Functional reconstitution of a chloride channel protein from bovine trachea.
We characterized the electrophysiological properties of a chloride channel protein isolated from bovine trachea after incorporation into planar lipid bilayers, and studied the effects of thiol-modulating agents on channel regulation both in bilayers and vesicular iodide uptake studies. Our experiments showed that this protein formed perfectly anion-selective channels in the bilayer, with an anion permeability sequence of I- (2.1) > NO3- (1.7) > Br- (1.2) > Cl- (1.0). The conductance of this channel was 25-30 picosiemens in 150 mM Cl-, and saturated with increasing chloride concentration. This channel could be completely inhibited by 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate. Immunoblot analysis, using polyclonal antibodies (anti-p38), revealed one major band at 140 kDa. Upon reduction with dithiothreitol, 64- and 38-kDa polypeptides were observed. Functional experiments showed that reduction was accompanied by loss of 125I- uptake and single-channel activity. In the presence of dithiothreitol, only the low molecular mass protein forms (64 and 38 kDa) were detected by anti-p38 antibodies on Western blots. Cross-linking of S-S bonds with Cu(2+)-o-phenanthroline led to activation of chloride channels in vesicles and bilayers. Over-aggregation of chloride channels by this S-S cross-linking reagent caused inhibition of 125I- uptake by 80-100% and the abolishment of single-channel activity. We propose that the native chloride channel from bovine trachea can exist in vivo in different structural and functional forms depending upon its thiol-disulfide oxidation reduction status. The oxidized form has a molecular mass of 140 kDa and represents a fully active chloride channel. Inactivation of this channel might occur by over-aggregation of protein subunits, or by dissociation of the 140-kDa subunit by disulfide bond reduction.
Ran
S
S
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
Fuller
C M
CM
Arrate
M P
MP
Latorre
R
R
Benos
D J
DJ
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D018118:Chloride Channels; D004229:Dithiothreitol / Q000494:pharmacology; D004553:Electric Conductivity; D004591:Electrophoresis, Polyacrylamide Gel; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*; D009130:Muscle, Smooth / Q000502:physiology; D011510:Proteolipids / Q000378:metabolism; D013997:Time Factors; D014132:Trachea / Q000502:physiology*
D018118:Chloride Channels; D007473:Ion Channels; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011510:Proteolipids; C020485:proteoliposomes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D004229:Dithiothreitol