Reconstitution of a kidney chloride channel and its identification by covalent labeling.
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.
Breuer
W
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Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
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
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