Reconstitution, identification, and purification of the Torpedo californica electroplax chloride channel complex.
A voltage-gated chloride channel was identified in the electric organ of the marine ray Torpedo californica by White and Miller (J. Biol. Chem. 254, 10161-10166 (1979)). The experiments reported here concern the purification and identification of this channel which was accomplished by solubilization of electric organ plasma membranes and reconstitution of the channel into vesicles made of phosphatidylethanolamine, phosphatidylserine, and cholesterol. Channel activity was measured in these vesicles by assaying 36Cl- uptake against an outwardly directed chloride chemical gradient as described by Garty et al. (J. Biol. Chem. 258, 13094-13099 (1983)). Maximal uptake occurred by 15 s. Addition of valinomycin after 10 min released intravesicular 36Cl- suggesting that chloride is moving through a channel. Channel activity was inhibited by DIDS (K0.5 of 56 mM) and NBD chloride (K0.5 of 176 mM). In a 40 lipid/1 protein (w/w) reconstitution, approx. 30% of the vesicles contained a functional chloride channel, based upon uptake done in the presence of chlorotriphenyltin (an anion ionophore), indicating that the Torpedo electric organ is an enriched source as shown by White and Miller (Biophys. J. 35, 455-462 (1981)). The chloride channel was purified approx. 40-fold by sedimentation velocity. In this purified preparation, four polypeptides (210, 95, 55, and 40 kDa) were visible by silver-staining after nonreducing SDS-PAGE. Of the four polypeptides, the largest (210 kDa) is not sufficient for Cl- channel activity by itself, but it is labeled by DIDS, an inhibitor of channel activity. Channel activity was approx. 20-fold greater in material that bound to concanavalin A compared to the concanavalin A flow-through; all four polypeptides were present in the bound materia. It is possible that some of these polypeptides are subunits of the chloride channel.
Rosenthal
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Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Guidotti
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D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D009327:4-Chloro-7-nitrobenzofurazan; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D003208:Concanavalin A / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism*; D010455:Peptides / Q000302:isolation & purification; D011868:Radioisotopes; D014101:Torpedo / Q000378:metabolism*
D018118:Chloride Channels; D002712:Chlorides; D010455:Peptides; D011868:Radioisotopes; D003208:Concanavalin A; D009327:4-Chloro-7-nitrobenzofurazan; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid