Solubilization and functional reconstitution of a chloride channel from Torpedo californica electroplax.
Chloride channels were detergent-extracted from Torpedo electroplax plasma membrane vesicles and reconstituted into liposomes by rapid detergent removal and a freeze-thaw-sonication procedure. Concentrative uptake of 36Cl-, driven by a Cl- gradient was used to determine conductance properties of reconstituted channels. Chloride flux assayed by this method is strongly selective for Cl- over cations, is blocked by SCN-, inactivated by treatment with DIDS, and exhibits an anion selectivity sequence Cl- greater than Br- greater than F- greater than SO4(2-), as does the voltage-gated Cl- channel from Torpedo observed in planar lipid bilayers. The channels are localized to the noninnervated face of the electrocyte, and a novel trapped-volume method is used to estimate a channel density on the order of 500 pmol/mg protein. An initial fractionation of the membrane extract by anion exchange chromatography yields fivefold enrichment of the channel activity.
Goldberg
A F
AF
Howard Hughes Medical Institute, Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts.
Miller
C
C
D000818:Animals; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D014101:Torpedo
D018118:Chloride Channels; D002712:Chlorides; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins