Short Hairpin RNA–mediated Selective Knockdown of NaV1.8 Tetrodotoxin-resistant Voltage-gated Sodium Channel in Dorsal Root Ganglion Neurons

Abstract

Background Voltage-gated sodium channels comprise a family of closely related proteins, each subserving different physiologic and pathologic functions. NaV1.8 is an isoform of voltage-gated sodium channel implicated in the pathogenesis of inflammatory and neuropathic pain, but currently, there is no isoform-specific inhibitor of any voltage-gated sodium channels. The authors explored the possibility of short hairpin RNA-mediated selective knockdown of NaV1.8 expression. Methods DNA constructs designed to transcribe short hairpin RNA targeting NaV1.8 were created and incorporated into recombinant lentiviruses. The virus-induced selective knockdown of NaV1.8 was examined at the protein, messenger RNA, and functional levels using Western blot, immunohistochemistry, reverse-transcription polymerase chain reaction, and patch clamp electrophysiology. Results Transduction of HEK293 cells stably expressing NaV1.8 or primary dorsal root ganglion neurons with lentivirus expressing short hairpin RNA resulted in the knockdown of NaV1.8 protein and messenger RNA concentrations. Whole cell patch clamp recordings confirmed decrease in the NaV1.8-mediated current density without changes in other biophysical properties. Conclusions A selective knockdown of NaV1.8 expression in dorsal root ganglion neurons can be attained by short hairpin RNA delivered with lentivirus. This method may provide a new gene therapy approach to controlling neuronal hyperexcitability and pathologic pain.