Functional assay of voltage-gated sodium channels using membrane potential-sensitive dyes.
The discovery of novel therapeutic agents that act on voltage-gated sodium channels requires the establishment of high-capacity screening assays that can reliably measure the activity of these proteins. Fluorescence resonance energy transfer (FRET) technology using membrane potential-sensitive dyes has been shown to provide a readout of voltage-gated sodium channel activity in stably transfected cell lines. Due to the inherent rapid inactivation of sodium channels, these assays require the presence of a channel activator to prolong channel opening. Because sodium channel activators and test compounds may share related binding sites on the protein, the assay protocol is critical for the proper identification of channel inhibitors. In this study, high throughput, functional assays for the voltage-gated sodium channels, hNa(V)1.5 and hNa(V)1.7, are described. In these assays, channels stably expressed in HEK cells are preincubated with test compound in physiological medium and then exposed to a sodium channel activator that slows channel inactivation. Sodium ion movement through open channels causes membrane depolarization that can be measured with a FRET dye membrane potential-sensing system, providing a large and reproducible signal. Unlike previous assays, the signal obtained in the agonist initiation assay is sensitive to all sodium channel modulators that were tested and can be used in high throughput mode, as well as in support of Medicinal Chemistry efforts for lead optimization.
Department of Ion Channels, Merck Research Laboratories, Rahway, NJ 07065, USA.
John P
JP
Felix
Brande S
BS
Williams
Birgit T
BT
Priest
Richard M
RM
Brochu
Ivy E
IE
Dick
Vivien A
VA
Warren
Lizhen
L
Yan
Robert S
RS
Slaughter
Gregory J
GJ
Kaczorowski
McHardy M
MM
Smith
Maria L
ML
Garcia
D002460:Cell Line; D004396:Coloring Agents / Q000032:analysis* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009124:Muscle Proteins / Q000032:analysis / Q000502:physiology; D062554:NAV1.5 Voltage-Gated Sodium Channel; D062556:NAV1.7 Voltage-Gated Sodium Channel; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels / Q000032:analysis* / Q000502:physiology*; D014701:Veratridine / Q000494:pharmacology
D004396:Coloring Agents; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D062556:NAV1.7 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; C568322:Scn5a protein, rat; C503480:Scn9a protein, rat; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D014701:Veratridine