Transport-specific isolation of large channels reconstituted into lipid vesicles.

The Journal of Membrane Biology
A L HarrisJ Zimmerberg

Abstract

To develop a technique for purifying and identifying pore-forming membrane proteins, we used a transport-specific increase in buoyant density to select for lipid vesicles containing voltage-dependent anion channels (VDAC). Monodisperse, single-walled vesicles were formed by gel filtration from a detergent-solubilized mixture of lipid and protein in a urea buffer. The vesicles were layered on a linear iso-osmolar density gradient formed of urea and sucrose buffers. Since VDAC is open at zero trans-membrane voltage and is permeable to urea and sucrose, vesicles containing functional VDAC should become more dense as sucrose enters and urea leaves, while those lacking open channels should maintain their original density. Vesicles formed in the absence of VDAC migrated to a characteristic density, while vesicles formed in the presence of VDAC fractionated into two populations in the gradients, one migrating to the same density as the vesicles formed without VDAC, and one at a significantly greater density. In contrast to the lower density vesicles, the higher density vesicles showed a high permeability to calcein, and contained functional VDAC channels (shown by electrophysiological recordings following fusion with a planar bilayer)...Continue Reading

References

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Citations

Oct 1, 1992·Brain Research. Molecular Brain Research·A L HarrisJ Zimmerberg
Dec 19, 2003·Cell Communication & Adhesion·Darren LockeAndrew L Harris
Nov 21, 2018·Artificial Cells, Nanomedicine, and Biotechnology·Preeti KourAmit Kumar Goyal
Apr 8, 2006·The Journal of Biological Chemistry·Wafaa A AyadAndrew L Harris
Nov 11, 2006·Molecular Pharmacology·Liang Tao, Andrew L Harris
Jan 28, 1999·The Journal of Biological Chemistry·C G Bevans, A L Harris
Feb 20, 2004·The Journal of Biological Chemistry·Xiaoyong BaoGuillermo A Altenberg
Mar 27, 2004·The Journal of Biological Chemistry·Darren LockeAndrew L Harris

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