Interaction Between AKAP150 And SNARE Family Proteins In Spinal Dorsal Horn And Its Regulatory Mechanism

Objective:Excitatory glutamate receptors trafficking to plasma membrane and then delivery into synapses through exocytosis are involved in the regulation of synaptic transmission and plasticity. Exocytosis depends on the membrane fusion mediated by Soluble Nethylmaleimide sensitive factor attachment protein recptor(SNAREs)family proteins. It is known that synaptic trafficking of excitatory glutamate receptors is regulated by protein kinase A(PKA) and protein kinase C(PKC), as well as by SNAREs-mediated exocytosis. A kinase anchoring protein 150(AKAP150), as an anchoring protein of PKA, PKC and PP2 B, can directly determine the location and distribution of these kinases and their catalytic efficiency. In this study, we investigated the interaction between AKAP150 and SNARE family proteins, and explore the mechanisms for the regulation of their interaction.Method:Co precipitation(CO-IP) and Western Blot were utilized to investigate the interaction between AKAP150 and SNARE family proteins. NMDA receptor, PKA and PKC were activated by intrathecal injection of NMDA, forskolin and PMA, respectively. Complete Freund’s Adjuvan(CFA) in 10-μl volumes was injected into the plantar surface of the hindpaws of mice to induce inflammatory pain and to discuss the possible changes of AKAP150 interaction with SNARE family proteins during chronic pain.Results:(1)Munc 18-1, syntaxin 3 and SNAP25 were coimmunoprecipitated by anti-AKAP150 antibody from homogenates of spinal dorsal horn in mice;(2)In the homogenates of spinal dorsal horn in mice, anti-SNAP25 antibody coimmunoprecipitated AKAP150;(3)In the spinal dorsal horn of mice, syntaxin 3 and SNAP25 were coimmunoprecipitated by anti-Munc 18-1 antibody; Munc 18-1 and SNAP 25 were coimmunoprecipitated by anti-syntaxin 3 antibody; Munc 18-1 and syntaxin 3 were coimmunoprecipitated by anti- SNAP 25 antibody. These results suggested that Munc 18-1, syntaxin 3 and SNAP 25 formed a SNARE complex which bound to AKAP150 in spinal dorsal horn of mice;(4)Activation of NMDA receptor by intrathecal application NMDA(0.07μg) dramatically reduced the amounts of Munc 18-1, syntaxin 3 and SNAP25 coimmunoprecipitated by anti-AKAP150 antibody, indicating that the interaction between AKAP150 and SNARE was decreased by activation of NMDA receptor;(5) Intrathecal application forskolin(2.5μg)reduced the amounts of syntaxin 3 coimmunoprecipitated by anti-AKAP150 antibody, whereas intrathecal application PMA(1.0μg)reduced the amounts of syntaxin 3 and SNAP 25 coimmunoprecipitated by anti-AKAP150 antibody;(6)The amounts of Munc 18-1, syntaxin 3 and SNAP25 coimmunoprecipitated by anti-AKAP150 antibody showed remarkably reduction by injecting CFA into the plantar surface of the hindpaws of mice, suggested that the SNARE complex dissociation was likely associated with inflammatory pain.Conclusion:In the spinal dorsal horn, AKAP150 complexed with SNAREs protein Munc 18-1, syntaxin 3 and SNAP25. The interaction between AKAP150 with Munc 18-1, syntaxin 3 and SNAP25 could be disrupted after activation of NMDA receptor and inflammation pain. The AKAP150 dissociation with SNAREs proteins was correlated with inflammatory pain.