Nicotinic acetylcholine receptors are ligand-gated ion channels that mediate fast chemical neurotransmission at the neuromuscular junction and have diverse signalling roles in the central nervous ...system. The nicotinic receptor has been a model system for cell-surface receptors, and specifically for ligand-gated ion channels, for well over a century. In addition to the receptors' prominent roles in the development of the fields of pharmacology and neurobiology, nicotinic receptors are important therapeutic targets for neuromuscular disease, addiction, epilepsy and for neuromuscular blocking agents used during surgery. The overall architecture of the receptor was described in landmark studies of the nicotinic receptor isolated from the electric organ of Torpedo marmorata. Structures of a soluble ligand-binding domain have provided atomic-scale insights into receptor-ligand interactions, while high-resolution structures of other members of the pentameric receptor superfamily provide touchstones for an emerging allosteric gating mechanism. All available high-resolution structures are of homopentameric receptors. However, the vast majority of pentameric receptors (called Cys-loop receptors in eukaryotes) present physiologically are heteromeric. Here we present the X-ray crystallographic structure of the human α4β2 nicotinic receptor, the most abundant nicotinic subtype in the brain. This structure provides insights into the architectural principles governing ligand recognition, heteromer assembly, ion permeation and desensitization in this prototypical receptor class.
The ability of oligomeric membrane proteins to assemble in different functional ratios of subunits is a common feature across many systems. Recombinant expression of hetero-oligomeric proteins with ...defined stoichiometries facilitates detailed structural and functional analyses, but remains a major challenge. Here we present two methods for overcoming this challenge: one for rapid virus titration and another for stoichiometry determination. When these methods are coupled, they allow for efficient dissection of the heteromer stoichiometry problem and optimization of homogeneous protein expression. We demonstrate the utility of the methods in a system that to date has proved resistant to atomic-scale structural study, the nicotinic acetylcholine receptor. Leveraging these two methods, we have successfully expressed, purified, and grown diffraction-quality crystals of this challenging target.
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•Streamlined bacmam virus titration method•Fluorescent protein fusion approach for estimation of subunit ratio in a heteromer•Expression and crystallization of a defined nicotinic receptor stoichiometry
Morales-Perez et al. developed a pair of methods to efficiently express defined stoichiometries of heteromeric membrane proteins. Application of this approach to a nicotinic acetylcholine receptor that can assemble in multiple functional ratios of subunits has yielded diffraction-quality crystals of the receptor.
Fast inhibitory neurotransmission in the brain is principally mediated by the neurotransmitter GABA (γ-aminobutyric acid) and its synaptic target, the type A GABA receptor (GABA
receptor). ...Dysfunction of this receptor results in neurological disorders and mental illnesses including epilepsy, anxiety and insomnia. The GABA
receptor is also a prolific target for therapeutic, illicit and recreational drugs, including benzodiazepines, barbiturates, anaesthetics and ethanol. Here we present high-resolution cryo-electron microscopy structures of the human α1β2γ2 GABA
receptor, the predominant isoform in the adult brain, in complex with GABA and the benzodiazepine site antagonist flumazenil, the first-line clinical treatment for benzodiazepine overdose. The receptor architecture reveals unique heteromeric interactions for this important class of inhibitory neurotransmitter receptor. This work provides a template for understanding receptor modulation by GABA and benzodiazepines, and will assist rational approaches to therapeutic targeting of this receptor for neurological disorders and mental illness.
The α7 nicotinic acetylcholine receptor plays critical roles in the central nervous system and in the cholinergic inflammatory pathway. This ligand-gated ion channel assembles as a homopentamer, is ...exceptionally permeable to Ca2+, and desensitizes faster than any other Cys-loop receptor. The α7 receptor has served as a prototype for the Cys-loop superfamily yet has proven refractory to structural analysis. We present cryo-EM structures of the human α7 nicotinic receptor in a lipidic environment in resting, activated, and desensitized states, illuminating the principal steps in the gating cycle. The structures also reveal elements that contribute to its function, including a C-terminal latch that is permissive for channel opening, and an anionic ring in the extracellular vestibule that contributes to its high conductance and calcium permeability. Comparisons among the α7 structures provide a foundation for mapping the gating cycle and reveal divergence in gating mechanisms in the Cys-loop receptor superfamily.
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•Structures of the human α7 nicotinic acetylcholine receptor•Structures of the three major conformations of the gating cycle•Identification of a C-terminal substructure involved in gating•Identification of an extracellular residue key to calcium permeability
Cryo-EM structures of the α7 nicotinic acetylcholine receptor in three major conformational states of the gating cycle—resting, activated, and desensitized—along with functional studies reveal components involved in calcium conductance and allosteric coupling of ligand binding to channel opening.
Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABA
) receptors to dampen neuronal activity in the brain
. However, direct ...structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABA
receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABA
receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABA
receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.
The nicotinic acetylcholine receptor, a pentameric ligand-gated ion channel, converts the free energy of binding of the neurotransmitter acetylcholine into opening of its central pore. Here we ...present the first high-resolution structure of the receptor type found in muscle-endplate membrane and in the muscle-derived electric tissues of fish. The native receptor was purified from Torpedo electric tissue and functionally reconstituted in lipids optimal for cryo-electron microscopy. The receptor was stabilized in a closed state by the binding of α-bungarotoxin. The structure reveals the binding of a toxin molecule at each of two subunit interfaces in a manner that would block the binding of acetylcholine. It also reveals a closed gate in the ion-conducting pore, formed by hydrophobic amino acid side chains, located ∼60 Å from the toxin binding sites. The structure provides a framework for understanding gating in ligand-gated channels and how mutations in the acetylcholine receptor cause congenital myasthenic syndromes.
•High-resolution structure of a native muscle-type nicotinic acetylcholine receptor•Previously unresolved structural elements contribute to neurotoxin binding•Channel is stabilized in a closed conformation by α-bungarotoxin from snake venom•Transduction mechanism and myasthenic disease mutations
Rahman et al. report the high-resolution single-particle cryo-EM structure of a native muscle-type nicotinic acetylcholine receptor from the Torpedo electric ray, in complex with α-bungarotoxin from the banded krait. The structure was obtained in a lipidic environment shown to support channel function and reveals a closed, hydrophobic ion channel gate.
Here we begin by briefly reviewing landmark structural studies on the nicotinic acetylcholine receptor. We highlight challenges that had to be overcome to push through resolution barriers, then focus ...on what has been gleaned in the past few years from crystallographic and single particle cryo-EM studies of different nicotinic receptor subunit assemblies and ligand complexes. We discuss insights into ligand recognition, ion permeation, and allosteric gating. We then highlight some foundational aspects of nicotinic receptor structural biology that remain unresolved and are areas ripe for future exploration.
This article is part of the special issue on ‘Contemporary Advances in Nicotine Neuropharmacology’.
Autoantibodies targeting neuronal membrane proteins can cause encephalitis, seizures, and severe behavioral abnormalities. While antibodies for several neuronal targets have been identified, ...structural details on how they regulate function are unknown. Here we determined cryo-electron microscopy structures of antibodies derived from an encephalitis patient bound to the γ-aminobutyric acid type A (GABA
A
) receptor. These antibodies induced a severe encephalitis by directly inhibiting GABA
A
function, resulting in nervous system hyperexcitability. The structures reveal mechanisms of GABA
A
inhibition and pathology. One antibody directly competes with neurotransmitter and locks the receptor in a resting-like state. The second antibody targets the subunit interface involved in binding benzodiazepines and antagonizes diazepam potentiation. We identify key residues in these antibodies involved in specificity and affinity and confirm structure-based hypotheses for functional effects using electrophysiology. Together these studies define mechanisms of direct functional antagonism of neurotransmission underlying autoimmune encephalitis in a human patient.
Structural analysis of antibodies from an encephalitis patient bound to the GABA
A
receptor reveals mechanisms of channel inhibition and pathology that drive autoimmune encephalitis in humans.
Nicotinic acetylcholine receptors are pentameric ion channels that mediate fast chemical neurotransmission. The α3β4 nicotinic receptor subtype forms the principal relay between the central and ...peripheral nervous systems in the autonomic ganglia. This receptor is also expressed focally in brain areas that affect reward circuits and addiction. Here, we present structures of the α3β4 nicotinic receptor in lipidic and detergent environments, using functional reconstitution to define lipids appropriate for structural analysis. The structures of the receptor in complex with nicotine, as well as the α3β4-selective ligand AT-1001, complemented by molecular dynamics, suggest principles of agonist selectivity. The structures further reveal much of the architecture of the intracellular domain, where mutagenesis experiments and simulations define residues governing ion conductance.
•The α3β4 ganglionic nicotinic receptor is a target for anti-addiction therapeutics•Receptor structures were determined in a functionally supportive lipidic environment•Comparisons with the α4β2 subtype suggest principles underlying ligand selectivity•Lateral portals in the intracellular domain facilitate ion permeation
Gharpure et al. combine single-particle cryo-electron microscopy, electrophysiology, and molecular dynamics simulations to interrogate the structure and function of the α3β4 nicotinic acetylcholine receptor in a lipidic environment.
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