Nicotinic acetylcholine receptors (nAChRs) are ligand- gated ion channels that mediate rapid neurotransmission in the central and peripheral nervous systems. The acetylcholine binding protein (AChBP) is a soluble structural and functional surrogate of the extracellular, ligand-binding domain of the nAChR that allows for studies not amenable to study of the nAChR as an integral membrane protein. In particular, AChBP provides a system in which to study solution dynamics and conformational changes related to ligand binding. To deduce ligand binding sites and infer conformational changes, cysteine mutants of AChBP were generated and labeled with the solvent- sensitive fluorophore, acrylodan. The fluorescence emission spectra from acrylodan-labeled AChBP mutants were examined in the absence and presence of nicotinic ligands. Binding of small molecules and large peptide toxins caused acrylodan conjugated to Q178C to move into environments of opposite polarities. From this, I proposed a hinge position at Q178 that allows for flexibility of the C-loop, such that it can expand or contract as a rigid body to accommodate bound ligand. Distinctive changes in acrylodan emission were also observed in the F-loop, indicating that this region of the protein likely plays a role in ligand binding, a finding not evident from existing structures. Complementary hydrodynamic and fluorescence anisotropy studies of AChBP free in solution and in complex with an 8 kD three-fingered alpha-neurotoxin indicated that the bound alpha-toxin has minimal effects on the translational and rotational diffusion properties of AChBP. Anisotropy experiments showed that, when bound, the alpha-toxin is highly dynamic. Its central finger, that contains beta- sheet structure and interacts with the agonist binding site, is the most rigid portion. The segmental flexibility of AChBP was studied by measuring decay of fluorescence anisotropy from fluorescein-labeled AChBP mutants. The results revealed that AChBP exhibits wide regional variation in alpha-carbon backbone flexibility, with the C -loop being overall most rigid in the picosecond- nanosecond time domain. In studying the effect of ligand binding on conformational flexibility, we found that the F -loop conformational change associated with ligand binding is pharmacologically correlated. Solution-based structural studies of conformation related to ligand binding should facilitate structure-guided drug design and increase understanding of nAChR function

General anesthetics and neuromuscular blockers are used together during surgery to stabilize patients in an unconscious state. Anesthetics act mainly by potentiating inhibitory ion channels and inhibiting excitatory ion channels, with the net effect of dampening nervous system excitability. Neuromuscular blockers act by antagonizing nicotinic acetylcholine receptors at the motor endplate; these excitatory ligand-gated ion channels are also inhibited by general anesthetics. The mechanisms by which anesthetics and neuromuscular blockers inhibit nicotinic receptors are poorly understood but underlie safe and effective surgeries. Here we took a direct structural approach to define how a commonly used anesthetic and two neuromuscular blockers act on a muscle-type nicotinic receptor. We discover that the intravenous anesthetic etomidate binds at an intrasubunit site in the transmembrane domain and stabilizes a non-conducting, desensitized-like state of the channel. The depolarizing neuromuscular blocker succinylcholine also stabilizes a desensitized channel but does so through binding to the classical neurotransmitter site. Rocuronium binds in this same neurotransmitter site but locks the receptor in a resting, non-conducting state. Together, this study reveals a structural mechanism for how general anesthetics work on excitatory nicotinic receptors and further rationalizes clinical observations in how general anesthetics and neuromuscular blockers interact.

Methaqualone, a quinazolinone marketed commercially as Quaalude, is a central nervous system depressant that was used clinically as a sedative-hypnotic, then became a notorious recreational drug in the 1960s-80s. Due to its high abuse potential, medical use of methaqualone was eventually prohibited, yet it persists as a globally abused substance. Methaqualone principally targets GABAA receptors, which are the major inhibitory neurotransmitter-gated ion channels in the brain. The restricted status and limited accessibility of methaqualone have contributed to its pharmacology being understudied. Here, we use cryo-EM to localize the GABAA receptor binding sites of methaqualone and its more potent derivative, PPTQ, to the same intersubunit transmembrane sites targeted by the general anesthetics propofol and etomidate. Both methaqualone and PPTQ insert more deeply into subunit interfaces than the previously-characterized modulators. Binding of quinazolinones to this site results in widening of the extracellular half of the ion-conducting pore, following a trend among positive allosteric modulators in destabilizing the hydrophobic activation gate in the pore as a mechanism for receptor potentiation. These insights shed light on the underexplored pharmacology of quinazolinones and further elucidate the molecular mechanisms of allosteric GABAA receptor modulation through transmembrane binding sites.

Chemotactile receptors (CRs) are a cephalopod-specific innovation that allow octopuses to explore the seafloor via taste by touch1. 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.

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 CRs1 and nicotinic receptors2. 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.