Solubilization, partial purification and functional reconstitution of a sheep brain endoplasmic reticulum anion...

1. An intracellular anion channel, known to be co-localized in brain endoplasmic reticulum membranes with ryanodine-sensitive calcium-release channels, was incorporated into voltage-clamped planar lipid bilayers from sheep brain microsomal membrane vesicles. 2. Single channels, which displayed a main open-state conductance of 80-100 pS in symmetric 450 mM choline Cl, reduced to approximately 20 pS in symmetric 225 mM (choline)2SO4 (the solutions also contained 10 mM Tris-HCl, pH 7.4), discriminated poorly between Cl- and choline+ (relative permeability ratio, PCl-/Pcholine+, 2.5). 3. Sheep brain microsomal membrane proteins were solubilized in the zwitterionic detergent CHAPS, and subjected to sequential anion-exchange and size-exclusion chromatography; the solubilizate, and partially-purified protein fractions, were then incorporated into large unilamellar liposomes by freeze-thaw sonication. 4. Reconstituted passive anion (Cl-)-transport, which was reduced by approximately 60% in the presence of SO4(2-), was assayed by measuring the efflux of entrapped 36Cl- (compared to the efflux of [3H]inulin), and also by monitoring the fluorescence quenching of entrapped SPQ by Cl(-)-influx. 5. Cl(-)-transporting activity was enriched up to 200-fold after two stages of purification, and the partially-purified channel protein was incorporated from reconstituted proteoliposomes into planar lipid bilayers, where its permeation behaviour remained very similar to that observed for the native channel.