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 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.
Silvestro
A M
AM
Department of Biochemistry, University of Edinburgh, U.K.
Ashley
R H
RH
D000818:Animals; D002540:Cerebral Cortex / Q000378:metabolism* / Q000648:ultrastructure; D018118:Chloride Channels / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D002793:Cholic Acids / Q000737:chemistry; D002794:Choline / Q000737:chemistry / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000502:physiology; D017136:Ion Transport / Q000502:physiology; D008051:Lipid Bilayers / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D012433:Ryanodine / Q000494:pharmacology; D012756:Sheep; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013431:Sulfates / Q000737:chemistry
D018118:Chloride Channels; D002712:Chlorides; D002793:Cholic Acids; D003902:Detergents; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D013431:Sulfates; C057561:1-palmitoyl-2-oleoylphosphatidylethanolamine; D012433:Ryanodine; C050294:1-palmitoyl-2-oleoylglycero-3-phosphoserine; D002794:Choline; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate