Phosphorylation of α3 Glycine Receptors Inducesa Conformational Change in the Glycine-Binding Site

摘要

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 extracellularconformation changes. We show using both voltage-clamp fluorometryand pharmacology that Ser346 phosphorylation elicits structural changesin the α3 glycine-binding site. These results provide the firstdirect evidence for phosphorylation-mediated extracellular conformationalchanges in pentameric ligand-gated ion channels, and thus suggestnew loci for investigating how phosphorylation modulates structureand function in this receptor family. More importantly, by demonstratingthat phosphorylation alters α3 GlyR glycine-binding site structure,they raise the possibility of developing analgesics that selectivelytarget inflammation-modulated GlyRs.