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Whole-GUV patch-clamping.

Matthias Garten1,2,3, Lars D Mosgaard4, Thomas Bornschlögl1,2

  • 1Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, 75005 Paris, France.

Proceedings of the National Academy of Sciences of the United States of America
|December 23, 2016
PubMed
Summary
This summary is machine-generated.

Researchers developed a stable whole-giant unilamellar vesicle (GUV) patch-clamp method. This technique enables precise control over membrane properties for studying ion transport and voltage-dependent processes in vitro.

Keywords:
biomimetic systemelectrophysiologygiant unilamellar vesiclelipid–glass interactionpatch clamp

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Area of Science:

  • Biophysics
  • Membrane Biology
  • Electrophysiology

Background:

  • Studying membrane protein function in vitro is hindered by cellular regulation of membrane properties and limitations of existing artificial systems.
  • Giant unilamellar vesicles (GUVs) are ideal for in vitro electrophysiology, but achieving a stable whole-GUV configuration for current measurements has been unsuccessful.
  • Previous attempts faced challenges with GUV rupture during patch detachment and irreversible GUV shrinkage due to membrane adhesion.

Purpose of the Study:

  • To develop a robust and stable whole-GUV patch-clamp configuration for in vitro electrophysiological studies.
  • To overcome challenges related to GUV rupture and shrinkage during patch-clamp experiments.
  • To enable controlled studies of ion transport and voltage-dependent processes in GUVs with defined membrane properties.

Main Methods:

  • Identified and resolved two key challenges: precise pressure matching to prevent GUV rupture and a dynamic passivation mechanism to prevent GUV shrinkage.
  • Encapsulated beta-casein into GUVs to mimic the passivation effect observed in cell-derived giant plasma membrane vesicles (GPMVs).
  • Utilized specific membrane capacitance measurements to confirm solvent-free membranes and controlled membrane tension, and tested ion transport with gramicidin.

Main Results:

  • Successfully established a stable, high-resistance (>1 GΩ) whole-GUV patch-clamp configuration applicable to various membrane compositions.
  • Demonstrated solvent-free membranes and physiological control over membrane tension.
  • Validated the system's potential for ion transport studies using gramicidin and performed voltage-clamp fluorometry.

Conclusions:

  • The developed whole-GUV patch-clamp technique provides a powerful new tool for in vitro electrophysiology.
  • This method allows for precise control over membrane composition, tension, and shape, facilitating detailed studies of membrane proteins.
  • Enables investigation of ion transport and other voltage-dependent processes in a controlled, artificial membrane environment.