Ryan E. Hibbs’s research while affiliated with University of California System and other places

The neuronal α7 nicotinic acetylcholine receptor (α7-nAChR) and muscle-type nicotinic acetylcholine receptor (mt-nAChR) are pivotal in synaptic signaling within the brain and the neuromuscular junction respectively. Additionally, they are both targets of a wide range of drugs and toxins. Here, we utilize cryoEM to delineate structures of these nAChRs in complex with the conotoxins ImI and ImII from Conus imperialis. Despite nominal sequence divergence, ImI and ImII exhibit discrete binding preferences and adopt drastically different conformational states upon binding. ImI engages the orthosteric sites of the α7-nAChR, while ImII forms distinct pore-bound complexes with both the α7-nAChR and mt-nAChR. Strikingly, ImII adopts a compact globular conformation that binds as a monomer to the α7-nAChR pore and as an oblate dimer to the mt-nAChR pore. These structural characterizations advance our understanding of nAChR-ligand interactions as well as the subtle sequence variations that result in dramatically altered functional outcomes in small peptide toxins. Importantly, these results further elucidate the broad nature of cone snail toxin activities and highlight how targeted molecular evolution can give rise to functionally similar activities with surprisingly diverse mechanisms of action.

CryoEM increasingly attempts to incorporate time-resolved techniques to bridge the gap between static images and dynamic processes. Here we describe simple LED-based photo-flash system designed to simplify sample preparation for cryogenic sample time-resolved electron microscopy (cryoTREM). This user-friendly system offers flexibility in its operation, is cost-effective, and achieves uniform light exposure with minimal heat impact on the samples before plunge freezing. We describe the mechanical, electronic and optical components, that are tailored for easy plug and play operation for both the TFS Vitrobot and Leica GP2 plunge freezers. We will also present some initial results using this system to study a number of dynamic systems.

Microbial communities coat nearly every surface in the environment and have co-existed with animals throughout evolution. Whether animals exploit omnipresent microbial cues to navigate their surroundings is not well understood. Octopuses use “taste by touch” chemotactile receptors (CRs) to explore the seafloor, but how they distinguish meaningful surfaces from the rocks and crevices they encounter is unknown. Here, we report that secreted signals from microbiomes of ecologically relevant surfaces activate CRs to guide octopus behavior. Distinct molecules isolated from specific bacterial strains located on prey or eggs bind single CRs in subtly different structural conformations to elicit distinct mechanisms of receptor activation, ion permeation and signal transduction, and maternal care and predation behavior. Thus, microbiomes on ecological surfaces act at the level of primary sensory receptors to inform behavior. Our study demonstrates that uncovering interkingdom interactions is essential to understanding how animal sensory systems evolved in a microbe rich world. Highlights Chemotactile receptors (CRs) detect microbiomes of prey and progeny Diverse microbial signals bind single CRs with distinct structural conformations Distinct microbial signals activate single CRs to permeate different ions Environmental microbes elicit octopus predatory and maternal behaviors

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that plays an important role in neuronal signaling throughout the nervous system. Its implication in neurological disorders and inflammation has spurred the development of numerous compounds that enhance channel activation. However, the therapeutic potential of these compounds has been limited by the characteristically fast desensitization of the α7 receptor. Using recent high-resolution structures from cryo-EM, and all-atom molecular dynamic simulations augmented by Markov state modeling, here we explore the mechanism of α7 receptor desensitization and its implication on allosteric modulation. The results provide a precise characterization of the desensitization gate and illuminate the mechanism of ion-pore opening/closing with an agonist bound. In addition, the simulations reveal the existence of a short-lived, open-channel intermediate between the activated and desensitized states that rationalizes the paradoxical pharmacology of the L247T mutant and may be relevant to type-II allosteric modulation. This analysis provides an interpretation of the signal transduction mechanism and its regulation in α7 receptors.

Type A GABA (γ-aminobutyric acid) receptors (GABAA receptors) mediate most fast inhibitory signalling in the brain and are targets for drugs that treat epilepsy, anxiety, depression and insomnia and for anaesthetics1,2. These receptors comprise a complex array of 19 related subunits, which form pentameric ligand-gated ion channels. The composition and structure of native GABAA receptors in the human brain have been inferred from subunit localization in tissue1,3, functional measurements and structural analysis from recombinant expression4, 5, 6–7 and in mice⁸. However, the arrangements of subunits that co-assemble physiologically in native human GABAA receptors remain unknown. Here we isolated α1 subunit-containing GABAA receptors from human patients with epilepsy. Using cryo-electron microscopy, we defined a set of 12 native subunit assemblies and their 3D structures. We address inconsistencies between previous native and recombinant approaches, and reveal details of previously undefined subunit interfaces. Drug-like densities in a subset of these interfaces led us to uncover unexpected activity on the GABAA receptor of antiepileptic drugs and resulted in localization of one of these drugs to the benzodiazepine-binding site. Proteomics and further structural analysis suggest interactions with the auxiliary subunits neuroligin 2 and GARLH4, which localize and modulate GABAA receptors at inhibitory synapses. This work provides a structural foundation for understanding GABAA receptor signalling and targeted pharmacology in the human brain.

Skeletal muscle contraction is mediated by acetylcholine (ACh) binding to its ionotropic receptors (AChRs) at neuromuscular junctions. In myasthenia gravis (MG), autoantibodies target AChRs, disrupting neurotransmission and causing muscle weakness. Despite available treatments, patient responses vary, suggesting pathogenic heterogeneity. Current information on molecular mechanisms of autoantibodies is limited due to the absence of structures of an intact human muscle AChR. Here, we overcome challenges in receptor purification and present high-resolution cryo-EM structures of the human adult AChR in different functional states. Using a panel of six MG patient-derived monoclonal antibodies, we mapped distinct epitopes involved in diverse pathogenic mechanisms, including receptor blockade, internalization, and complement activation. Electrophysiological and binding assays further defined how these autoantibodies impair AChR function. These findings provide critical insights into MG immunopathology, revealing previously unrecognized antibody epitope diversity and mechanisms of receptor inhibition, offering a foundation for personalized therapies targeting antibody-mediated autoimmune disorders.

A recently reported behavioral screen in larval zebrafish for phenocopiers of known anesthetics and associated drugs yielded an isoflavone. Related isoflavones have also been reported as GABA A potentiators. From this, we synthesized a small library of isoflavones and incorporated an in vivo phenotypic approach to perform structure-behavior relationship studies of the screening hit and related analogs via behavioral profiling, patch-clamp experiments, and whole brain imaging. This revealed that analogs effect a range of behavioral responses, including sedation with and without enhancing the acoustic startle response. Interestingly, a subset of compounds effect sedation and enhancement of motor responses to both acoustic and light stimuli. Patch clamp recordings of cells with a human GABA A receptor confirmed that behavior-modulating isoflavones modify the GABA signaling. To better understand these molecules' nuanced effects on behavior, we performed whole brain imaging to reveal that analogs differentially effect neuronal activity. These studies demonstrate a multimodal approach to assessing activities of neuroactives. Neuroactive compounds (neuroactives) constitute a large group of clinically relevant yet poorly understood molecules that perturb nervous systems by engaging one or multiple targets. The polypharmacology of these molecules challenges the effectiveness of single-target in vitro studies, which are common in drug discovery and chemical biology. Even the assessment of multiple targets in vitro provides limited insights into their potential effects on neural circuits and behaviors. Thus, phenotypic approaches, like behavioral profiling, are valuable for exploring neuroactives with unknown targets or mechanisms. 1 These methods have revealed that drugs with similar pharmacological properties often induce similar behavioral patterns. 2−4 To complement these approaches, in vivo imaging of fluorescent markers using next-generation microscopy can assess neuronal activities within the central nervous system (CNS), underlying these behaviors. 5 Advanced behavioral screening platforms now allow precise control, capture, and quantification of animal behaviors influenced by neuroactives. 4,6,7 Zebrafish (Danio rerio) are an ideal model for connecting drug effects to behavior and neural activity changes. Larval zebrafish are particularly useful for in vivo studies of neuroactives due to the ease of imaging their CNS activity and behaviors. Our custom platform can generate and record responses from multiple animals per well in a 96-well plate, making it suitable for high-throughput experiments, including neuroactive compound testing 2,4 (Figure 1a). At 7 days post fertilization (dpf), larval zebrafish exhibit stereotyped sensor-imotor responses to stimuli (Figure 1b). For these studies, we performed behavioral profiling experiments involving the exposure of larval zebrafish to acoustic and light stimuli, recording their responses, and quantifying their movements as a motion index (Figure 1b, y-axis). Remarkably, unique behavioral profiles often correlate with distinct pharmacological treatments. 2,4,6 This platform is a powerful tool for identifying novel compounds that mimic clinically used drugs. Previous studies have successfully screened over ten thousand compounds to identify anesthetic-related compounds 2 and drugs that phenocopy first-generation typical antipsychotics like haloperidol. 8 By connecting behavioral patterns from phenotypic screens with known neuroactives, new hypotheses can be generated.