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

Abstract

• 1.

  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.

  2. Single channels, which displayed a main open-state conductance of 80–100 pS in symmetric 450 mM choline Cl, reduced to ~20pS 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_chlorine+, 2.5).

• 3.

  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.

  4. Reconstituted passive anion (Cl₋)-transport, which was reduced by ~60% in the presence of SO₄²⁻, 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.

  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.