Phosphorylation state–dependent modulation of spinal glycine receptors alleviates inflammatory pain
Mario A. Acuña, Gonzalo E. Yévenes, William T. Ralvenius, Dietmar Benke, Alessandra Di Lio, Cesar O. Lara, Braulio Muñoz, Carlos F. Burgos, Gustavo Moraga-Cid, Pierre-Jean Corringer, Hanns Ulrich Zeilhofer
Mario A. Acuña, Gonzalo E. Yévenes, William T. Ralvenius, Dietmar Benke, Alessandra Di Lio, Cesar O. Lara, Braulio Muñoz, Carlos F. Burgos, Gustavo Moraga-Cid, Pierre-Jean Corringer, Hanns Ulrich Zeilhofer
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Research Article Neuroscience

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

Abstract

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 E2- (PGE2-) and PKA-dependent phosphorylation of a specific subtype of glycine receptors (GlyRs) that contain α3 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 αβGlyRs containing phosphorylated α3 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 α3 subunit. In native murine spinal cord tissue, 2,6-DTBP modulated synaptic, presumably αβ heteromeric, GlyRs only after priming with PGE2. This observation is consistent with results obtained from molecular modeling of the α-β subunit interface and suggests that in α3βGlyRs, 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 α3GlyR-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.

Authors

Mario A. Acuña, Gonzalo E. Yévenes, William T. Ralvenius, Dietmar Benke, Alessandra Di Lio, Cesar O. Lara, Braulio Muñoz, Carlos F. Burgos, Gustavo Moraga-Cid, Pierre-Jean Corringer, Hanns Ulrich Zeilhofer

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Figure 5

Pretreatment with PGE2 renders synaptic α3βGlyRs susceptible to modulation by 2,6-DTBP.

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Pretreatment with PGE2 renders synaptic α3βGlyRs susceptible to modulati...
(A) Normalized amplitudes (mean ± SEM) (top panel) and decay time constants (bottom panel) of light-evoked Gly-IPSC traces versus time, before and during the application of PGE2 (1 μM) and in the additional presence of 2,6-DTBP (100 μM). Insets are example traces averaged from 10 consecutive stimulations under the three conditions (n = 12). (B) Same as (A) but preincubated with H-89 (5 μM) to prevent phosphorylation of GlyRs by PGE2 (n = 7). (C) Same as (A) but experiments done in slices taken from GlyRα3–/– mice. In these experiments, Gly-IPSCs were evoked by electrical field stimulation, n = 12. (D) Left panel: PGE2 reduced Gly-IPSC amplitudes in control slices but not in H89-treated slices or in slices from GlyRα3–/– mice, while 2,6-DTBP had no significant effects on Gly-IPSC amplitudes in either condition. **P = 0.01, significant versus control (CTRL). ANOVA followed by Bonferroni post-hoc test. F(2,35) = 8.58, F(2,18) = 1.76, and F(2,30) = 0.12 for Gly-IPSC amplitudes shown in A–C, respectively. Right panel: 2,6-DTBP significantly prolonged Gly-IPSC decay time courses in PGE2-treated wild-type slices, but not in slices pretreated with H89, or in slices prepared from GlyRα3–/– mice. ANOVA followed by Bonferroni post-hoc test. †††P < 0.001, significant versus PGE2; ***P < 0.001 significant versus control. F(2,35) = 15.66, F(2,18) = 0.20, and F(2,30) = 0.62 for experiments shown in A–C, respectively. n = 7–12 cells per group. All scale bars: 50 ms, 400 pA.