title: Comparison of the pharmacological properties of rat Na(V)1.8 with rat Na(V)1.2a and human Na(V)1.5 voltage-gated sodium channel subtypes using a membrane potential sensitive dye and FLIPR.
abstract: A novel, membrane potential sensitive dye and a fluorescence imaging plate reader (FLIPR) have been used to characterize the pharmacological properties of rat Na(v)1.8 voltage-gated sodium channels (VGSC) in parallel with rat Na(v)1.2a and human Na(v)1.5 VGSC subtypes, respectively. The sensitivity of recombinant Na(v)1.2a-CHO, Na(v)1.5-293-EBNA, and Na(v)1.8-F-11 cells to VGSC activators was subtype dependent. Veratridine evoked depolarization of Na(v)1.2a-CHO and Na(v)1.5-293-EBNA cells with pEC(50) values of 4.78 +/- 0.13 and 4.84 +/- 0.12, respectively (n = 3), but had negligible effect on Na(v)1.8-F-11 cells (pEC(50) < 4.5). Type I pyrethroids were without significant effect at all subtypes. In contrast, the type II pyrethroids deltamethrin and fenvalerate evoked direct depolarization of Na(v)1.8-F-11 and Na(v)1.5-293-EBNA cells. Deltamethrin potentiated the veratridine-evoked response in Na(v)1.8-F-11 cells by > or =20-fold, in contrast to a <or =3-fold potentiation of the response in Na(v)1.2a, and Na(v)1.5 cells. Tetrodotoxin (TTX) inhibited VGSC activator-evoked depolarization of Na(v)1.8-F-11 cells with a biphasic concentration-response curve. The calculated pIC(50) values were 8.05 +/- 0.25 (n = 4) and 4.32 +/- 0.21 (n = 4), corresponding to TTX inhibition of endogenous TTX-sensitive (TTX-S), and recombinant Na(v)1.8 TTX-resistant (TTX-R) VGSCs, respectively. With the exception of TTX, the potencies of a number of ion channel blockers for the Na(v)1.8, Na(v)1.2a, and Na(v)1.5 VGSC subtypes were similar. In summary, these high-throughput FLIPR assays represent a valuable tool for the determination of the relative potencies of compounds at different VGSC subtypes and may prove useful for the identification of novel subtype-selective inhibitors.
authors.affiliation: Theravance Inc., 901 Gateway Boulevard, South San Francisco, CA 94080, USA. rvickery@theravance.com
authors.forename: R G
authors.initials: RG
authors.lastname: Vickery
authors.forename: S M
authors.initials: SM
authors.lastname: Amagasu
authors.forename: R
authors.initials: R
authors.lastname: Chang
authors.forename: N
authors.initials: N
authors.lastname: Mai
authors.forename: E
authors.initials: E
authors.lastname: Kaufman
authors.forename: J
authors.initials: J
authors.lastname: Martin
authors.forename: J
authors.initials: J
authors.lastname: Hembrador
authors.forename: M D
authors.initials: MD
authors.lastname: O'Keefe
authors.forename: C
authors.initials: C
authors.lastname: Gee
authors.forename: D
authors.initials: D
authors.lastname: Marquess
authors.forename: J A M
authors.initials: JA
authors.lastname: Smith
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes; D006801:Humans; D009124:Muscle Proteins / Q000378:metabolism*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D062559:NAV1.8 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism*; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D062559:NAV1.8 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins; C568317:SCN10A protein, human; C568320:SCN5A protein, human; C568319:Scn10a protein, rat; C568322:Scn5a protein, rat; D015222:Sodium Channels; D012964:Sodium