title: Phosphorylation of α3 glycine receptors induces a conformational change in the glycine-binding site.
abstract: Inflammatory pain sensitization is initiated by prostaglandin-induced phosphorylation of α3 glycine receptors (GlyRs) that are specifically located in inhibitory synapses on spinal pain sensory neurons. Phosphorylation reduces the magnitude of glycinergic synaptic currents, thereby disinhibiting nociceptive neurons. Although α1 and α3 subunits are both expressed on spinal nociceptive neurons, α3 is a more promising therapeutic target as its sparse expression elsewhere implies a reduced risk of side-effects. Here we compared glycine-mediated conformational changes in α1 and α3 GlyRs to identify structural differences that might be exploited in designing α3-specific analgesics. Using voltage-clamp fluorometry, we show that glycine-mediated conformational changes in the extracellular M2-M3 domain were significantly different between the two GlyR isoforms. Using a chimeric approach, we found that structural variations in the intracellular M3-M4 domain were responsible for this difference. This prompted us to test the hypothesis that phosphorylation of S346 in α3 GlyR might also induce extracellular conformation changes. We show using both voltage-clamp fluorometry and pharmacology that Ser346 phosphorylation elicits structural changes in the α3 glycine-binding site. These results provide the first direct evidence for phosphorylation-mediated extracellular conformational changes in pentameric ligand-gated ion channels, and thus suggest new loci for investigating how phosphorylation modulates structure and function in this receptor family. More importantly, by demonstrating that phosphorylation alters α3 GlyR glycine-binding site structure, they raise the possibility of developing analgesics that selectively target inflammation-modulated GlyRs.
authors.affiliation: Queensland Brain Institute and ‡School of Biomedical Sciences, The University of Queensland , Brisbane QLD 4072, Australia.
authors.forename: Lu
authors.initials: L
authors.lastname: Han
authors.forename: Sahil
authors.initials: S
authors.lastname: Talwar
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Shan
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Lynch
mesh_terms: D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics; D005110:Extracellular Space / Q000235:genetics; D005260:Female; D005998:Glycine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D018836:Inflammation Mediators / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D018009:Receptors, Glycine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D018836:Inflammation Mediators; D020033:Protein Isoforms; D018009:Receptors, Glycine; C113965:glycine receptor alpha3 subunit; D017868:Cyclic AMP-Dependent Protein Kinases; D005998:Glycine