PURIFICATION, RECONSTITUTION, AND SUBUNIT COMPOSITION OF A VOLTAGE-GATED CHLORIDE CHANNEL FROM TORPEDO ELECTROPLAX

The voltage-gated Cl- channel from Torpedo electroplax was purified in functional form by an immunoaffinity procedure. Channel activity was assayed by Cl-36(-) uptake into reconstituted liposomes and by direct recording after insertion into planar lipid bilayers. The purified channel displays the same ''double-barreled'' gating kinetics observed with native membranes, as well as the correct single-channel permeation characteristics. Preparations of active channels consist of a 90-kDa polypeptide, as expected from the known cDNA sequence. No associated subunits are present in the purified material. Direct protein sequencing confirms the absence of a cleavable signal sequence and demonstrates an N-terminus at Ser-2 of the cDNA-derived, sequence. This ''ClC-O'' protein is lightly glycosylated, losing only approximate to 2 kDa of sugar upon treatment with endoglycosidase H or N-glycanase. Most if not all of this glycosylation is found on Asn-365. This result necessitates revision of current transmembrane topology proposals, which have placed this residue on the cytoplasmic side of the membrane. Sedimentation in sucrose density gradients under activity-preserving conditions suggests the ClC-O channel is slightly larger than the Na/K-ATPase alpha/beta-protomer (similar or equal to 150 kDa) and substantially smaller than the reduced form bf the nicotinic acetylcholine receptor (similar or equal to 300 kDa). The detergent-solubilized ClC-O channel, which invariably displays two Cl- diffusion pores in the active complex, is therefore built most likely as a homodimer of the 90-kDa protein purified here.