Explore the vast range of titles available.

Fast chemical communication in the nervous system is mediated by neurotransmitter-gated ion channels. The prototypical member of this class of cell surface receptors is the cation-selective nicotinic acetylcholine receptor. As with most ligand-gated ion channels, nicotinic receptors assemble as oligomers of subunits, usually as hetero-oligomers and often with variable stoichiometries 1 . This intrinsic heterogeneity in protein composition provides fine tunability in channel properties, which is essential to brain function, but frustrates structural and biophysical characterization. The α4β2 subtype of the nicotinic acetylcholine receptor is the most abundant isoform in the human brain and is the principal target in nicotine addiction. This pentameric ligand-gated ion channel assembles in two stoichiometries of α- and β-subunits (2α:3β and 3α:2β). Both assemblies are functional and have distinct biophysical properties, and an imbalance in the ratio of assemblies is linked to both nicotine addiction 2 , 3 and congenital epilepsy 4 , 5 . Here we leverage cryo-electron microscopy to obtain structures of both receptor assemblies from a single sample. Antibody fragments specific to β2 were used to ‘break’ symmetry during particle alignment and to obtain high-resolution reconstructions of receptors of both stoichiometries in complex with nicotine. The results reveal principles of subunit assembly and the structural basis of the distinctive biophysical and pharmacological properties of the two different stoichiometries of this receptor. Cryo-electron microscopy structures of two stoichiometries of heteromeric acetylcholine receptors in complex with nicotine reveal principles of subunit assembly and the structural basis of the distinctive biophysical and pharmacological properties of the different stoichiometries.

The intracellular domain (ICD) of Cys-loop receptors mediates diverse functions. To date, no structure of a full-length ICD is available due to challenges stemming from its dynamic nature. Here, combining nuclear magnetic resonance (NMR) and electron spin resonance experiments with Rosetta computations, we determine full-length ICD structures of the human α7 nicotinic acetylcholine receptor in a resting state. We show that ~57% of the ICD residues are in highly flexible regions, primarily in a large loop (loop L) with the most mobile segment spanning ~50 Å from the central channel axis. Loop L is anchored onto the MA helix and virtually forms two smaller loops, thereby increasing its stability. Previously known motifs for cytoplasmic binding, regulation, and signaling are found in both the helices and disordered flexible regions, supporting the essential role of the ICD conformational plasticity in orchestrating a broad range of biological processes. The intracellular domain (ICD) of Cys-loop receptors mediates many of their functions, but no complete structure of a Cys-loop receptor ICD is available to date. Here, the authors combine NMR and ESR spectroscopy to determine the full-length ICD structures of the human α7 nicotinic acetylcholine receptor (α7nAChR).

The α6β4 nicotinic acetylcholine receptor (nAChR) is found in the sensory neurons of dorsal root ganglia. It is a promising therapeutic target for pain. However, the difficultly of heterologous functional expression of α6β4 receptor has hindered the discovery of drugs that target it. Here, we functionally express the human α6β4 receptor and determine the cryo-EM structures of α6β4 receptor in complex with its agonists, nicotine and the preclinical drug tebanicline. These structures were captured in non-conducting desensitized states. We elucidate that the stoichiometry of α- and β- subunits in the α6β4 receptor is 2α6:3β4. Furthermore, we identify the binding pockets for nicotine and tebanicline, demonstrating the essential residues contributing to ligand affinity and providing detailed molecular insights into why these agonists have different binding affinities despite both occupying the orthosteric site of the α6β4 receptor. These structures offer significant molecular insight into the function and ligand recognition of α6β4 receptor. The α6β4 receptor has been considered an appealing therapeutic target for pain. Here, the authors determined the structures of α6β4 bound with its agonists, nicotine and tebanicline, providing molecular insights into its function.

The α9α10 nicotinic cholinergic receptor (nAChR) is a ligand-gated pentameric cation-permeable ion channel that mediates synaptic transmission between descending efferent neurons and mechanosensory inner ear hair cells. When expressed in heterologous systems, α9 and α10 subunits can assemble into functional homomeric α9 and heteromeric α9α10 receptors. One of the differential properties between these nAChRs is the modulation of their ACh-evoked responses by extracellular calcium (Ca 2+ ). While α9 nAChRs responses are blocked by Ca 2+ , ACh-evoked currents through α9α10 nAChRs are potentiated by Ca 2+ in the micromolar range and blocked at millimolar concentrations. Using chimeric and mutant subunits, together with electrophysiological recordings under two-electrode voltage-clamp, we show that the TM2-TM3 loop of the rat α10 subunit contains key structural determinants responsible for the potentiation of the α9α10 nAChR by extracellular Ca 2+ . Moreover, molecular dynamics simulations reveal that the TM2-TM3 loop of α10 does not contribute to the Ca 2+ potentiation phenotype through the formation of novel Ca 2+ binding sites not present in the α9 receptor. These results suggest that the TM2-TM3 loop of α10 might act as a control element that facilitates the intramolecular rearrangements that follow ACh-evoked α9α10 nAChRs gating in response to local and transient changes of extracellular Ca 2+ concentration. This finding might pave the way for the future rational design of drugs that target α9α10 nAChRs as otoprotectants.

The existence of α7β2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer's disease, it is critical to determine whether α7β2 nAChRs are present in the human brain, in which brain areas, and whether they differ functionally from α7 nAChR homomers. We used α-bungarotoxin to affinity purify α7-containing nAChRs from surgically excised human temporal cortex, and found that α7 subunits co-purify with β2 subunits, indicating the presence of α7β2 nAChRs in the human brain. We validated these results by demonstrating co-purification of β2 from wild-type, but not α7 or β2 knock-out mice. The pharmacology and kinetics of human α7β2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293 cells. Notably, α7β2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with β2 subunits, and that human α7β2 nAChR heteromers respond to nAChR agonists with a unique pharmacology and kinetic profile. α7β2 nAChRs thus represent an alternative mechanism for the reported clinical efficacy of α7 nAChR ligands.

RationalePrevious neuroimaging studies of cognition involving nicotinic acetylcholine receptor (nAChR) agonist administration have repeatedly found enhanced task-induced deactivation of regions of the default mode network (DMN), a group of brain systems that is more active at rest and mediates task-independent thought processes. This effect may be related to pro-cognitive nAChR agonist effectsObjectivesThe present study sought to test whether nAChR modulation of the DMN is bi-directional, i.e., whether a nAChR antagonist would reduce task-induced deactivation.MethodsEighteen healthy non-smokers underwent functional magnetic resonance imaging while performing a letter N-back task. Scans were performed after nicotine administration (7 mg/24 h, transdermally), after administration of the nAChR antagonist mecamylamine (7.5 mg, p.o.), and after double placebo, in counterbalanced sequence. Blood-oxygen-level-dependent (BOLD) signal was analyzed within ventromedial prefrontal cortex (vmPFC) and posterior cingulate cortex (PCC) regions of interest—central hubs of the DMN in which consistent nAChR agonist–induced changes had previously been identified.ResultsNicotine enhanced hit rate in both the 0-back and 2-back condition, while mecamylamine slowed reaction time in the 2-back condition. Mecamylamine reduced task-induced deactivation of vmPFC and PCC. Nicotine had no significant effects on the BOLD signal.ConclusionsThe finding that nAChR tone reduction by mecamylamine weakened task-induced DMN deactivation indicates that a constant tone of nAChR activation helps regulate DMN activity in healthy individuals. This suggests that low nAChR tone may play a causal role in DMN dysregulation seen in conditions such as mild cognitive impairment or Alzheimer’s disease.

Genetic variation in CHRNA5, the gene encoding the α5 nicotinic acetylcholine receptor subunit, increases vulnerability to tobacco addiction and lung cancer, but the underlying mechanisms are unknown. Here we report markedly increased nicotine intake in mice with a null mutation in Chrna5 . This effect was ‘rescued’ in knockout mice by re-expressing α5 subunits in the medial habenula (MHb), and recapitulated in rats through α5 subunit knockdown in MHb. Remarkably, α5 subunit knockdown in MHb did not alter the rewarding effects of nicotine but abolished the inhibitory effects of higher nicotine doses on brain reward systems. The MHb extends projections almost exclusively to the interpeduncular nucleus (IPN). We found diminished IPN activation in response to nicotine in α5 knockout mice. Further, disruption of IPN signalling increased nicotine intake in rats. Our findings indicate that nicotine activates the habenulo-interpeduncular pathway through α5-containing nAChRs, triggering an inhibitory motivational signal that acts to limit nicotine intake. Anti-smoking drug target Genetic association studies implicate variation in CHRNA5 , the gene for the α5 neuronal nicotinic acetylcholine receptor (nAChR) subunit, in susceptibility to tobacco dependence, lung cancer and chronic obstructive pulmonary disease. The mechanisms linking this gene to behaviour are unknown. Using knockout mice, lentiviral rescue and RNAi knockdown in rats, Fowler et al . show that manipulating the levels of this subunit alters the drive to obtain nicotine, particularly at high doses. Altering activity levels in the habenulo-interpeduncular tract of the brain, where this subunit is highly expressed, changes the amount of nicotine the animals consume. This work identifies α5-containing nAChRs as potential targets for smoking-cessation therapies. In humans, vulnerability to tobacco addiction has been linked to variations in the gene encoding the α5 nicotinic acetylcholine receptor subunit, but the functional mechanisms linking gene to behaviour are unknown. Using a combination of knockout mice, lentiviral rescue, and RNAi knockdown in rats, this study shows that manipulating the levels of this subunit alters the drive to obtain nicotine, particularly at high doses that are aversive to wild-type animals. Furthermore, these subunits are implicated in the projection between medial habenula and interpeduncular nucleus in integrating negative side effects of high doses of nicotine and reward signals. It is proposed that this projection provides a negative motivational signal that limits nicotine consumption.

Activation of the cholinergic anti-inflammatory pathway (CAP) via vagus nerve stimulation has been shown to improve acute kidney injury in rodent models. While alpha 7 nicotinic acetylcholine receptor (α7nAChR) positive macrophages are thought to play a crucial role in this pathway, their in vivo significance has not been fully understood. In this study, we used macrophage-specific α7nAChR-deficient mice to confirm the direct activation of α7nAChRs in macrophages. Our findings indicate that the administration of GTS-21, an α7nAChR-specific agonist, protects injured kidneys in wild-type mice but not in macrophage-specific α7nAChR-deficient mice. To investigate the signal changes or cell reconstructions induced by α7nAChR activation in splenocytes, we conducted single-cell RNA-sequencing of the spleen. Ligand-receptor analysis revealed an increase in macrophage-macrophage interactions. Using macrophage-derived cell lines, we demonstrated that GTS-21 increases cell contact, and that the contact between macrophages receiving α7nAChR signals leads to a reduction in TNF-α. Our results suggest that α7nAChR signaling increases macrophage-macrophage interactions in the spleen and has a protective effect on the kidneys. The signaling through α7 nicotinic acetylcholine receptor (α7nAChR) in mouse macrophages has been revealed to enhance macrophage-macrophage interactions in the spleen and has a protective effect on the kidneys.

The evolution of new traits enables expansion into new ecological and behavioural niches. Nonetheless, demonstrated connections between divergence in protein structure, function and lineage-specific behaviours remain rare. Here we show that both octopus and squid use cephalopod-specific chemotactile receptors (CRs) to sense their respective marine environments, but structural adaptations in these receptors support the sensation of specific molecules suited to distinct physiological roles. We find that squid express ancient CRs that more closely resemble related nicotinic acetylcholine receptors, whereas octopuses exhibit a more recent expansion in CRs consistent with their elaborated ‘taste by touch’ sensory system. Using a combination of genetic profiling, physiology and behavioural analyses, we identify the founding member of squid CRs that detects soluble bitter molecules that are relevant in ambush predation. We present the cryo-electron microscopy structure of a squid CR and compare this with octopus CRs 1 and nicotinic receptors 2 . These analyses demonstrate an evolutionary transition from an ancestral aromatic ‘cage’ that coordinates soluble neurotransmitters or tastants to a more recent octopus CR hydrophobic binding pocket that traps insoluble molecules to mediate contact-dependent chemosensation. Thus, our study provides a foundation for understanding how adaptation of protein structure drives the diversification of organismal traits and behaviour. Octopus and squid use cephalopod-specific chemotactile receptors to sense their respective marine environments, but structural adaptations in these receptors support the sensation of specific molecules suited to distinct physiological roles.

Chemotactile receptors (CRs) are a cephalopod-specific innovation that allow octopuses to explore the seafloor via ‘taste by touch’ 1 . CRs diverged from nicotinic acetylcholine receptors to mediate contact-dependent chemosensation of insoluble molecules that do not readily diffuse in marine environments. Here we exploit octopus CRs to probe the structural basis of sensory receptor evolution. We present the cryo-electron microscopy structure of an octopus CR and compare it with nicotinic receptors to determine features that enable environmental sensation versus neurotransmission. Evolutionary, structural and biophysical analyses show that the channel architecture involved in cation permeation and signal transduction is conserved. By contrast, the orthosteric ligand-binding site is subject to diversifying selection, thereby mediating the detection of new molecules. Serendipitous findings in the cryo-electron microscopy structure reveal that the octopus CR ligand-binding pocket is exceptionally hydrophobic, enabling sensation of greasy compounds versus the small polar molecules detected by canonical neurotransmitter receptors. These discoveries provide a structural framework for understanding connections between evolutionary adaptations at the atomic level and the emergence of new organismal behaviour. Cryo-electron microscopy analyses reveal adaptations that facilitate the octopus chemotactile receptor’s evolutionary transition from an ancestral role in neurotransmission to detecting greasy environmental agonists for ‘taste by touch’ sensory behaviour.