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Protein reconstitution into freestanding planar lipid membranes for electrophysiological characterization

Nature Protocols volume 10, pages 188–198 (2015)Cite this article

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Abstract

The reconstitution of channel-forming proteins into planar lipid bilayers enables their functional characterization at very low (sometimes below attomolar) concentrations. We describe the three main approaches used in our laboratories (the Mueller-Rudin technique, in which the bilayers contain an organic solvent, the Montal-Mueller or solvent-free technique, and a method for membrane reconstitution via liposome formation), and we discuss their respective advantages and limitations. Despite the differences in the reconstitution procedures, subsequent protein characterization is based on the same electrophysiological technique. A transmembrane electric field is applied, inducing an ion current and allowing conclusions to be drawn on apparent pore sizes, or suggesting functional properties such as channel opening and closing upon ligand binding, pH-induced conformational changes, ion selectivity or substrate specificity.

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Figure 1: Two methods for forming lipid membrane patches.
Figure 2: Typical experimental bilayer setup.
Figure 3: Adapted patch-clamp setup for the formation of small lipid bilayers on nanopipettes12.
Figure 4: Lipid ion channel events in a synthetic lipid membrane made of a mixture of di-myristoyl-phosphatidyl-choline (DMPC) and di-lauroyl-phosphatidyl-choline (DLPC) in a 150 mM NaCl solution.
Figure 5: Reconstitution of a single trimeric maltoporin channel into a virtually solvent-free planar lipid bilayer and application of −150 mV transmembrane voltage cause an ion current.

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Acknowledgements

The research leading to these results was conducted as part of the Translocation consortium, and it has received support from the Innovative Medicines Joint Undertaking under grant agreement no. 115525, resources which are composed of financial contributions from the EU's seventh framework program (FP7/2007-2013) and European Federation of Pharmaceutical Industries and Associations (EFPIA) companies' in-kind contribution, as well as grant no. Wi2278/18-1 from the Deutsche Forschungsgemeinschaft (DFG). U.K. acknowledges funding from a European Research Council starting grant.

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Authors and Affiliations

  1. Division of Biophysics, Research Center Borstel, Center for Medicine and Bioscience, Borstel, Germany

    Thomas Gutsmann & Mathias Winterhalter

  2. Membrane Biophysics Group, Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark

    Thomas Heimburg

  3. Cavendish Laboratory, University of Cambridge, Cambridge, UK

    Ulrich Keyser

  4. Department of Chemistry, University of Oxford, Oxford, UK

    Kozhinjampara R Mahendran

  5. Jacobs University Bremen, Bremen, Germany

    Mathias Winterhalter

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Correspondence to Mathias Winterhalter.

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Gutsmann, T., Heimburg, T., Keyser, U. et al. Protein reconstitution into freestanding planar lipid membranes for electrophysiological characterization. Nat Protoc 10, 188–198 (2015). https://doi.org/10.1038/nprot.2015.003

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