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. Ch em. 254, 10161-10166 (1979)). The experiments reported here concern th e purification and identification of this channel which was accomplish ed by solubilization of electric organ plasma membranes and reconstitu tion of the channel into vesicles made of phosphatidylethanolamine, ph osphatidylserine, and cholesterol. Channel activity was measured in th ese vesicles by assaying Cl-36(-) uptake against an outwardly directed chloride chemical gradient as described by Garty et al. (J. Biol. Che m. 258, 13094-13099 (1983)). Maximal uptake occurred by 15 s. Addition of valinomycin after 10 min released intravesicular Cl-36(-) suggesti n that chloride is moving through a channel. Channel activity was inhi bited by DIDS (K-0.5 of 56 mM) and NBD chloride (K-0.5 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 th e presence of chlorotriphenyltin (an anion ionophore), indicating that the Torpedo electric organ is an enriched source as shown by White an d Miller (Biophys. J. 35, 455-462 (1981)). The chloride channel was pu rified approx. 40-fold by sedimentation velocity. In this purified pre paration, 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 it self, but it is labeled by DIDS, an inhibitor of channel activity. Cha nnel activity was approx. 20-fold greater in material that bound to co ncanavalin A compared to the concanavalin A flow-through; all four pol ypeptides were present in the bound materia. It is possible that some of these polypeptides are subunits of the chloride channel.