Study Of Mechanism Of Episodic Pain With Na_v1.9 Channel Mutations

Voltage-gated sodium channel 1.9（Na_v1.9）encoded by SCN11A is highly expressed in peripheral sensory neurons and modulates resting membrane potential（RMP）and amplifies subthreshold stimuli.As of 2013,Na_v1.9 mutations were firstly reported in two large Chinese families with episodic pain by our group.Subsequently,Na_v1.9 mutations were reported in episodic pain,small fiber neuropathy,and postoperation pain.In this study,we collected one new Chinese family with episodic pain,which included 38 members and 12affected individuals.The intense pain was localized principally to the distal lower extremities;the frequency of episodic pain gradually decreased as the subject aged;the pain was triggered by exhaustion and cold.Alcohol could trigger episodic pain in three patients in this family.After the informed consent was obtained from participants,we identified the c.664C>A（p.Arg222Ser）mutation of SCN11A in all patients.Alcohol-aggravated pain syndromes was also confirmed in one patient from previously reported Chinese episodic pain family with SCN11A mutations（c.2423C>G/p.Ala808Gly）,but at that time we did not further study this trigger.The main ingredient of wine is ethanol besides water.The ethanol metabolic pathway in humans mainly depends on two enzymatic steps.Firstly,ethanol is oxidized by alcohol dehydrogenase（ADH）to generate acetaldehyde.Then aldehyde dehydrogenase-2（ALDH2）is the crucial enzyme that eliminates most of acetaldehyde through the oxidation of acetaldehyde into acetate.It has been shown that the p.Glu504Lys polymorphism of ALDH2 could significantly decrease ALDH2 enzyme activity and result in enhanced accumulation of acetaldehyde in the blood.Whether the four affected individuals carried the p.Glu504Lys polymorphism?Thus,the enhanced accumulation of acetaldehyde in the blood after drinking trigger episodic pain.The p.Glu504Lys polymorphism of ALDH2 was detected by Sanger sequencing in the four affected individuals,rather than other patients.The p.Glu504Lys polymorphism may be causative of alcohol-aggravated pain in patients with SCN11A mutation from genetically.To model human pain channelopathy in vivo and further elucidate the molecular mechanism for the new trigger in patients,we generated a Scn11a^A796G knock-in mouse carrying the mutation h Na_v1.9 p.Ala808Gly（orthologous mutation m Na_v1.9 p.Ala796Gly in mice）by homologous recombination.Compared with WT channels,the activation of Scn11a^+/A796G channels displayed a significant hyperpolarizing shift of 6 m V,while Scn11a^A796G/A796G channels showed a larger hyperpolarizing shift of 11.7 m V in activation.The slope factor also remained unaffected.No significant differences were observed for the midpoint or the slope factor of fast inactivation curve.The Scn11a^A796G/A796G channels,but not the Scn11a^+/A796G channels,significantly increased the residual current through non-inactivating channels compared with WT channels.The combination of activation and steady-state fast inactivation resulted in increased overlap between activation and fast inactivation,which predicts a larger window current for Scn11a^A796G/A796G channels compared with Scn11a^+/A796G and WT channels.These results show that the homozygous channel produced higher electrical activities than that of WT and heterozygous mutation.To assess the influence of mutation on Scn11a^A796G mouse DRG neuronal excitability,we found that DRG neurons from Scn11a^A796G/A796G mice evoked a significantly higher frequency of action potential firing than that of WT mice by current clamp technique.The firing frequency was slightly higher in DRG neurons of Scn11a^+/A796G mice than that of WT mice,but the difference was not significant.The RMP and voltage threshold were not significantly different in DRG neurons among the genotypes.In addition,the proportion of spontaneous firing cells for Scn11a^A796G/A796G DRG neurons is higher than that of WT or Scn11a^+/A796GDRG neurons.These results showed that the homozygous alteration induced hyperexcitability of DRG neurons.To confirm whether the phenotype of Scn11a knock-in mice similar to that of patients with episodic pain,we examined the basal pain behaviors of Scn11a^A796G knock-in mice.Compared with WT mice,Scn11a^A796G/A796G mice showed mechanical,heat and cold hypersensitivity,indicated that Scn11a^A796G/A796G mice recapitulated many clinical features of patients.While Scn11a^+/A796G mice only exhibited heat hypersensitivity and the heat pain threshold of Scn11a^+/A796G mice was higher than that of Scn11a^A796G/A796G mice.To determine the effect of acetaldehyde accumulation on the pain behavioral response,we conducted intraplantar injection of acetaldehyde in Scn11a^A796G knock-in mice.We found that the total time spent in painful behaviors for Scn11a^A796G/A796G mice was significantly increased compared with that of WT mice and acetaldehyde injection potently extended the duration of nociceptive behaviors in Scn11a^A796G/A796G mice compared with WT mice.This extended duration of nociceptive behaviors in Scn11a^+/A796G mice was only observed in mechanical threshold.Following intraplantar formalin injection,a significant increase in paw licking and lifting time was observed in Scn11a^A796G/A796G mice compared with WT mice.While the painful behaviors of Scn11a^+/A796G mice were not affected compared with those of WT mice.In addition,acetaldehyde increased the mutant m Na_v1.9 channel current and enhanced the action potential firing of Scn11a^A796G/A796Gmouse DRG neurons.These results suggest that Scn11a^A796G/A796G mice showed hypersensitivity to aldehydes compared with WT mice,which was similar to the alcohol-aggravated pain symptoms in the four patients.These results demonstrated that the p.Glu504Lys polymorphism could be causative of alcohol-aggravated pain in individuals with the Na_v1.9 mutation.The episodic pain was relieved by oral administration of ibuprofen in patients,we found that ibuprofen increased heat pain threshold in Scn11a^A796G/A796G mice,although significant differences were not observed.Ibuprofen is a nonseclective COX inhibitor,parecoxib（COX2 selective inhibitor）was selected and injected intravenously into the tail vein.We found that COX2 selective inhibitor parecoxib significantly blunted heat hypersensitivity in Scn11a^A796G/A796G mice.And parecoxib effectively inhibited mutant m Na_v1.9 current and excitability of DRG neurons after 1 h pretreatment,rather than in the acute application.Thus,inflammatory signaling pathways regulated by COX2 can be used as new sight to explore the targeted medication for episodic pain.In this study,we collected one Chinese family with episodic pain and identified SCN11A mutation（c.664C>A/p.Arg222Ser）.In addition,we found alcohol-aggravated pain symptoms in four patients from two family with episodic pain and identified that the ALDH2polymorphism（c.1510G>A/p.Glu504Lys）may be causative of alcohol-aggravated pain.And we successfully generated the Scn11a^A796G/A796G mouse that recapitulated many clinical features of patients,found that the homozygous alteration induced hyperexcitability of DRG neurons.Thus,the Scn11a^A796G/A796G mice showed mechanical,heat and cold hypersensitivity.We confirmed that the Scn11a^A796G/A796G mice showed hypersensitivity to aldehydes and COX2 inhibitor significantly relieved the heat hypersensitivity in Scn11a^A796G/A796G mice.These results also provide new insight into accurately diagnose of complex clinical symptoms and theoretical basis for developing the targeted analgesics.