Structural characterization of a full-length heteromeric glycine receptor in multiple functional states

Glycine receptors (GlyR) are chloride-conducting ligand-gated ion channels that provide inhibitory neuronal input, particularly in the spinal cord. Synaptic GlyR is a heteromeric protein complex composed of five homologous subunits arranged pseudo-symmetrically about the channel pore. Individual subunits come in two varieties, α and β. Here, we present the structures of full-length zebrafish α1/β GlyR solved without any fiducial marker that may alter the functional state of the protein. This was done in the presence of an antagonist (strychnine), agonist (glycine) and agonist with an allosteric modulator (glycine and ivermectin). These structures confirm other recent findings regarding the stoichiometry of heteromeric GlyR (4α:1β) and also reveal previously uncharacterized subunit-specific features in the extracellular and intracellular domain. The functional state of each structure was assessed by molecular dynamics simulations. The strychnine-bound pore is in a closed state that is mostly symmetric about the five-fold axis. The glycine structures with and without ivermectin both adopted desensitized conformations, but the ivermectin structure has significant asymmetry at the intracellular entrance to the channel pore. Consistent with this observation, ligand density near the ivermectin binding pocket was only observed at the sole α/β interface, and not at α/α interfaces as observed in past homomeric GlyR structures. Simulations show a small number of permeation events for the ivermectin-bound state, suggestive of an asymmetric mechanism for channel opening. Together, these results highlight distinct structural features of heteromeric GlyR and demonstrate the functional range of this physiologically important protein channel.