A High-Capacity Membrane Potential FRET-Based Assay for Na V 1.8 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 (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.