| Objective:Protein Kinase A (PKA, also known as the cAMP-dependent protein kinase) catalyzes the phosphorylation of a variety of ion channels including N-methyl-D-aspartate (NMDA)-subtype glutamate receptor (NMDAR), which plays an important role in synaptic plasticity. The PKA catalytic efficacy and substrate specificity are critically dependent on its cellular localization. A kinase anchoring proteins (AKAPs) have been identified as the scaffolding proteins that interact with PKA and target PKA to the close vicinity of substrates. Although PKA has been widely described in the regulation of NMDAR-dependent spinal sensitization, the roles of AKAPs in these processes are largely unknown as yet. In this study, the mechanism and function of AKAPs in NMDAR-mediated nociceptive transmission were investigated by disruption of AKAPs/PKA interaction with stearated St-Ht31 peptide (St-N-DLIEEAASRIVDAVIEQVKAAGAYC) in spinal dorsal horn.Methods:Inflammatory pain model was established by intraplantar injection of Complete Freund’s Adjuvant (CFA) in mice. Nociceptive behavior test, electrophysiological patch clamp recordings and immunoblotting were utilized to investigate the inhibitory effects of St-Ht31 peptide on inflammatory pain and the underlying molecular mechanism.Results:(1) St-Ht31 dose-dependently alleviated CFA-induced inflammatory pain, while no effect was observed with its stearated control St-Ht31p peptide (St-N-DLIEEAASRPVDAVPEQVKAAGAYC). (2) The results of electrophysiological recordings showed that CFA significantly potentiated the amplitudes of excitatory postsynaptic currents (EPSCs) mediated by NMDAR in spinal dorsal horn neurons. Consistent with these results, the ratios of a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid subtype glutamate receptor (AMPAR)-mediated EPSCs amplitudes to NMDAR-EPSCs amplitudes (AMPAR/NMDAR ratios) were significantly reduced after CFA injection. The potentiated NMDAR-EPSCs and the reduced AMPAR/NMDAR ratios in CFA mice were reversed by postsynaptic loading of St-Ht31 (50.0μM) in spinal dorsal horn neurons. These results indicated that St-Ht31 generated an inhibitory effect on NMDAR-mediated nociceptive transmission in inflammatory mice. (3) In spinal slices from CFA-injected mice, the reductions in the magnitudes of NMDAR-EPSCs induced by extracellular perfusion of ifenprodil (3.0μM) were significantly suppressed by pretreatment with St-Ht31 peptide (50.0μM), implicating that St-Ht31 peptide inhibited synaptic transmission by reducing the synaptic function of GluN2B subunit-containing NMDAR (GluN2B-NMDA receptor). Consistent with these results, intrathecal application of St-Ht31 (7.0μg) effectively reversed the synaptic hyper-expression of GluN2B-NMDA receptor induced by CFA injection. These results indicated that synaptic inhibition mediated by St-Ht31 peptide might be related to the reduction in the number of GluN2B-NMDA receptor at synapses. (4) Direct activation of postsynaptic PKA by perfusion of 8-Br-cAMP (100.0μM) increased the amplitudes of NMDAR-EPSCs in spinal dorsal horn neurons of intact mice, which, however, could be blocked by loading of nocodazole (10.0μM), a Microtubule destabilizer. In addition, postsynaptic loading of nocodazole (10.0μM) also dramatically occluded the inhibitory effect of St-Ht31 on NMDAR-EPSCs in spinal slices from CFA-injected mice. These results indicated that St-Ht31 decreased the number of synaptic GIuN2B-NMDA receptor and the amplitudes of NMDAR-EPSCs in inflamed mice by inhibiting NMDA receptor transport along Microtubules. (5) Postsynaptic introduction of dynasore (80.0μM), a cell-permeable dynamin GTPase activity inhibitor, didn’t change the amplitudes of NMDAR-EPSCs in inflamed mice. However, dynasore effectively blocked the decrease of NMDAR-EPSCs amplitudes induced by St-Ht31. These results indicated that the St-Ht31 exerted inhibitory effect on synaptic responses by interfering with the dynamin-dependent endocytosis of NMDA receptor. (6) In the spinal dorsal horn, the level of cofilin phosphorylation at Serine 3 (cofilin-Ser3) significantly increased one day after CFA injection. Intrathecal application of St-Ht31 (7.0μg) peptide could repress CFA-induced cofilin-Ser3 phosphorylation. Postsynaptic loading of phalloidin (100.0μM), an actin cytoskeleton stabilizer, didn’t influence NMDA receptor synaptic currents in CFA-injected mice. However, pretreatment of spinal slices by phalloidin (100.0μM) clearly suppressed inhibitory effect of St-Ht31 on NMDAR-EPSCs in CFA-injected mice. These results suggested that St-Ht31 might repress NMDAR-mediated synaptic transmission by disturbing the synaptic NMDA receptor stabilization.Conclusions:St-Ht31 peptide might ameliorate the inflammatory pain by suppressing NMDAR-mediated nociceptive transmission. The molecular mechanisms underlying the synaptic inhibition by St-Ht31 might involve the interference with several NMDAR trafficking steps, including Microtubule-based intracellular transport toward synaspes, dynamin-dependent receptor endocytosis and F-actin mediated stabilization. AKAPs-bound PKA played an important role in NMDAR-mediated nociceptive transmission by manipulating multiple steps involved in the receptor trafficking process. |
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