title: A high-capacity membrane potential FRET-based assay for NaV1.8 channels.
abstract: Clinical treatment of neuropathic pain can be achieved with a number of different drugs, some of which interact with all members of the voltage-gated sodium channel (NaV1) family. However, block of central nervous system and cardiac NaV1 channels can cause dose-limiting side effects, preventing many patients from achieving adequate pain relief. Expression of the tetrodotoxin-resistant NaV1.8 subtype is restricted to small-diameter sensory neurons, and several lines of evidence indicate a role for NaV1.8 in pain processing. Given these features, NaV1.8 subtype-selective blockers are predicted to be efficacious in the treatment of neuropathic pain and to be associated with fewer adverse effects than currently available therapies. To facilitate the identification of NaV1.8-specific inhibitors, we stably expressed the human NaV1.8 channel together with the auxiliary human beta1 subunit (NaV beta1) in human embryonic kidney 293 cells. Heterologously expressed human NaV1.8/NaV beta1 channels display biophysical properties that are similar to those of tetrodotoxin-resistant channels present in mouse dorsal root ganglion neurons. A membrane potential, fluorescence resonance energy transfer-based functional assay on a fluorometric imaging plate reader (FLIPR-Tetra, Molecular Devices, Sunnyvale, CA) platform has been established. This highcapacity assay is sensitive to known state-dependent NaV1 modulators and can be used to identify novel and selective NaV1.8 inhibitors.
authors.affiliation: Department of Ion Channels, Merck Research Laboratories, Rahway, NJ 07065, USA.
authors.forename: Chou J
authors.initials: CJ
authors.lastname: Liu
authors.forename: Birgit T
authors.initials: BT
authors.lastname: Priest
authors.forename: Randal M
authors.initials: RM
authors.lastname: Bugianesi
authors.forename: Paula M
authors.initials: PM
authors.lastname: Dulski
authors.forename: John P
authors.initials: JP
authors.lastname: Felix
authors.forename: Ivy E
authors.initials: IE
authors.lastname: Dick
authors.forename: Richard M
authors.initials: RM
authors.lastname: Brochu
authors.forename: Hans-Guenther
authors.initials: HG
authors.lastname: Knaus
authors.forename: Richard E
authors.initials: RE
authors.lastname: Middleton
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kaczorowski
authors.forename: Robert S
authors.initials: RS
authors.lastname: Slaughter
authors.forename: Maria L
authors.initials: ML
authors.lastname: Garcia
authors.forename: Martin G
authors.initials: MG
authors.lastname: Köhler
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004594:Electrophysiology / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D007668:Kidney; D008564:Membrane Potentials / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D062559:NAV1.8 Voltage-Gated Sodium Channel; D009475:Neurons, Afferent / Q000502:physiology*; D010446:Peptide Fragments / Q000276:immunology; D011487:Protein Conformation; D011817:Rabbits; D015222:Sodium Channels / Q000235:genetics / Q000502:physiology*
chemical_list: D017931:DNA Primers; D062559:NAV1.8 Voltage-Gated Sodium Channel; D010446:Peptide Fragments; C568317:SCN10A protein, human; C568318:Scn10a protein, mouse; D015222:Sodium Channels