal or (؉)-face subunit, whereas three azidonicotinoid probes derivatized only Y192. Both the neonicotinoid and nicotinoid probes labeled Ac-AChBP at only one position at the interface between loop C Y195 and loop E M116. These findings were used to establish structural models of the two AChBP subtypes. In the Ac-AChBP, the neonicotinoids and nicotinoids are nestled in similar bound conformations. Intriguingly, for the Ls-AChBP, the neonicotinoids have two bound conformations that are inverted relative to each other, whereas nicotinoids appear buried in only one conserved conformation as seen for the Ac-AChBP subtype. Accordingly, the subtype selectivity is based on two disparate bound conformations of nicotinic agonists, thereby establishing an atypical concept for neonicotinoid versus nicotinoid selectivity between insect and vertebrate nAChRs.acetylcholine-binding protein ͉ imidacloprid ͉ neonicotinoids ͉ nicotinic receptor ͉ photoaffinity labeling T he nicotinic acetylcholine (ACh) receptor (nAChR) is critically important in synaptic neurotransmission and the target of potential therapeutic agents for neurological dysfunction and of major neonicotinoid insecticides for crop protection and animal health. The drug-binding sites are localized at subunit interfaces of the nAChR pentameric structure. Specific vertebrate and insect subunit combinations make up diverse nAChR subtypes that differ in pharmacological profiles (1, 2). Highly subtype-selective nicotinic agents are required for development of therapeutics and insecticides. A family of peptide antagonists, the ␣-conotoxins, serve as important probes for studying structural determinants of subtype selectivity (3-5). However, the molecular mechanism of selectivity for small agonist molecules is not resolved because most of the key amino acids in the nAChR-binding pocket are conserved in all of the receptor subtypes and species and the binding region for antagonists extends over a large interfacial surface. Understanding drugnAChR interactions was greatly facilitated by the discovery and crystallization of mollusk homopentamer...

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Unnatural Amino Acid Mutagenesis of the GABA_AReceptor Binding Site Residues Reveals a Novel Cation–π Interaction between GABA and β₂Tyr97

Cited by 106 publications

References 30 publications

A Cation-π Interaction at a Phenylalanine Residue in the Glycine Receptor Binding Site Is Conserved for Different Agonists

A Cation-π Interaction at a Phenylalanine Residue in the Glycine Receptor Binding Site Is Conserved for Different Agonists

Crystal Structures of a Cysteine-modified Mutant in Loop D of Acetylcholine-binding Protein

Atypical nicotinic agonist bound conformations conferring subtype selectivity

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Unnatural Amino Acid Mutagenesis of the GABAAReceptor Binding Site Residues Reveals a Novel Cation–π Interaction between GABA and β2Tyr97

Cited by 106 publications

References 30 publications

A Cation-π Interaction at a Phenylalanine Residue in the Glycine Receptor Binding Site Is Conserved for Different Agonists

A Cation-π Interaction at a Phenylalanine Residue in the Glycine Receptor Binding Site Is Conserved for Different Agonists

Crystal Structures of a Cysteine-modified Mutant in Loop D of Acetylcholine-binding Protein

Atypical nicotinic agonist bound conformations conferring subtype selectivity

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Unnatural Amino Acid Mutagenesis of the GABA_AReceptor Binding Site Residues Reveals a Novel Cation–π Interaction between GABA and β₂Tyr97