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Biochim Biophys Acta Biomembr. 2017 Dec;1859(12):2373-2380. doi: 10.1016/j.bbamem.2017.09.002. Epub 2017 Sep 6.

Functional reconstitution of cell-free synthesized purified Kv channels.

Author information

1
Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France; CNRS, IBS, F-38044 Grenoble, France; CEA, IBS, F-38044 Grenoble, France.
2
Synthelis, 5 avenue du Grand Sablon, 38700 La Tronche, France. Electronic address: sandra.cortes@synthelis.fr.
3
Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France; CNRS, IBS, F-38044 Grenoble, France; CEA, IBS, F-38044 Grenoble, France. Electronic address: beate.bersch@ibs.fr.
4
Inserm UMR 1087/CNRS UMR 6291, Institut du Thorax, 44007 Nantes, France; Smartox Biotechnology, 570 Rue de la Chimie, 38400 Saint-Martin d'Hères, France. Electronic address: michel.dewaard@univ-nantes.fr.
5
Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France; CNRS, IBS, F-38044 Grenoble, France; CEA, IBS, F-38044 Grenoble, France. Electronic address: beatrice.schaack@ibs.fr.

Abstract

The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active Kv1.1 and Kv1.3 channels and their efficient insertion into liposomes. Two complementary methods for the determination of the electrical activity of the proteoliposome-embedded channels were compared using Kv1.1 as a model system: (1) single channel recordings in droplet interface bilayers (DIB) and (2) measurement of the membrane voltage potential generated by a potassium ion diffusion potential using the voltage-sensitive fluorescent dye oxonol VI. Single channel recordings in DIBs proved unreliable because of the non-reproducible fusion of proteoliposomes with an artificial membrane. Therefore, the use of the optical indicator oxonol VI was adapted for 96 well microtiter plates using the ionophore valinomycin as a positive control. The activity of Kv1.1 and Kv1.3 channels was then monitored in the absence and presence of different venom toxins, demonstrating that fluorescent dyes can be used very efficiently when screening small molecules for their channel blocking activity.

KEYWORDS:

DIB; K(v) channels; Oxonol VI; Proteoliposomes

PMID:
28888365
DOI:
10.1016/j.bbamem.2017.09.002
[Indexed for MEDLINE]
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