Abstract
The long QT syndrome (LQTS) is a cardiac dysfunction that prolongs the heart repolarisation interval, leading to cardiac arrhythmia or failure. This pathology can be induced by off-target effects of drugs which block the human ether-a-go-go related gene (hERG) potassium channels located in the myocardium cell membranes. To reduce the risks of this acquired LQTS, regulatory authorities demand in vitro testing of all new drug entities for hERG-blocking potential. As the pore domain of the hERG channel is an important target of LQTS-prone drugs, the objective of our work was to express and purify this region (Asp540-Tyr673) in E. coli to allow biophysical and electrophysiological studies. The detergent sarkosyl was employed for the solubilisation and a His6 N-terminal tag was used to isolate the transmembrane pore domain of hERG with yields of approximately 0.5-1 mg per liter of LB media. Mass spectrometry and Western blot confirmed the identity of the protein and circular dichroism showed that the majority of the hERG pore domain segments adopt an α-helix structure as was expected from sequence homology with other K+ channels. The functionality of the channel is proven by incorporating it into lipid bilayers formed using Montal Mueller method.
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