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Article Dans Une Revue Biochimica et Biophysica Acta:Biomembranes Année : 2017

Functional reconstitution of cell-free synthesized purified K v channels

Sandra Cortès
  • Fonction : Auteur
  • PersonId : 835938
Beate Bersch
Xavier Henry
  • Fonction : Auteur
  • PersonId : 774206
  • IdRef : 034133232
Béatrice Schaack

Résumé

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.

Dates et versions

hal-01593501 , version 1 (26-09-2017)

Identifiants

Citer

Stéphane Renauld, Sandra Cortès, Beate Bersch, Xavier Henry, Michel de Waard, et al.. Functional reconstitution of cell-free synthesized purified K v channels. Biochimica et Biophysica Acta:Biomembranes, 2017, 1859 (12), pp.2373 - 2380. ⟨10.1016/j.bbamem.2017.09.002⟩. ⟨hal-01593501⟩
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