A Functional Na_V1.7-Na_VAb Chimera with a Reconstituted High-Affinity ProTx-II Binding Site

The Na_V1.7 voltage-gated sodium channel is implicated in human pain perception by genetics. Rare gain of function mutations in Na_V1.7 lead to spontaneous pain in humans whereas loss of function mutations results in congenital insensitivity to pain. Hence, agents that specifically modulate the function of Na_V1.7 have the potential to yield novel therapeutics to treat pain. The complexity of the channel and the challenges to generate recombinant cell lines with high Na_V1.7 expression have led to a surrogate target strategy approach employing chimeras with the bacterial channel Na_VAb. In this report we describe the design, synthesis, purification, and characterization of a chimera containing part of the voltage sensor domain 2 (VSD2) of Na_V1.7. Importantly, this chimera, DII S1-S4, forms functional sodium channels and is potently inhibited by the Na_V1.7 VSD2 targeted peptide toxin ProTx-II. Further, we show by [¹²⁵I]ProTx-II binding and surface plasmon resonance that the purified DII S1-S4 protein retains high affinity ProTx-II binding in detergent. We employed the purified DII S1-S4 protein to create a scintillation proximity assay suitable for high-throughput screening. The creation of a Na_V1.7-Na_VAb chimera with the VSD2 toxin binding site provides an important tool for the identification of novel Na_V1.7 inhibitors and for structural studies to understand the toxin-channel interaction.