Reconstitution and partial purification of an amiloride-sensitive, cation channel from rabbit kidney.
The aim of the present study was to reconstitute and purify an epithelial potassium channel from rabbit kidney. Renal brush border membrane vesicles (BBMV) were found to contain a potassium conductance which was inhibited by amiloride, 5-(N-methyl-N-isobutyl)amiloride (MIA) and by barium. Membrane vesicle proteins were solubilized and reconstituted in proteoliposomes. Channel activity was assayed using Acridine orange and the voltage sensitive dye, 3,3'-diethylthiadicarbocyanine iodide (DiSC2(5)). Both methods yielded similar results which indicated the presence of an amiloride-sensitive, cation channel in the proteoliposomes. This channel was more permeable to K than to Na and its activity was increased in reconstituted proteoliposomes as compared to native brush border membranes. We conclude that rabbit BBMV possess an amiloride sensitive cation channel. Channel activity was successfully reconstituted in proteoliposomes and the protein was partially purified during reconstitution.
Desir
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Yale University School of Medicine, Department of Medicine, New Haven, CT 06510.
D000165:Acridine Orange; D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D001485:Basement Membrane / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry; D004225:Dithiazanine; D007687:Kidney Tubules, Proximal / Q000737:chemistry*; D008297:Male; D008565:Membrane Proteins / Q000032:analysis*; D015221:Potassium Channels / Q000737:chemistry* / Q000187:drug effects; D011510:Proteolipids / Q000032:analysis; D011817:Rabbits
D008565:Membrane Proteins; D015221:Potassium Channels; D011510:Proteolipids; C020485:proteoliposomes; D004225:Dithiazanine; D000584:Amiloride; D000165:Acridine Orange