title: Reconstitution of a kidney chloride channel and its identification by covalent labeling.
abstract: The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is characterized by its high content of Na+/K(+)-ATPase and a Cl- conductance, which function in parallel in salt reabsorption. In order to reconstitute the Cl- channels, TALH membrane vesicles were solubilized in 1% sodium cholate in buffer containing 200 mM KCl, followed by dilution with soybean lipids (final ratio of protein/detergent/lipid of 1:3:15 in mg) and removal of the detergent by gel filtration on Sephadex G-50. Cl- channel activity in the liposomes was determined by a 36Cl- uptake assay where the accumulation of the radioactive tracer against its chemical gradient is driven by the membrane potential (positive inside) generated by an outward Cl- gradient. The 36Cl- uptake by the KCl-loaded liposomes was dependent on the inclusion of membrane protein and was abolished by valinomycin, indicating the involvement of a conductive pathway. It was also inhibited by 36% by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Solubilization of the Cl- channels in cholate was optimal in the presence of 200 mm KCl, but was found to decrease markedly at low ionic strength. SDS-PAGE analysis of the proteins extracted by cholate at high and low salt concentrations showed that the Cl- channel-containing high KCl extract was enriched in the 96 and 55 kDa alpha- and beta-subunits of the Na+/K(+)-ATPase (the major proteins in the membrane preparation) and several minor protein bands. Treatment of the membrane vesicles with the radioactive analogue of DIDS, [3H]2DIDS, labeled primarily a 65 and a 31 kDa protein. The solubilization of the 31 kDa protein by cholate depended markedly on the ionic strength and thus paralleled the solubilization pattern of Cl- channel activity. Furthermore, the labeling of the 31 kDa protein was prevented by nonradioactive DIDS and by NPPB but not by other compounds, indicating that it may be a Cl- channel component.
authors.affiliation: Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: W
authors.initials: W
authors.lastname: Breuer
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D002713:Chlorine / Q000378:metabolism; D002850:Chromatography, Gel; D007684:Kidney Tubules / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D008138:Loop of Henle / Q000378:metabolism*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism*; D009579:Nitrobenzoates / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011868:Radioisotopes; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D012995:Solubility; D013552:Swine; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D018118:Chloride Channels; D002712:Chlorides; D008081:Liposomes; D008565:Membrane Proteins; D009579:Nitrobenzoates; D011868:Radioisotopes; D014634:Valinomycin; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D002713:Chlorine; D011189:Potassium Chloride; D000254:Sodium-Potassium-Exchanging ATPase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid