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.