Purification of a stilbene sensitive chloride channel and reconstitution of chloride conductivity into phospholipid vesicles.
A protein conferring passive chloride permeability was isolated from a N-octylglucoside solubilized extract of partially purified H(+)-transporting osteoclast cell membranes. Purification was achieved by binding of solubilized protein to an amine-linked 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) Sepharose 4B column and elution with 50 mM KCl. A major protein, with MR = 60 kD on 10% SDS-PAGE, was obtained, which was further purified to homogeneity by HPLC gel filtration. This protein introduced 36Cl- permeability when reconstituted in phospholipid membranes by equilibrium dialysis. The Cl- transport recovered in reconstituted membranes retained sensitivity to DIDS confirming the identity of the isolated protein as a stilbene-sensitive chloride channel.
Blair
H C
HC
Department of Pathology, Jewish Hospital of St. Louis, Missouri.
Schlesinger
P H
PH
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives; D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D002645:Chickens; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005260:Female; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification / Q000502:physiology*; D008854:Microscopy, Electron; D010010:Osteoclasts / Q000502:physiology*; D013267:Stilbenes / Q000494:pharmacology*
D018118:Chloride Channels; D002712:Chlorides; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid