Article: article from journal or magazin.
The cellular pool of Na+ channels in the amphibian cell line A6 is not altered by mineralocorticoids. Analysis using a new photoactive amiloride analog in combination with anti-amiloride antibodies.
Journal of Biological Chemistry
An amiloride-sensitive Na+ channel is found in the apical plasma membrane of high resistance, Na+ transporting epithelia. We have developed a method for the identification of this channel based on the use of a new high affinity photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), and anti-amiloride antibodies to identify photolabeled polypeptides. NMBA specifically labels the putative Na+ channel in bovine kidney microsomes. A 130-kDa polypeptide is detected on immunoblots with anti-amiloride antibodies. NMBA is a potent inhibitor of Na+ transport in the established amphibian kidney epithelial cell line A6, and specifically labels a 130-kDa polypeptide. We utilized both NMBA photolabeling and [3H]benzamil binding in order to examine the cellular pool of putative channels following hormonal regulation of Na+ transport. This pool is not significantly altered by the mineralocorticoid agonist aldosterone or antagonist spironolactone, despite a 3.8-fold difference in transepithelial Na+ transport.
Affinity Labels, Aldosterone/metabolism, Amiloride/analogs & derivatives, Amiloride/immunology, Animals, Blotting, Western, Cattle, Cell Line, Kidney/metabolism, Microsomes/metabolism, Mineralocorticoids/pharmacology, Photochemistry, Sodium Channels/drug effects, Sodium Channels/metabolism, Spectrophotometry, Ultraviolet, Spironolactone/metabolism, Xenopus laevis
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