Adenosine A1 Receptor Regulated The Phosphorylation Of Glycine Receptor α1^ins Subunit In Spinal Dorsal Horn

Objective:Glycine receptorα1^inssubunit is located at the inhibitory synapses of superficial dorsal horn of spinal and is engaged in the glycinergic inhibitory synaptic transmission.Following peripheral inflammation,the active extracellular signal-regulated kinase（ERK）phosphorylatesα1^inssubunit at Ser380,which impairs glycinergic synaptic transmission and contributes to spinal disinhibition.Previous studies have shown that activation of Adenosine A1 Receptor（A1R）is able to potentiate glycinergic transmission.However,the mechanisms underlying A1R action remain to be elucidated.The purpose of the current study was to investigate the regulatory effect of A1R onα1^insphosphorylation.Method:Biotinylation assays were conducted to examine the expression ofα1^inson plasma membrane.Theα1^insphosphorylation at Ser380 was investigated by using western blot.Glycine receptor currents were recorded under whole-cell voltage-clamp configuration.The co-immunoprecipitation was conducted to examine protein-protein interaction.The pain thresholds and motor function were measured by behavioral tests.Results:（1）Intrathecal injection of adenosine（20,40,60 nmol）in mice dose-dependently relieved the mechanical allodynia and thermal hyperalgesia caused by peripheral inflammation.（2）The analgesic action of adenosine correlated with the decreasedα1^insphosphorylation at Ser380 and increased expression ofα1^inson plasma membrane.（3）Among multiple adenosine receptors,we identified A1R as the subtype that reducedα1^insphosphorylation at Ser380 and inhibited inflammatory pain.（4）By inhibiting Ser380 phosphorylation,A1R was found to enhanceα1^inscurrents.（5）When activated by adenosine,the G protein-coupled A1R anchored protein phosphatase-1（PP1）in the close vicinity ofα1^insthrough Gβγdimmers.（6）PP1 was able to dephosphorylateα1^insat Ser380 directly.（7）Sequestration of Gβγdimer impaired the ability of A1R to dephosphorylateα1^insat Ser380.（8）Sequestration of Gβγdimer also attenuated the analgesic efficacy of A1R against inflammatory pain.（9）In addition to enhance the PP1 catalytic efficacy,A1R activation also inhibited ERK activity,both of which contributed to Ser380 dephosphorylation.（10）Knockdown of spinalα1^inssubunit significantly attenuated the analgesic action of A1R.Conclusion:Glycine receptorα1^inssubunit served as the key target for A1R to exert pain-relief action.Through Gβγ/PP1 and ERK signaling pathways,A1R reducedα1^insphosphorylation at Ser380,augmentedα1^ins-mediated glycinergic synaptic transmission,and alleviated inflammatory pain.