A Glra3 phosphodeficient mouse mutant establishes the critical role of protein kinase A–dependent phosphorylation and inhibition of glycine receptors in spinal inflammatory hyperalgesia

Abstract

Glycinergic neurons and glycine receptors (GlyRs) exert a critical control over spinal nociception. Prostaglandin E2 (PGE2), a key inflammatory mediator produced in the spinal cord in response to peripheral inflammation, inhibits a certain subtype of GlyRs (α3GlyR) that is defined by the inclusion of α3 subunits and distinctly expressed in the lamina II of the spinal dorsal horn, ie, at the site where most nociceptive nerve fibers terminate. Previous work has shown that the hyperalgesic effect of spinal PGE2 is lost in mice lacking α3GlyRs and suggested that this phenotype results from the prevention of PGE2-evoked protein kinase A (PKA)-dependent phosphorylation and inhibition of α3GlyRs. However, direct proof for a contribution of this phosphorylation event to inflammatory hyperalgesia was still lacking. To address this knowledge gap, a phospho-deficient mouse line was generated that carries a serine to alanine point mutation at a strong consensus site for PKA-dependent phosphorylation in the long intracellular loop of the GlyR α3 subunit. These mice showed unaltered spinal expression of GlyR α3 subunits. In behavioral experiments, they showed no alterations in baseline nociception, but were protected from the hyperalgesic effects of intrathecally injected PGE2 and exhibited markedly reduced inflammatory hyperalgesia. These behavioral phenotypes closely recapitulate those found previously in GlyR α3-deficient mice. Our results thus firmly establish the crucial role of PKA-dependent phosphorylation of α3GlyRs in inflammatory hyperalgesia.

Glycine Receptors in Spinal Nociceptive Control—An Update

“…In HEK293 cells cotransfected with cDNAs encoding for GlyRα3 and EP2 receptors, application PGE 2 led to a decrease in glycinergic membrane currents with no change in their inactivation kinetics or plasma membrane expression. In single channel recording experiments, activation of PKA progressively reduced single current amplitudes to about 66%, which is close to the inhibition of glycinergic synaptic currents observed in mouse spinal cord slices [5,8,14]. Introducing the phospho-mimicking serine to glutamate (S→E) mutation reduced single channel conductance to a similar degree with no effect on single channel open probabil-ity.…”

“…The recent generation of a genetically engineered S346A point mutated mouse line allowed filling this gap (Figure 2). This mouse carries a serine to alanine amino acid exchange at position 346 (S346A mutation) in the PKA consensus sequence of the GlyR α3 subunit [8]. Electrophysiological recordings from substantia gelatinosa neurons in spinal cord slices of these mice demonstrated not only that the point-mutated α3GlyRs were resistant to inhibition by PGE 2 but also confirmed the critical contribution of phosphorylation at this site to inflammatory hyperalgesia.…”

“…Both wild-type mice and GlyR α3 deficient mice developed prolonged thermal and mechanical hyperalgesia following the constriction injury of the sciatic nerve, suggesting that the phosphorylation of GlyR α3 subunits is dispensable for the development of hyperalgesia following peripheral nerve injury. This finding was recently confirmed in experiments with S346A point mutated mice, which also developed unaltered hyperalgesia following peripheral nerve injury [8]. It should be stressed that the normal development of neuropathic hyperalgesia in the GlyR α3 deficient or S346A point mutated mice does not exclude that α3 GlyRs still control Additional insights have also been gained into the molecular mechanisms that link phosphorylation at S346 to decreased glycinergic currents [13].…”

“…Furthermore, a screening of the human genetic variation database (www.ncbi.nlm.nih.gov/variation/) for loss of function (non-sense) variants revealed few hits. All of which occurred with frequencies too low to allow systematic clinical trials in such persons (for a discussion on this topic, see [8]).…”

Glycine Receptor Subtypes and Their Roles in Nociception and Chronic Pain

“…Using recombinant expression of mutant GlyRs, Harvey et al (2004) identified the S346 residue within the ICD of α3GlyRs as the residue targeted by PKA. The recent generation of a knock-in mouse line carrying the S346A mutation in the Glra3 gene allowed direct examination of the relevance of this chronic pain mechanism in vivo ( Werynska et al, 2021 ). Behavioral experiments demonstrated that α3GlyR-S346A mice are devoid of pain sensitization elicited by intrathecal PGE 2 and display a diminished inflammatory pain hyperalgesia.…”