The Role Of Spinal AKAP150 And Its Palmitoylation In Inflammatory Pain

Objective:A-Kinase anchoring protein 150(AKAP150)regulates ion channel receptors such as α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor(AMPAR)through anchoring protein kinases A(PKA),protein kinases C(PKC)and protein phosphatase 2B / calcineurin(PP2B/Ca N)and thereby participates in synaptic plasticity.AKAP150 itself undergoes S-palmitoylation,which profoundly influences the spatial distribution and function of the protein.This study aims to reveal the possible roles of AKAP150 and its palmitoylation in spinal AMPAR modification and behavioral sensitization after inflammation.Method: Chronic inflammatory pain model was established by subcutaneous injection of complete Freund’s adjuvant(CFA)into the hind plantar of mice.The subcellular fractions were separated by gradient centrifugation combined with detergent lysis to investigate protein subcellular distribution and synaptic expression.The acylbiotin exchange(ABE)method was used to detect protein palmitoylation level.Western Blot was used to detect protein expression and phosphorylation level.Coimmunoprecipitation was used to study the interaction between proteins.By constructing and transfecting AKAP150(C36,123S)plasmid,the contributions of AKAP150 palmitoylation in AKAP150 distribution,AMPAR synaptic expression and pain sensitization were investigated.By constructing and transfecting siRNA,the effects of knockdown of AKAP150 and ZDHHC2 on AMPAR and pain sensitization were studied.By detecting mechanical paw withdrawal thresholds(PWTs)and paw withdrawal latenicies(PWLs),the changes in pain behaviors were observed.Results:(1)After intraplantar injection of complete Freund’s adjuvant(CFA),the content of AKAP150 in the homogenates(H)from spinal dorsal horn was unchanged,but the content of AKAP150 in synaptosomal membrane fractions(P2)and PSD-enriched fractions(P3)significantly increased,which was accompanied by a reduction of AKAP150 content in cytosolic fractions(S2),suggesting that peripheral inflammation caused synaptic redistribution of AKAP150.(2)CFA increased the interaction between AKAP150 and PSD95 in the spinal dorsal horn,and significantly enhanced PKA and PKC,while reduced Ca N binding to AKAP150,suggesting that peripheral inflammation might cause a reorganization of the AKAP150 signaling complex.(3)CFA significantly increased the palmitoylation level of AKAP150.Spinal expression of palmitoylation-deficient mutant AKAP150(C36,123S)significantly inhibit the CFA-induced synaptic expression of AKAP150,suggesting that the palmitoylation of AKAP150 induced by peripheral inflammation was an important factor for the synaptic redistribution of AKAP150.(4)CFA specifically increased synaptic expression of GluA1-containing AMPAR.Knock down the expression of AKAP150 could significantly inhibit the CFA-induced synaptic hyperexpression of GluA1 by intrathecal administration of AKAP150-siRNA.Spinal expression of wild-type AKAP150 directly increased the synaptic level of GluA1.These results suggested that AKAP150 played an important role in synaptic trafficking of GluA1 during inflammation.(5)Intrathecal application of St-Ht31 peptide to interrupt interaction of PKA with AKAPs or intrathecal injection of TAT-AKAP150(31-51)peptide to interfere with the binding of PKC with AKAP150 could effectively reverse the CFA-induced synaptic expression of GluA1.Spinal expression of the AKAP150ΔPKA or AKAP150ΔPKC significantly inhibited CFA-induced GluA1 accumulation,suggesting that the AKAP150-anchored protein kinases played important roles in the synaptic recruitment of GluA1 under inflammatory conditions.(6)Intrathecal applications of a broad-spectrum palmitoylation inhibitor 2-BP(200 ng)or spinal expression of AKAP150(C36,123S)significantly inhibited the synaptic recruitment and phosphorylation of GluA1 at Ser845 and Ser831 in inflamed mice,indicating that AKAP150 palmitoylation was essential for synaptic recruitment of GluA1.(7)CFA significantly increased the total and synaptic levels of ZDHHC2 in the spinal dorsal horn,meanwhile increased the binding of ZDHHC2 to AKAP150.Transfection of ZDHHC2-siRNA in the spinal dorsal horn significantly inhibited the CFA-induced synaptic recruitment and phosphorylation of GluA1,indicating that ZDHHC2 was important for AKAP150 palmitoylation and the synaptic incorporation of GluA1 induced by CFA.(8)Expression of AKAP150(C36,123S)or ZDHHC2-siRNA in the spinal dorsal horn effectively alleviated the mechanical allodynia and thermal hyperalgesia in CFAinjected mice.Intrathecal administration of 2-BP could dose-dependently reduce the CFA-induced pain hypersensitivity.Conclusion: The AKAP150 signaling complex and its palmitoylation contributed to the development of peripheral inflammation-induced pain hypersensitivity via facilitating the synaptic recuriment of GluA1-containing AMPAR in the spinal dorsal horn.Inhibition of palmitoylation of AKAP150 can relieve chronic pain symptoms.