Purification and subunit structure of the [3H]phenamil receptor associated with the renal apical Na+ channel.
Sodium crosses the apical membrane of tight epithelia through a sodium channel, which is inhibited by the diuretic amiloride and by analogs such as phenamil. Target size analysis indicated that the functional size of the [3H]phenamil binding sites associated with the epithelial Na+ channel from pig kidney is 92 +/- 10 kDa. The [3H]phenamil receptor was solubilized by using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized material displayed the same properties of interaction with amiloride and its derivatives as the membrane-bound receptor. A two-step purification of the epithelial Na+ channel was achieved by using QAE Sephadex chromatography and affinity chromatography on a Bandeiraea simplicifolia lectin column. It results in an 1100-fold purification of the Na+ channel as compared to pig kidney microsomes with a yield of 15% +/- 5%. The maximal specific activity was 3.7 nmol/mg of protein. NaDodSO4/poly-acrylamide gel electrophoresis of the purified Na+ channel under nonreducing conditions showed the presence of a single major polypeptide chain of apparent molecular mass 185 kDa. Under disulfide-reducing conditions, the purified epithelial Na+ channel migrated as a single band of apparent molecular mass 105 kDa. It is suggested that the epithelial Na+ channel from pig kidney has a total molecular mass of 185 kDa and consists of two nearly identical 90- to 105-kDa polypeptide chains crosslinked by disulfide bridges.
D000584:Amiloride / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000032:analysis; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004848:Epithelium / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D007684:Kidney Tubules / Q000378:metabolism*; D007686:Kidney Tubules, Distal / Q000378:metabolism*; D008970:Molecular Weight; D011955:Receptors, Drug / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D015222:Sodium Channels; D013552:Swine
D007473:Ion Channels; D011955:Receptors, Drug; D015222:Sodium Channels; C047088:phenylamil; D000584:Amiloride