Structural Studies of Nicotinic Acetylcholine Receptors: Using Acetylcholine‐Binding Protein as a Structural Surrogate

Abstract

Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel superfamily that play important roles in the control of neurotransmitter release in the central and peripheral nervous system. These receptors are important therapeutic targets for the development of drugs against a number of mental health disorders and for marketed smoking cessation aids. Unfortunately, drug discovery has been hampered by difficulties in obtaining sufficiently selective compounds. Together with functional complexity of the receptors, this has made it difficult to obtain drugs with sufficiently high-target to off-target affinity ratios. The recent and ongoing progress in structural studies holds promise to help understand structure-function relationships of nAChR drugs at the atomic level. This will undoubtedly lead to the design of more efficient drugs with fewer side effects. As a high-resolution structure of a nAChR is yet to be determined, structural studies are to a large extent based on acetylcholine-binding proteins (AChBPs) that despite low overall sequence identity display a high degree of conservation of overall structure and amino acids at the ligand-binding site. Further, AChBPs reproduce relative binding affinities of ligands at nAChRs. Over the past decade, AChBPs have been used extensively as models for nAChRs and have aided the understanding of drug receptor interactions at nAChRs significantly.Pentameric ligand-gated ion channels (pLGICs) constitute a class of receptors that include the cation-selective nicotinic acetylcholine receptors (nAChRs) and 5-hydroxytryptamine (serotonin) type 3 receptors (5-HT 3 ), as well as the anionselective c-amino butyric acid type A/C receptors (GABA A/C ) and glycine receptors (GlyRs). These receptors mediate a rapid chemical neurotransmission in the central and peripheral nervous system and play important roles regulating the release of other neurotransmitters. The overall structure of pLGICs is known from electron microscopy structures of the Torpedo marmorata nAChR [1,2] and zebrafish a1 GlyR [3] and from X-ray structures of the eukaryotic glutamate-activated pLGIC from Caenorhabditis elegans (GluCl) [4,5], a human GABA Ab3 receptor [6], a mouse serotonin 5-HT 3 receptor [7] and a human a3 GlyR [8]. Further, mechanistic insight has been obtained from the studies of bacterial ion channels from Erwinia chrysanthemi (ELIC) [9] and Gloeobacter violaceus (GLIC) [10][11][12].The functional pLGICs are pentameric assemblies of either identical or homologous protein chains arranged symmetrically around a central ion-conducting pore. Each chain comprises an N-terminal extracellular ligand-binding domain that carries the neurotransmitter-binding sites, a transmembrane domain formed by four a-helices, a cytoplasmic loop between the 3rd and 4th transmembrane helices and a variable extracellular C-terminal part ( fig. 1). The metazoan members of the family also contain an intracellular domain.The nAChRs are classified into muscle-and neuronal-type re...

A Structural Rationale for N‐Methylbicuculline Acting as a Promiscuous Competitive Antagonist of Inhibitory Pentameric Ligand‐Gated Ion Channels

“…The recent cryo‐EM structure of the complex between bicuculline and the human GABA A ‐[α1] 2 [β3] 2 [γ] receptor offered the opportunity for comparison and further analysis. First a comment; there is an inconsistency in the description of what was used to derive the structure in PDB ID: 6HUK . The publication and PDB entry describe bicuculline methochloride being used, but the coordinates and chemical diagram indicate bicuculline in both β3[+]/α1[−] sites, and that is used in our comparisons.…”

“…The crystal was immersed in liquid nitrogen, then placed under a stream of nitrogen gas at around −170 °C, and diffraction data were collected with a Rigaku M007HF copper‐anode generator, Varimax Cu‐VHF optics, Saturn 944HG+ CCD detector and AFC‐11 4‐axis partial χ goniometer. The data were integrated with XDS and scaled with AIMLESS; the structure was solved by molecular replacement with PHASER, exploiting the already refined structure of GBP with glycine (PDB ID: 5OAN) . Multiple rounds of automated restrained refinement were completed by using REFMAC5, interspersed with model adjustment based on inspection of electron and difference density maps in COOT .…”

“…These receptors consist of intracellular, transmembrane and extracellular domains . The acetylcholine‐binding protein from Aplysia californica ( Ac AChBP) shares sequence identity and structural homology with the extracellular domain (ECD) of pLGICs, and has been exploited as a surrogate system . We engineered an AChBP derivative called glycine‐binding protein (GBP) as a surrogate for the physiologically relevant β[+]/α[−] heteromeric interface of GlyR .…”

5‐Membered Mesoionic Insecticides: Synthesis and Evaluation of 1,3,4‐Thiadiazol‐4‐ium‐2‐olates with High Affinity for the Insect Nicotinic Acetylcholine Receptor

“…Aplysia californica and Lymnaea stagnalis . AChBP is responsible for regulating acetylcholine concentration in snail cholergenic synapses and serves as a surrogate for the extracellular region of the full‐length nAChR . In the last 15 years, >100 wild‐type and mutant AChBP structures have been deposited in the Protein Databank (PDB)…”

“…AChBP is responsible for regulating acetylcholine concentration in snail cholergenic synapses and serves as a surrogate for the extracellular region of the fulllength nAChR. [22] In the last 15 years, >100 wild-type and mutant AChBP structures have been deposited in the Protein Databank (PDB). [23] In this study the coordinates of an AChBP crystal structure (PDB 3WTH) were utilized to understand the binding mode and protein-ligand interactions of compound 9.…”