The neuronal ␣4␤2 nicotinic acetylcholine receptors exist as two distinct subtypes, (␣4) 2 (␤2) 3 and (␣4) 3 (␤2) 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 ␣4 and ␤2 subunits and concatenated constructs, we demonstrate that a biphasic response is an intrinsic functional property of the (␣4) 3 (␤2) 2 receptor. In addition to two high-sensitivity sites at ␣4␤2 interfaces, the (␣4) 3 (␤2) 2 receptor contains a third low-sensitivity agonist binding site in the ␣4␣4 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 ␣4␤2 and ␣4␣4 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.

Background: Molecular features governing ␣4␤2 nAChRs efficacy have remained elusive. Results: Binding studies, electrophysiology, and structural data from co-crystallization with Ls-AChBP are reported for a series of ␣4␤2 agonists. Conclusion: Direct halogen bonds and an invariant Loop-C suggest that intersubunit bridge formation governs efficacy. Significance: The data provide a structural basis for understanding of efficacy levels at nAChRs.

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 GABAA receptor is made available as Model S1.

Open3DQSAR is a freely available open-source program aimed at chemometric analysis of molecular interaction fields. MIFs can be imported from different sources (GRID, CoMFA/CoMSIA, quantum-mechanical electrostatic potential or electron density grids) or generated by Open3DQSAR itself. Much focus has been put on automation through the implementation of a scriptable interface, as well as on high computational performance achieved by algorithm parallelization. Flexibility and interoperability with existing molecular modeling software make Open3DQSAR a powerful tool in pharmacophore assessment and ligand-based drug design.

An open-source, cross-platform software aimed at conformer generation and unsupervised rigid-body molecular alignment is presented. Different algorithms have been implemented to perform single and multi-conformation superimpositions on one or more templates. Alignments can be accomplished by matching pharmacophores, heavy atoms or a combination of the two. All methods have been successfully validated on eight comprehensive datasets previously gathered by Sutherland and co-workers. High computational performance has been attained through efficient parallelization of the code. The unsupervised nature of the alignment algorithms, together with its scriptable interface, make Open3DALIGN an ideal component of high-throughput, automated cheminformatics workflows.

Background: Cys loop receptors can be modulated by exogenous compounds. Results: By combining x-ray crystallography, homology modeling, quantum mechanical calculations, and functional studies on ␣4␤2 nAChRs, the binding mode and modulatory mechanism of the ␣4␤2 nAChR modulator NS9283 were revealed. Conclusion: Modulatory actions occur by mimicking agonists in the ␣4-␣4 ACh-binding pocket. Significance: Increased understanding of modulator actions open new possibilities for rational drug design.

Background: Positive allosteric modulators (PAMs) of ␣4␤2 nicotinic acetylcholine receptors have significant therapeutic potential. Results: Two PAMs, NS206 and NS9283, were observed to have differential and additive pharmacological actions due to binding at distinct receptor sites. Conclusion: Modulator binding activity is linked to the specific binding position in Cys-loop receptors. Significance: Diverse PAM profiles increase possibilities for rational drug design and understanding of receptor function.

Despite extensive studies on nicotinic acetylcholine receptors (nAChRs) and homologues, details of acetylcholine binding are not completely resolved. Here, we report the crystal structure of acetylcholine bound to the receptor homologue acetylcholine binding protein from Lymnaea stagnalis. This is the first structure of acetylcholine in a binding pocket containing all five aromatic residues conserved in all mammalian nAChRs. The ligand-protein interactions are characterized by contacts to the aromatic box formed primarily by residues on the principal side of the intersubunit binding interface (residues Tyr89, Trp143 and Tyr185). Besides these interactions on the principal side, we observe a cation-π interaction between acetylcholine and Trp53 on the complementary side and a water-mediated hydrogen bond from acetylcholine to backbone atoms of Leu102 and Met114, both of importance for anchoring acetylcholine to the complementary side. To further study the role of Trp53, we mutated the corresponding tryptophan in the two different acetylcholine-binding interfaces of the widespread α4β2 nAChR, i.e. the interfaces α4(+)β2(−) and α4(+)α4(−). Mutation to alanine (W82A on the β2 subunit or W88A on the α4 subunit) significantly altered the response to acetylcholine measured by oocyte voltage-clamp electrophysiology in both interfaces. This shows that the conserved tryptophan residue is important for the effects of ACh at α4β2 nAChRs, as also indicated by the crystal structure. The results add important details to the understanding of how this neurotransmitter exerts its action and improves the foundation for rational drug design targeting these receptors.