br Acknowledgements We thank LetPub www letpub com for its
Acknowledgements We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. This work was funded by National Natural Science Foundation of Beijing (No. 7122187), and Special fund for clinical research of Chinese Medical Association (No. 13040700455). The authors are very grateful to technicians from the Laboratory Animal Center for animal operational assistance, including Shenshen Kong, Guosheng Yang, Yinghong Tao, Yuanyuan Ma, and Yongyan Hu.
Introduction Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter receptors involved in fundamental cognitive processes such as learning, attention, and memory (Dehaene and Changeux, 1991). They are oligomeric protein assemblies that convert a chemical signal into an ion flux through the postsynaptic membrane, which mediates the transmission of an IKK Inhibitor VII (Cecchini and Changeux, 2015). At rest, the ion channel is closed. Binding of neurotransmitter elicits a rapid isomerization to the active state, which opens a transmembrane pore and allows the diffusion of anions or cations at rates approaching tens of millions per second. Prolonged exposure to high levels of agonist eventually results in a time-dependent decrease of ion conductance, or desensitization (Katz and Thesleff, 1957), which is thought to involve the transition to a structurally distinct closed-channel state (Gielen et al., 2015). The conformational transition bridging the resting state in the absence of agonist to the active state in the presence of agonist is commonly referred to as gating (Gielen and Corringer, 2018). Glycine receptors (GlyR) are pentameric ligand-gated chloride channel that mediate fast inhibitory synaptic transmission in the spinal cord and brainstem. They play a key role in motor coordination, essential sensory functions including vision and audition, and have been long recognized as pharmacological targets for chronic pain (Dutertre et al., 2012). Elucidating the mechanism of gating in this receptor family, i.e., the physiologically relevant states, the functional transitions, and their regulation by ligand binding, is likely to open novel therapeutic avenues against a range of neurological disorders including autism and hyperekplexia (startle disease) (Bode and Lynch, 2014). High-resolution structures of the zebrafish α1 and the human α3 GlyR in complex with ligands (agonist/antagonist/modulators) have been recently determined by cryoelectron microscopy (cryo-EM) (Du et al., 2015) and X-ray crystallography (Huang et al., 2015, Huang et al., 2017a, Huang et al., 2017b). These structural studies provided atomistic models for the open and closed states, demonstrating that binding of strychnine arrests GlyR in a closed-channel state, whereas the binding of glycine (Gly) and ivermectin (IVM) elicits a quaternary isomerization to the open state, which involves both receptor twisting and blooming (Cecchini and Changeux, 2015). Surprisingly, the EM reconstruction of GlyR in complex with Gly captured a previously unreported open state that features a pore configuration twice as large as of previously characterized open channels in GLIC at pH 4 (Bocquet et al., 2009, Sauguet et al., 2013) and GluCl with L-glutamate and IVM bound (Hibbs and Gouaux, 2011). Based on this observation, the Gly-bound wide-open structure was annotated as a truly agonist-activated open state, whereas the semi-open structure in complex with Gly and IVM was annotated as a low-conductance or possibly desensitized state (Du et al., 2015). Finally, two structures of GlyR α3 have been recently solved by X-ray crystallography in complex with Gly and the allosteric potentiator AM-3607 both in the presence (Huang et al., 2017b) and the absence of IVM (Huang et al., 2017a). These structures were described as consistent with the semi-open state of GlyR α1 and possibly representative of the desensitized state. Since the wide-open state was never observed in other pLGICs, its physiological significance in the context of the superfamily is debated (Nemecz et al., 2016).