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Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies
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Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

DOI:
10.3791/52281-v

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11:42 min

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January 22, 2015

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, , ,
1Institut Curie, Centre de Recherche, CNRS, UMR 168, PhysicoChimie Curie,Université Pierre et Marie Curie, 2Kavli Institute for Brain and Mind,University of California, San Diego, 3Molecular Physiology and Biophysics Section, National Institute for Neurological Disorders and Stroke,National Institute of Health

Chapters

  • 00:05Title
  • 01:44Giant Unilamellar Vesicle Growth by Electroformation
  • 04:29Giant Unilamellar Vesicle Growth by Gel-assisted Swelling
  • 06:07Harvesting and Transferring GUVs
  • 07:33Patch-clamping GUVs
  • 09:14Results: Microscopy and Patch Clamp Studies of KvAP GUVs
  • 11:03Conclusion

Summary

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The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods — electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Tags

Keywords: Transmembrane ProteinVoltage-gated Ion ChannelKvAPGiant Unilamellar Vesicles (GUVs)Biomimetic SystemMembrane Associated PhenomenaDehydration-rehydrationElectroformationGel-assisted SwellingSmall Unilamellar VesiclesRehydration BufferAC FieldAgarose GelFluorescence MicroscopyPatch-clampProtein Distribution

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