Spinophilin Negatively Controlled The Function Of TRPV1 In The Dorsal Root Ganglia Neurons

Objective: Transient receptor potential vanilloid 1(TRPV1)(also called capsaicin receptor)is an ion channel receptor and involves in the pain transmission and sensitization.Our previous study had illustrated that spinophilin regulates nociceptive synaptic transmission and plasticity in spinal cord dorsal horn by interacting with and recruiting protein phosphatase-1(PP1)to postsynaptic density.However,the function of spinophilin in dosal root ganglia(DRG)neurons remains to be elucidated.The current study was designed to investigate the location of spinophilin in nociceptors.We examined the effect of spinophilin on TRPV1 and the relationship between spinophilin and TRPV1 hyperfunction induced by peripheral inflammation.Method: Cellular distribution of spinophilin in DRGs of adult mice was examined by immunohistochemistry.The paw withdrawal latencies(PWLs)to heat stimuli,paw withdrawal thresholds(PWTs)to Von Frey stimuli,cold allodynia to acetone application and motor coordination of mice were measured to evaluate the role of spinophilin/PP1 complex in pain transmission.The effects of spinophilin/PP1 complex on TRPV1 and CaMKII phosphorylation were examined by immunoblotting.The TRPV1 contents on plasma membrane were determined by biotinylation assays.We performed co-immunoprecipitation to investigate the effect of peripheral inflammatory insults on the interaction of spinophilin/PP1 complex with TRPV1.Capsaicin or formalin was injected into the hindpaws of mice to induce spontaneous pain,and the regulatory effects of spinophilin/PP1 complex on peripheral sensitization were investigated.Results:(1)Spinophilin was prevalent in DRG neurons and co-localized with neuronal marker NeuN,calcitonin gene-related peptide(CGRP)and isolectin-B4(IB4).However,spinophilin did not co-localize with astrocyte marker GFAP,suggesting that both peptidergic and non-peptidergic C fiber nociceptors expressed spinophilin.(2)Expression of spinophilin(F451A)in DRGs did not affect the cold allodynia and motor function,but significantly reduced the PWTs and PWLs values,indicating that PP1 anchored by endogenous spinophilin negatively controlled the sensitivity of mice to heat and mechanical stimuli.(3)Spinophilin antibody faithfully pulled down TRPV1 and PP1 from DRG lysates,suggesting that spinophilin was able to direct PP1 to the vicinity of TRPV1.(4)Expression of spinophilin(F451A)led to a marked increase of TRPV1 phosphorylation at Ser502 and of CaMKII autophosphorylation at Thr286,suggesting that spinophilin-associated PP1 negatively controlled CaMKII phosphorylation of TRPV1.(5)Expression of Spinophilin(WT)had a tendency to decrease the contents of TRPV1 on plasma membrane,which,however,did not reach the statistical significance.By contrast,Spinophilin(F451A)significantly enhanced TRPV1 expression on the plasma membrane,implicating that spinophilin/PP1 complex was constitutively active in the inhibition of surface TRPV1.(6)Despite of the ineffectiveness of spinophilin(WT)on surface TRPV1 of intact animals,prior expression of spinophilin(WT)noticeably attenuated spontaneous painful behaviors induced by capsaicin,suggesting that spinophilin/PP1 complex inhibited TRPV1-dependent pain sensitization.(7)Formalin injection into the intraplantar surface of hindpaws disturbed the interaction of spinophilin with TRPV1 and caused a significant increase of TRPV1 phosphorylation at Ser502.(8)Prior expression of spinophilin(WT)in DRGs blocked Ser502 phosphorylation and biphasic spontaneous pain induced by formalin.Conclusion: Spinophilin-directed PP1 controlled the phosphorylation and surface expression of TRPV1 in DRG neurons.The dissociation of spinophilin with TRPV1 was critical for TRPV1 hyperfunction.The PP1 targeting by spinophilin to TRPV1 was beneficial for the alleviation of inflammatory pain sensitization.