Allosteric and hyperekplexic mutant phenotypes investigated on an α ₁ glycine receptor transmembrane structure

Abstract

The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α 1 GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α 1 GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α 1 GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a poreopening mechanism for the α 1 GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α 1 GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor.glycine receptor | hyperekplexia | allostery

Phosphorylation state–dependent modulation of spinal glycine receptors alleviates inflammatory pain

“…To this end, we developed a model of the α3GlyR based on the crystal structures of GluCl and GLICGlyR (27,28). The results of this modeling were not only consistent with F388 being located in the MA stretch close to transmembrane segment 4 ( Figure 3H), but also provided insights into the molecular composition of the putative acceptor site for 2,6-DTBP.…”

“…2,6-DTBP was co-applied with glycine, without pre-application. Singlechannel recordings from transfected cells were done as described (19,28). All recordings were performed in the cell-attached configuration at 19°C-21°C with thick-walled borosilicate glass pipettes (final resistance of 10-15 MΩ).…”

“…Antihyperalgesia was quantified for a time interval of length GlyR α3 subunit was created by homology modeling using GluCl [PDB: 2RIF] (27) as the template in Modeller 9v13 (52). Additional refinement was performed using the α1GlyR-GLIC structure (28). The final model was obtained after energy minimization with a conjugate gradient protocol in the software MacroModel (version 9.9, Schröding-er, LLC).…”

Length of the TM3-4 loop of the glycine receptor modulates receptor desensitization

“…Although X-ray analysis provided lots of information on the ECD structure of CLRs, the overall fold of the intracellular domain is not yet solved. Even recently solved structures of Glu-Cl and Lily (GLIC-GlyR chimera) lacked the large ICD [14,15]. Several reports described ␣-helical organization close to TM3 and TM4 [16,17] with basic residues being important determinants for channel properties.…”

Structure and Pharmacologic Modulation of Inhibitory Glycine Receptors

“…A more significant contribution for the understanding of GlyR structural characteristics was recently achieved by studying the crystallographic structure of the glutamategated chloride channels (GluCl) from Caenorhabditis elegans, a eukaryotic LGIC, which allowed the homology modeling of GlyR because it presented a higher percentage of identity (a1/ GluCl : 44%) (Hibbs and Gouaux, 2011). A key advancement in the resolution of GlyR structure was obtained from studies of homopentamers formed by a1 or a3 subunits and the chimeric receptor GLIC/a1 GlyR called Lily (Du et al, 2015;Huang et al, 2015;Moraga-Cid et al, 2015). All these recent studies have contributed to understand the nature of conformational changes that might occur during open-closed states, desensitization, and pharmacologic modulation.…”