Regulation Of Glycine Receptor By GPR39 In Spinal Cord Dorsal Horn

Objective:G protein-coupled receptor 39(GPR39)is involved in a series of pathophysiological processes.However,the role of GPR39 in the pain regulation is unclear as yet.The current study was designed to examine the effects of GPR39 on glycinergic synaptic transmission and investigate the mechanisms underlying GPR39action.Methods:Immunohistochemistry was performed to examine the distribution of GPR39 in the spinal cord.Behavioral tests were conducted to evaluate the role of GPR39 in the pain transmission.The effects of GPR39 on synaptic proteins were investigated by immunoprecipitation and GST-pull down assays.We performed the whole-cell patch clamp recordings in spinal somatostatin-positive(SOM⁺)neurons and HEK293T cells to examine the influence of GPR39 on glycinergic neurotransmission.Results:(1)GPR39 was distributed in the superficial dorsal horn of spinal cord and was predominantly expressed in the spinal excitatory interneurons.(2)Genetic knockout of GPR39 significantly increased the excitability of superficial dorsal horn neurons and specifically evoked dynamic allodynia.(3)Given the key role of spinal SOM⁺neurons in the transmission of dynamic allodynia,we virally expressed sh RNA against GPR39 selectively in SOM⁺neurons of SOM-Cre mice,and found that GPR39 knockdown elicited pronounced dynamic allodynia.(4)The electrophysiological recordings demonstrated that GPR39 knockdown in SOM⁺neurons had no effects on the excitatory synaptic currents mediated by?-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor(AMPAR)and N-Methyl-d-Aspartate receptor(NMDAR).No significant change was also observed with the inhibitory synaptic currents mediated by Type A?-aminobutyric acid receptor(GABA_AR).However,GPR39 deletion selectively reduced the amplitudes of miniature inhibitory postsynaptic currents(m IPSCs)mediated by Gly Rs.(5)To confirm the effect of GPR39 on glycinergic synaptic responses,we recorded the electrically evoked IPSCs mediated by Gly Rs(Gly Rs-IPSCs),and extracellularly perfused TC-G1008,a GPR39-selective agonist.In the spinal slices from intact mice,the activation of GPR39 produced no significant changes of Gly Rs-IPSCs amplitudes.In the slices from Complete Freund's Adjuvant(CFA)-injected inflamed mice,however,the GPR39 agonist noticeably enhanced Gly Rs-IPSCs amplitudes by means of blocking the endocytosis of postsynaptic Gly Rs,which might reverse the glycinergic disinhibition caused by peripheral inflammation.(6)The behavioral tests showed that intrathecal injection of TC-G1008 alleviated the dynamic allodynia induced by CFA.(7)Among the functional Gly Rs subunits,we identified the?1 isoform as the specific target for GPR39 regulation.(8)GPR39 is known as one of the G protein-coupled receptors.However,the potentiation of GlyRs-?1 currents by GPR39 persisted when we knocked down the G?s?G?q,G?11,G?12 and G?13 proteins,or sequestered G??subunits,or blocked the protein kinases downstream of G proteins,suggesting that GPR39 might regulate glycinergic currents in a manner independent of G protein signalings.(9)We found that the cytoplasmic tail of GPR39 directly bound to the intracellular large loop of GlyRs-?1,which disrupted the interaction of GPR39 with PTP1B,a non-receptor protein tyrosine phosphatase.(10)PTP1B functioned to dephosphorylate GlyRs-?1 at Tyr339,resulting in the decrease of GlyRs-?1 currents.(11)Peripheral inflammation significantly enhanced the protein level of PTP1B in the dorsal horn,which reduced GlyRs-?1 phosphorylation at Tyr339 and glycinergic inhibitory efficacy.(12)In the inflamed mice,the activation of GPR39 was able to remove PTP1B-mediated inhibition of GlyRs-?1 by competitively disturbing the association of PTP1B.As a result,GPR39 stimulation increased the phosphorylation level of GlyRs-?1 and potentiated glycinergic transmission.(13)Overexpression of the cytoplasmic tail of GPR39(GPR39ct)in SOM⁺neurons mimicked the action of GPR39 agonist by disrupting PTP1B interaction with GlyRs-?1,increasing Tyr339 phosphorylation,enhancing Gly Rs-IPSCs amplitudes and alleviating dynamic allodynia.(14)Similarly,the genetic knockdown of PTP1B expression in SOM⁺neurons effectively attenuated the dynamic allodynia caused by CFA.(15)As the endogenous ligand for GPR39,Zn²⁺engagement with GPR39 was tonically active in the maintenance of GlyRs-?1 phosphorylation state and normal glycinergic transmission.The deletion of GPR39 or PTP1B noticeably attenuated the regulatory effect of Zn²⁺on Gly Rs-IPSCs.Conclusion:GPR39 specifically regulated the inhibitory glycinergic synaptic transmission through mechanisms independent of G protein signalings.The activation of spinal GPR39 reversed spinal glycinergic disinhibition caused by peripheral inflammation and effectively alleviated dynamic allodynia.