Production of Native-Like Refolded NaV1.7 Voltage Sensing Domain As Shown by Toxin Binding Activity

Voltage gated sodium channels (VGSCs) are essential to the propagation of nerve cell impulses and thus communication in the nervous system. The Human voltage-gated sodium channel NaV1.7 has been shown to play a vital role in the nerve pathways that induce pain response. Many of the currently available pain therapeutic drugs are opiates, derivative molecules of opium. These drugs target receptors in the brain that result in many other effects in addition to pain relief, such as euphoria, which gives such drugs a high risk for misuse. Drugs that target and inhibit NaV1.7 on the other hand should only affect the pain signal and therefore lead to less addiction problems than opiate-based medications. Although the atomic structures of several bacterial VGSCs have been determined (and that of a bacterial-mammalian chimeric VGSC construct), the structure of native human NaV1.7 is currently unknown. In this study, we expressed, purified and reconstituted the voltage sensing domain (VSD) of repeat II of this channel with the aim of conducting ligand binding and structural studies via solution state NMR spectroscopy. We have successfully expressed the recombinant voltage sensing domain (VSD) of NaV1.7 using the Trp-Delta-Ldr inclusion body system. The fusion protein was cleaved with hydroxylamine using an added NG sequence between the fusion partners. The VSD was then refolded using the zwitterionic lipid, DMPC. Micro-scale thermophoresis was used to confirm binding of the recombinant VSD to Protoxin-II, which is a known inhibitor of NaV1.7. We are currently optimizing the membrane mimetic system for the recombinant VSD so that it will be suitable for solution state NMR studies.