title: Cell-free expression of a functional pore-only sodium channel.
abstract: Voltage-gated sodium channels participate in the propagation of action potentials in excitable cells. Eukaryotic Navs are pseudo homotetrameric polypeptides, comprising four repeats of six transmembrane segments (S1-S6). The first four segments form the voltage-sensing domain and S5 and S6 create the pore domain with the selectivity filter. Prokaryotic Navs resemble these characteristics, but are truly tetrameric. They can typically be efficiently synthesized in bacteria, but production in vitro with cell-free synthesis has not been demonstrated. Here we report the cell-free expression and purification of a prokaryotic tetrameric pore-only sodium channel. We produced milligram quantities of the functional channel protein as characterized by size-exclusion chromatography, infrared spectroscopy and electrophysiological recordings. Cell-free expression enables advanced site-directed labelling, post-translational modifications, and special solubilization schemes. This enables next-generation biophysical experiments to study the principle of sodium ion selectivity and transport in sodium channels.
authors.affiliation: Department of Chemistry, University of Gothenburg, P.O. Box 462, SE-40530 Gothenburg, Sweden.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Kovácsová
authors.affiliation: Department of Chemistry, University of Gothenburg, P.O. Box 462, SE-40530 Gothenburg, Sweden.
authors.forename: Emil
authors.initials: E
authors.lastname: Gustavsson
authors.affiliation: Nanion Technologies GmbH, Gabrielenstraße 9, 80636 Munich, Germany.
authors.forename: Jiajun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Nanion Technologies GmbH, Gabrielenstraße 9, 80636 Munich, Germany.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Kreir
authors.affiliation: Department of Chemistry, University of Gothenburg, P.O. Box 462, SE-40530 Gothenburg, Sweden. Electronic address: peukers@gmail.com.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Peuker
authors.affiliation: Department of Chemistry, University of Gothenburg, P.O. Box 462, SE-40530 Gothenburg, Sweden. Electronic address: westenho@chem.gu.se.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Westenhoff
mesh_terms: D000818:Animals; D002474:Cell-Free System / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D008869:Microtubule-Associated Proteins; C503386:Nav1 protein, mouse
keywords: Cell-free expression; Membrane protein; Sodium channel