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 36Cl- 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-0" protein is lightly glycosylated, losing only approximately 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-0 channel is slightly larger than the Na/K-ATPase alpha/beta-protomer (approximately equal to 150 kDa) and substantially smaller than the reduced form of the nicotinic acetylcholine receptor (approximately equal to 300 kDa). The detergent-solubilized ClC-0 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.
Middleton
R E
RE
Howard Hughes Medical Institute, Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Pheasant
D J
DJ
Miller
C
C
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D018118:Chloride Channels / Q000276:immunology / Q000302:isolation & purification*; D007163:Immunosorbent Techniques; D008051:Lipid Bilayers / Q000737:chemistry; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D017421:Sequence Analysis; D014101:Torpedo
D000906:Antibodies; D018118:Chloride Channels; D008051:Lipid Bilayers; D010455:Peptides