CONTROL OF MEMBRANE TRANSPORT BY OSMOTIC STRESS

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We measure the internal volume change during opening and closing of ionic transmembrane channels by adding impermeant solutes to both sides of the channel-containing membrane. The extra work of channel opening under osmotic stress is measured as a shift in the current-voltage curve or as a bias in the open/closed statistics of a channel. We are measuring volume changes in the channels mentioned below. We have developed a new lipid bilayer chamber to ensure good mixing of viscous solutions and decreased turbulence leading to improved membrane stability. A microcomputer data analysis system has been adapted for these measurements. The mitochondrial voltage-dependent anion channel (VDAC) inserted into planar lipid bilayers shows a volume change of 20 to 40 thousand cubic angstroms. This is a large change inconsistent with traditional blocking of local gating models but supporting models with major closure of the channel space. In addition, the role of VDAC as an intracellular osmoregulator is suggested. The gap junction is the locus of direct transfer of ions and small molecules from cell to cell. To measure volume changes we reconstitute junctions into planar membranes. Channels with many of the properties of junctional channels were successfully incorporated into planar membranes. A transport-specific purification of vesicles containing channels has been developed to improve the efficiency of the reconstitution. We expect to distinguish between two published models of the gap junction channel based upon our volume-change measurements.