In recent studies one serine residue (Ser-346) within the protein-kinase-A (R-E-S-R) consensus sequence of the GlyRα3 intracellular loop has proven to be an essential target for prostaglandin-E₂-mediated phosphorylation, which further modulates spinal nociceptive transmission and central inflammatory pain sensitization. In the present study we investigated the effect of Ser-346 phosphorylation and Ser-346 mutation on receptor kinetics and function using whole-cell patch-clamp recordings in transfected HEK 293 T cells. We compared biophysical properties of wild type GlyRα3 and two site-directed mutants, where Ser-346 was replaced by alanine or aspartate, in the absence and presence of prostaglandin-E₂. The mutation to alanine was accompanied by significantly altered dose-response and desensitization properties. Mutation to aspartate had only minor effects on receptor kinetics and function. Phosphorylation of Ser-346 slowed desensitization and decreased glycinergic currents in GlyRα3/mEP2 transfected cells. In addition, we demonstrated that prostaglandin-E₂ also had an effect on the GlyRα2 subunit. Exposure to prostaglandin-E₂ decreased the maximum peak current amplitude of glycinergic currents in GlyRα2/mEP2 transfected cells in the same manner as phosphorylation of the GlyRα3 subunit. It led to a significant increase of the desensitization time constants and thus significantly affected the desensitization behaviour. These results indicate that the GlyRα2 and the GlyRα3 subunits act as important subunits for the modulation of glycine receptor kinetics and function.