Cell-free expression of a functional pore-only sodium channel

Protein Expression and Purification
Gabriela KovácsováSebastian Westenhoff

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.

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Citations

Mar 1, 2018·The Journal of General Physiology·Laura-Marie WintersteinGerhard Thiel
Mar 22, 2020·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Srujan Kumar DondapatiStefan Kubick
Dec 29, 2020·The Journal of Biological Chemistry·Elena D NosyrevaRuhma Syeda
Jan 10, 2021·The Journal of Biological Chemistry·Elena D NosyrevaRuhma Syeda
Oct 24, 2018·The Journal of Physical Chemistry. B·Karen M Callahan, Benoît Roux

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Methods Mentioned

BETA
NMR
infrared spectroscopy
size exclusion chromatography
chip

Software Mentioned

PatchControl
Nanion
Clampfit

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