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Assaying the proton transport and regulation of UCP1 using solid supported membranes

Abstract

The uncoupling protein 1 (UCP1) is a mitochondrial protein that carries protons across the inner mitochondrial membrane. It has an important role in non-shivering thermogenesis, and recent evidence suggests its role in human adult metabolism. Using rapid solution exchange on solid supported membranes, we succeeded in measuring electrical currents generated by the transport activity of UCP1. The protein was purified from mouse brown adipose tissue, reconstituted in liposomes and absorbed on solid supported membranes. A fast pH jump activated the ion transport, and electrical signals could be recorded. The currents were characterized by a fast rise and a slow decay, were stable over time, inhibited by purine nucleotides and activated by fatty acids. This new assay permits direct observation of UCP1 activity in controlled cell-free conditions, and opens up new possibilities for UCP1 functional characterization and drug screening because of its robustness and its potential for automation.

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Acknowledgments

We thank Dr. Petr Obrdlik for help and advice concerning the current measurements. The work was supported by the Institut Universitaire de France, by the Agence Nationale de la Recherche (Trans-MIT and MIT-2 M projects) and by EU project EDICT no. 211800.

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Correspondence to Bruno Miroux or Eva Pebay-Peyroula.

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Blesneac, I., Ravaud, S., Machillot, P. et al. Assaying the proton transport and regulation of UCP1 using solid supported membranes. Eur Biophys J 41, 675–679 (2012). https://doi.org/10.1007/s00249-012-0844-2

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Keywords

  • Mitochondrial carriers
  • Uncoupling protein
  • Proteoliposomes
  • Solid supported membranes
  • Electrophysiology