SOLUBILIZATION, PARTIAL-PURIFICATION AND FUNCTIONAL RECONSTITUTION OF A SHEEP BRAIN ENDOPLASMIC-RETICULUM ANION CHANNEL

1. An intracellular anion channel, known to be co-localized in brain endoplsmic 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 similar to 20 pS in symmetric 225 mM (choline)(2)SO4 (the solutions also contained 10 mM Tris-HCl, pH 7.4), discriminated poorly between Cl- and choline(+) (relative permeability ratio, P-Cl-/P-choline+, 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 similar to 60% in the presence of SO42-, was assayed by measuring the efflux of entrapped Cl-36(-) (compared to the efflux of [H-3]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.