Solubilization and reconstitution of a chloride transporter from tracheal apical membrane.
Electrogenic Cl- transport in bovine tracheal membrane vesicles was studied by measuring the kinetics of Cl- efflux. Efflux from KCl-loaded vesicles was assayed as the appearance of Cl- in the medium using an anion-specific electrode. Maximal sensitivity was obtained by passage of the vesicles through a Sephadex column immediately before the assay to remove external Cl-. Two components of Cl- transport could be detected. Vesicles exhibited a characteristic spontaneous Cl- efflux that appears to be limited by the cation permeability of the membrane. Addition of the K+-conducting ionophore to increase selectively the permeability to K+ resulted in a stimulation of Cl- efflux. The electrically neutral ionophore nigericin caused only a slight transient increase in Cl- efflux. The valinomycin-dependent flux may be assumed to represent flux through a parallel conductive pathway. Valinomycin-dependent electrogenic Cl- flux was 240 +/- 36 nmol X mg protein-1 X min-1 (n = 4). Soluble extracts obtained with the nonionic detergent octyl glucoside were reconstituted by the freeze-thaw procedure. The reconstituted vesicles exhibited a number of similarities to the native vesicles. Efflux was maximally stimulated with the ionophore valinomycin in the presence of an outwardly directed K+ gradient. Nigericin had no effect on efflux, suggesting the reconstituted transporter is a conductive rather than exchange mechanism. The initial rate of the valinomycin-dependent component was 896 +/- 63 nmol X mg protein-1 X min-1 (n = 5), representing approximately a fourfold increase in specific activity compared with the native membrane vesicle.
D000818:Animals; D000838:Anions; D001692:Biological Transport / Q000187:drug effects; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D002712:Chlorides / Q000378:metabolism*; D004553:Electric Conductivity; D007700:Kinetics; D009550:Nigericin / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D014132:Trachea / Q000378:metabolism*; D014634:Valinomycin / Q000494:pharmacology
D000838:Anions; D002712:Chlorides; D014634:Valinomycin; D009550:Nigericin; D011188:Potassium