![]() In each, we confirm the 4α:1β stoichiometry by identifying subunit-specific features. ![]() Here we report three structures of full-length zebrafish (ZF) heteromeric GlyR composed of α 1 and β Β subunits absent of any exogenous fiduciary markers. However, functional interpretation was limited by the use of protein alterations or antibodies, which aid in cryo-EM image processing but potentially alter native channel properties 19. These structures agreed with the surprising finding that heteromeric GlyR has a 4α:1β stoichiometry, contrary to prior predictions from biochemical analyses, which indicated a 2α:3β or 3α:2β stoichiometry 13, 14, 15, 16, 17, 18. Recent studies have reported structures of heteromeric GlyR expressed in HEK cells (α 2β) and from native porcine brain tissue (α 1β) 11, 12. The majority of adult GlyRs are composed of α 1 and β subunits 9, 10. However, synaptic localization requires the β subunit as it solely interacts with gephyrin, a synaptic-scaffold protein 8. There are four genes encoding α subunits (α1–α4), each capable of forming functional homomeric channels. Structurally, GlyRs assemble as pentamers composed of homologous α and β subunits. As such, GlyR-positive allosteric modulators (PAMs) have promise as therapeutics for chronic and inflammatory pain 7. ![]() GlyRs also play an important modulatory role in visual, auditory, and pain perception 4, 5, 6. Their inhibitory input is essential for coordinated muscle movement, as manifested in hyperekplexia 3, a neurological disorder where mutations affecting GlyR clustering or function result in sporadic muscle movements. Glycine receptors (GlyRs) are chloride-conducting, pentameric ligand-gated ion channels (pLGIC) primarily found in spinal cord neurons where they mediate fast inhibitory neurotransmission 1, 2. Together, our results show distinct compositional and conformational properties of α 1βGlyR and provide a framework for further study of this physiologically important channel. We also report features of the extracellular and intracellular domains. Subunit-specific features were sufficient to solve structures without a fiduciary marker and to confirm the 4α:1β stoichiometry recently observed. Ivermectin binds at all five interfaces, but in a distinct binding pose at the β-α interface. Molecular dynamic simulations found the structures were in a closed (strychnine) and desensitized states (glycine and glycine/ivermectin). Each structure shows a distinct pore conformation with varying degrees of asymmetry. Here we present cryo-EM structures of full-length zebrafish α1β BGlyR in the presence of an antagonist (strychnine), agonist (glycine), or agonist with a positive allosteric modulator (glycine/ivermectin). Synaptic GlyRs are a heteromeric assembly of α and β subunits. Glycine Receptors (GlyRs) provide inhibitory neuronal input in the spinal cord and brainstem, which is critical for muscle coordination and sensory perception.
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