Recombinant Expression of a Voltage Sensing Domain from Human NaV1.7

Abstract

Voltage gated sodium channels (VGSCs) are involved with excitatory cell signal response and propagation. The human voltage-gated sodium channel NaV1.7 has been shown to be involved with neuronal pain response. The current standard for pain relief medications involves opiates, derivative molecules of opium, which have a high risk of addiction and misuse. Therefore, drugs that can inhibit NaV1.7 as an alternative target could have great potential as a new class of pain medications that would not pose the problem of addiction. The three-dimensional structure of NaV1.7 is currently unknown. Knowledge of such a high-resolution structure could lead to major breakthroughs in the field of rational drug design for pain therapeutics. In this study, we intend to gain structural information about this channel via NMR spectroscopy. Due to the size constraints of solution NMR, we are focusing our investigation on the isolated voltage sensing domain (VSD) from repeat II of the channel. We have successfully expressed this VSD using the Trp-Δ-LE inclusion body system. We designed a fusion construct containing an Asn-Gly sequence in between the fusion partners for hydroxylamine cleavage. We are currently working on refolding the cleaved VSD by exchanging denaturing detergent with phospholipids as membrane mimetics. The NaV1.7 repeat II VSD reconstituted into lipid bicelles will then be used for NMR structural studies and for the discovery of toxins targeting this particular domain of the channel.