title: A cell-sparing electric field stimulation technique for high-throughput screening of voltage-gated ion channels.
abstract: The Trans Cell Layer Electrical Field Stimulation (TCL-EFS) system has been developed for high-throughput screening (HTS) of voltage-gated ion channels in microplate format on a Voltage-Ion Probe Reader (VIPR) platform. In this design, a wire electrode is placed above the cell layer of each filter well, and a whole plate perimeter electrode resides beneath the filter layer. This configuration allows the electrodes to be placed away from the cell layer to minimize the near electrode field effects on cell function and dye bleaching observed with other existing designs. Mathematical simulation indicates that the electric field at the cell layer becomes uniform as the top electrode is raised to a position near the surface of the solution in the well. Using the TCL-EFS system and membrane potential fluorescence resonance energy transfer (FRET) dyes, the sensitivity of voltage-gated sodium channels to tetrodotoxin and other channel inhibitors was found to be similar to those determined by established electrophysiological and more conventional VIPR techniques. A good correlation was also observed with the TCL-EFS system for inhibition of Cav2.2 by omega-conotoxin-GVIA and for block of Cav1.2 by known small molecule inhibitors. Thus, the TCLEFS system is suitable for both quantitative analysis and HTS of voltage-gated sodium and calcium channels, without the liabilities of previously reported EFS methodologies.
authors.affiliation: Department of Ion Channels, Merck Research Laboratories, Rahway, NJ 07065, USA. randal_bugianesi@merck.com
authors.forename: Randal M
authors.initials: RM
authors.lastname: Bugianesi
authors.forename: Paul R
authors.initials: PR
authors.lastname: Augustine
authors.forename: Karim
authors.initials: K
authors.lastname: Azer
authors.forename: Claude
authors.initials: C
authors.lastname: Dufresne
authors.forename: James
authors.initials: J
authors.lastname: Herrington
authors.forename: Gary S
authors.initials: GS
authors.lastname: Kath
authors.forename: Owen B
authors.initials: OB
authors.lastname: McManus
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Napolitano
authors.forename: Alison
authors.initials: A
authors.lastname: Rush
authors.forename: Jeff
authors.initials: J
authors.lastname: Sachs
authors.forename: Neal
authors.initials: N
authors.lastname: Simpson
authors.forename: Michael K
authors.initials: MK
authors.lastname: Wismer
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kaczorowski
authors.forename: Robert S
authors.initials: RS
authors.lastname: Slaughter
mesh_terms: D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D003198:Computer Simulation; D004558:Electric Stimulation; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology*; D009124:Muscle Proteins / Q000187:drug effects / Q000502:physiology*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D015222:Sodium Channels / Q000187:drug effects / Q000502:physiology*; D013779:Tetrodotoxin / Q000494:pharmacology; D020866:omega-Conotoxin GVIA / Q000494:pharmacology
chemical_list: D002121:Calcium Channel Blockers; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; D015222:Sodium Channels; D013779:Tetrodotoxin; D020866:omega-Conotoxin GVIA