Whole-GUV patch-clamping.
作者： Matthias Garten , Lars D. Mosgaard , Thomas Bornschlögl , Stéphane Dieudonné , Patricia Bassereau
关键词: Patch clamp 、 Ion channel 、 Materials science 、 Biophysics 、 Vesicle 、 Pipette 、 Nanotechnology 、 Semipermeable membrane 、 Membrane 、 Ion transporter 、 Membrane potential
摘要: Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate properties, whereas existing in vitro systems have limitations, such as residual solvent unphysiologically high tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for electrophysiology, but efforts to measure current of intact GUVs been unsuccessful. In this work, two challenges obtaining "whole-GUV" patch-clamp configuration were identified resolved. First, unless patch pipette GUV pressures are precisely matched GUV-attached configuration, breaking also ruptures GUV. Second, shrink irreversibly membrane/glass adhesion creating high-resistance seal (>1 GΩ) continuously pulls into pipette. contrast, cell-derived plasma (GPMVs), allows GPMV contents passivate surface, thereby dynamically blocking spreading whole-GMPV mode. To mimic dynamic passivation mechanism, beta-casein was encapsulated GUVs, yielding a stable, high-resistance, whole-GUV range compositions. Specific capacitance measurements confirmed that membranes truly solvent-free tension could controlled over physiological range. Finally, potential transport studies tested using model channel, gramicidin, voltage-clamp fluorometry performed with voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping other processes studied while controlling composition, tension, shape.
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