Expression and optimization of voltage-gated sodium channels Na_V1.8 in Xenopus laevis oocytes

Voltage-gated sodium channels (VGSCs, Na_V) are essential for the generation and conduction of action potentials in excitable cells. The Na_V1.8 subtype is predominantly expressed in sensory neurons, particularly in small-diameter neurons of the dorsal root ganglia (DRG), and plays a critical role in the signal transduction of chronic, neuropathic, and inflammatory pain. Due to its tetrodotoxin-resistant properties and electrophysiological characteristics of slow activation and inactivation, Na_V1.8 is a key target for analgesic drug development, facilitating repeated neuronal firing. Consequently, establishing an in vitro drug screening model based on Na_V1.8 is crucial for identifying lead compounds with analgesic potential. However, heterologous expression of Na_V1.8 remains challenging, primarily due to low expression levels in non-neuronal cell lines, which hinders effective drug screening. In this study, we established an in vitro expression model of rat Na_V1.8 using Xenopus laevis oocytes as the expression system. Channel property was characterized using the two electrode voltage clamp (TEVC) technique, providing a platform for screening active compounds. Quantitative polymerase chain reaction confirmed that rat Na_V1.8 is predominantly expressed in the DRG and trigeminal ganglia (Guangxi University Ethics Committee Review and approval number: GXU-2022-159). We cloned the full-length Na_V1.8 gene and constructed two chimeric variants, Na_V1.8-N and Na_V1.8-C. Following in vitro transcription of the corresponding capped RNA, it was injected into Xenopus laevis oocytes for heterologous expression. TEVC recordings were used to detect Na_V1.8 and chimera expression, and channel activity was evaluated through I-V curves, steady-state activation curves, and the specific Na_V1.8 antagonists A-803467 and VX-548. The chimera Na_V1.8-C resolved the problem in stable expression of Na_V1.8 in vitro and exhibited similar pharmacological properties with native Na_V1.8, offering a valuable tool for future screening of compounds targeting voltage-gated sodium channel.