A high-capacity membrane potential FRET-based assay for Nav1.8 channels

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 (Na(V)1) family. However, block of central nervous system and cardiac Na(V)1 channels can cause dose-limiting side effects, preventing many patients from achieving adequate pain relief. Expression of the tetrodotoxin-resistant Na(V)1.8 subtype is restricted to small-diameter sensory neurons, and several lines of evidence indicate a role for Na(V)1.8 in pain processing. Given these features, Na(V)1.8 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 Na(V)1.8-specific inhibitors, we stably expressed the human Na(V)1.8 channel together with the auxiliary human beta 1 subunit (Na-V beta 1) in human embryonic kidney 293 cells. Heterologously expressed human Na(V)1.8/Na-V beta 1 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(R)-Tetra, Molecular Devices, Sunnyvale, CA) platform has been established. This high-capacity assay is sensitive to known state-dependent Na(V)1 modulators and can be used to identify novel and selective Na(V)1.8 inhibitors.