The neuronal α4β2 nicotinic acetylcholine receptors exist as two distinct subtypes, (α4)₂(β2)₃ and (α4)₃(β2)₂, and biphasic responses to acetylcholine and other agonists have been ascribed previously to coexistence of these two receptor subtypes. We offer a novel and radical explanation for the observation of two distinct agonist sensitivities. Using different expression ratios of mammalian α4 and β2 subunits and concatenated constructs, we demonstrate that a biphasic response is an intrinsic functional property of the (α4)₃(β2)₂ receptor. In addition to two high-sensitivity sites at α4β2 interfaces, the (α4)₃(β2)₂ receptor contains a third low-sensitivity agonist binding site in the α4α4 interface. Occupation of this site is required for full activation and is responsible for the widened dynamic response range of this receptor subtype. By site-directed mutagenesis, we show that three residues, which differ between the α4β2 and α4α4 sites, control agonist sensitivity. The results presented here provide a basic insight into the function of pentameric ligand-gated ion channels, which enables modulation of the receptors with hitherto unseen precision; it becomes possible to rationally design therapeutics targeting subpopulations of specific receptor subtypes.

We present a full-length α₁β₂γ₂ GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate-gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode similar to the binding mode of glutamate in the GluCl X-ray structure. Key interactions are predicted with residues α₁R66, β₂T202, α₁T129, β₂E155, β₂Y205 and the backbone of β₂S156. Muscimol is predicted to bind similarly, however, with minor differences rationalized with quantum mechanical energy calculations. Muscimol key interactions are predicted to be α₁R66, β₂T202, α₁T129, β₂E155, β₂Y205 and β₂F200. Furthermore, we argue that a water molecule could mediate further interactions between muscimol and the backbone of β₂S156 and β₂Y157. DZP is predicted to bind with interactions comparable to those of the agonists in the orthosteric site. The carbonyl group of DZP is predicted to interact with two threonines α₁T206 and γ₂T142, similar to the acidic moiety of GABA. The chlorine atom of DZP is placed near the important α₁H101 and the N-methyl group near α₁Y159, α₁T206, and α₁Y209. We present a binding mode of DZP in which the pending phenyl moiety of DZP is buried in the binding pocket and thus shielded from solvent exposure. Our full length GABA_A receptor is made available as Model S1.

One of the most frequent protein–protein interaction modules in mammalian cells is the postsynaptic density 95/discs large/zonula occludens 1 (PDZ) domain, involved in scaffolding and signaling and emerging as an important drug target for several diseases. Like many other protein–protein interactions, those of the PDZ domain family involve formation of intermolecular hydrogen bonds: C-termini or internal linear motifs of proteins bind as β-strands to form an extended antiparallel β-sheet with the PDZ domain. Whereas extensive work has focused on the importance of the amino acid side chains of the protein ligand, the role of the backbone hydrogen bonds in the binding reaction is not known. Using amide-to-ester substitutions to perturb the backbone hydrogen-bonding pattern, we have systematically probed putative backbone hydrogen bonds between four different PDZ domains and peptides corresponding to natural protein ligands. Amide-to-ester mutations of the three C-terminal amides of the peptide ligand severely affected the affinity with the PDZ domain, demonstrating that hydrogen bonds contribute significantly to ligand binding (apparent changes in binding energy, ΔΔG = 1.3 to >3.8 kcal mol^–1). This decrease in affinity was mainly due to an increase in the dissociation rate constant, but a significant decrease in the association rate constant was found for some amide-to-ester mutations suggesting that native hydrogen bonds have begun to form in the transition state of the binding reaction. This study provides a general framework for studying the role of backbone hydrogen bonds in protein–peptide interactions and for the first time specifically addresses these for PDZ domain–peptide interactions.