Functional expression of the chimera proteins of Nav1.7 and NavAb in Escherichia coli

Na_v1.7 is a eukaryotic voltage-dependent Na channel (Na_v) family membrane protein and has four channel domains and four voltage sensor domains (VSD-I–IV). It is involved in pain perception, and VSDs that differ significantly by Na_v subtype are targeted in the development of Na_v1.7-specific inhibitors. This is expected to result in neuropathic pain treatments with fewer side effects. We previously reported on intra-periplasm secretion and selection (PERISS), a peptide drug discovery system that targets membrane proteins by co-expressing a peptide library and a target membrane protein. For PERISS screening of VSD-specific new Na_v1.7 inhibitors, the chimera protein (Na_vAb/1.7VSD) of Na_v from prokaryotic Arcobacter butzleri (Na_vAb), in which extracellular loops of VSD were replaced with homologous loops from Na_v1.7, serves as an effective model. This is because Na_vAb harbors only one VSD and the biological activity of Na_vAb/1.7VSD was previously confirmed. To date, Na_vAb/1.7VSD has only been found to be expressed in insect cells. In this study, we report on the expression and channel activity of Na_vAb/1.7VSD-II in Escherichia coli (E. coli). The expression of this protein in the inner membrane of E. coli was confirmed by western blotting. Channel activity was assessed by measuring the channel currents of the purified recombinant proteins and inhibition using a Na_v1.7-specific peptide inhibitor. The results indicate that Na_vAb/1.7VSD-II was functionally expressed in E. coli, providing empirical support for the discovery of new VSD-specific Na_v1.7 inhibitors using the PERISS screening method.