title: Inhibition of voltage-gated Na⁺ channels by the synthetic cannabinoid ajulemic acid.
abstract: BACKGROUND
  The synthetic cannabinoid ajulemic acid has been demonstrated to alleviate pain in patients suffering from chronic neuropathic pain. Cannabinoids interact with several molecules within the pain circuit, including a potent inhibition of voltage-gated sodium channels. In this study, we closely characterized this property on neuronal and nonneuronal sodium channels.
  
  
  METHODS
  The inhibition of sodium inward currents by ajulemic acid was studied in vitro. Human embryonic kidney 293t cells were used as the expression system for Nav1.2, 1.3, 1.4, 1.5, 1.5N406K, 1.5F1760A, and 1.7; Nav1.8 was transiently expressed in ND7/23 cells. Nav1.2, Nav1.3, and Nav 1.8 were from rats, and Nav1.4, Nav1.5, and Nav1.7 were of human origin. Sodium currents were analyzed by means of the whole cell patch-clamp technique. The investigated concentrations of ajulemic acid were 0.1, 0.3, 1, 3, 10, and 30 μmol/L.
  
  
  RESULTS
  Ajulemic acid reversibly and concentration-dependently inhibited all voltage-gated sodium channel (Nav) isoforms investigated in this study, including Nav1.2, 1.3, 1.4, 1.5, 1.7, and 1.8. Tonic block of resting channels yielded half-maximal inhibitory concentration values between 2 and 9 μmol/L and was strongly enhanced on inactivated channels, suggesting state-dependent inhibition by ajulemic acid. Tonic block did not differ significantly when comparing Nav1.2 and Nav1.3, Nav1.4 and Nav1.5, and Nav1.7 and Nav1.8. Statistical analysis of other combinations of subunits (e.g., Nav1.2 and Nav1.4) by analysis of variance yielded a significant difference in block. Although we did not observe any relevant use-dependent block, ajulemic acid induced a strong hyperpolarizing shift of the voltage dependency of fast inactivation and modest shift of slow inactivation. The local anesthetic-insensitive Nav1.5 constructs N406K and F1760A displayed a preserved sensitivity to block by ajulemic acid. Finally, we found that low concentrations of ajulemic acid efficiently inhibited Navβ4 peptide-mediated resurgent currents in Nav1.5.
  
  
  CONCLUSIONS
  Our data suggest that block of sodium channels can be a relevant mechanism by which ajulemic acid alleviates neuropathic pain. The potent inhibition of resurgent currents and the preserved block on local anesthetic-insensitive channels indicates that ajulemic acid interacts with a conserved but yet unknown site of sodium channels.
authors.affiliation: From the *Department of Anesthesia and Critical Care Medicine, and † Department of Neurology and Clinical Neurophysiology, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Nilufar
authors.initials: N
authors.lastname: Foadi
authors.forename: Christian
authors.initials: C
authors.lastname: Berger
authors.forename: Igor
authors.initials: I
authors.lastname: Pilawski
authors.forename: Carsten
authors.initials: C
authors.lastname: Stoetzer
authors.forename: Matthias
authors.initials: M
authors.lastname: Karst
authors.forename: Gertrud
authors.initials: G
authors.lastname: Haeseler
authors.forename: Florian
authors.initials: F
authors.lastname: Wegner
authors.forename: Andreas
authors.initials: A
authors.lastname: Leffler
authors.forename: Jörg
authors.initials: J
authors.lastname: Ahrens
mesh_terms: D000465:Algorithms; D000779:Anesthetics, Local / Q000378:metabolism; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D013759:Dronabinol / Q000031:analogs & derivatives* / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D018408:Patch-Clamp Techniques; D026941:Sodium Channel Blockers; D061566:Voltage-Gated Sodium Channels / Q000187:drug effects* / Q000235:genetics
chemical_list: D000779:Anesthetics, Local; D026941:Sodium Channel Blockers; D061566:Voltage-Gated Sodium Channels; D013759:Dronabinol; C471849:lenabasum