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

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.

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

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.

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

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.

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

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).

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

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.

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

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.