Transport-specific isolation of large channels reconstituted into lipid vesicles

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). Thus, vesicles containing open channels were separable from those that did not by a transport-specific shift in density. This technique may be useful for the enrichment of channels of known permeability properties from impure material.