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

Diminished inhibitory neurotransmission in the superficial dorsal horn of the spinal cord is thought to contribute to chronic pain. In inflammatory pain, reductions in synaptic inhibition occur partially through prostaglandin E-2-(PGE(2)-) and PICA-dependent phosphorylation of a specific subtype of glycine receptors (GlyRs) that contain alpha 3 subunits. Here, we demonstrated that 2,6-di-tert-butylphenol (2,6-DTBP), a nonanesthetic propofol derivative, reverses inflammation-mediated disinhibition through a specific interaction with heteromeric alpha beta GlyRs containing phosphorylated alpha 3 subunits. We expressed mutant GlyRs in HEK293T cells, and electrophysiological analyses of these receptors showed that 2,6-DTBP interacted with a conserved phenylalanine residue in the membrane-associated stretch between transmembrane regions 3 and 4 of the GlyR alpha 3 subunit. In native murine spinal cord tissue, 2,6-DTBP modulated synaptic, presumably alpha beta heteromeric, GlyRs only after priming with PGE(2). This observation is consistent with results obtained from molecular modeling of the alpha-beta subunit interface and suggests that in alpha 3 beta GlyRs, the binding site is accessible to 2,6-DTBP only after PKA-dependent phosphorylation. In murine models of inflammatory pain, 2,6-DTBP reduced inflammatory hyperalgesia in an alpha 3GlyR-dependent manner. Together, our data thus establish that selective potentiation of GlyR function is a promising strategy against chronic inflammatory pain and that, to our knowledge, 2,6-DTBP has a unique pharmacological profile that favors an interaction with GlyRs that have been primed by peripheral inflammation.