CONTROL OF MEMBRANE TRANSPORT BY OSMOTIC STRESS

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To measure the internal volume change during opening and closing of ionic transmembrane channels, we have been subjecting perfused preparations to positive and negative osmotic stress. 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. Suppression and enhancement of potassium channel conductance in the squid giant axon correspond nicely to the response one expects to osmotic stress. The data do not fit a simple blocking model. The channel volume inferred has an upper bound of 1300 cubic angstroms. The mitochondrial voltage-dependent anion channel (VDAC) inserted into planar lipid bilayers shows a volume change of 20 to 40 thousand cubic angstroms. These are large changes inconsistent with traditional blocking or local gating models but supporting models with major closure of the channel space. A microcomputer data analysis system has been further adapted for these measurements. The gap junction is the locus of direct transfer of ions and small molecules from cell to cell. We have (1) incorporated material from isolated gap junctions into vesicles, (2) applied a density shift technique to select vesicles containing large open channels, and (3) incorporated those channels into planar bilayers. A transport-specific purification of vesicles containing channels has been developed to improve the efficiency of the reconstitution. Predominantly three sizes of conductance changes were seen. They were 20-30, 60-70, and 110-130 pS, each having different degrees of voltage sensitivity, asymmetry, and kinetics. Membranes containing several channels were anion selective.