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.
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.
Kleyman
T R
TR
Department of Medicine, Columbia University, New York 10032.
Cragoe
E J
EJ
Kraehenbuhl
J P
JP
D000345:Affinity Labels; D000450:Aldosterone / Q000378:metabolism; D000584:Amiloride / Q000031:analogs & derivatives* / Q000276:immunology; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D007668:Kidney / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008901:Mineralocorticoids / Q000494:pharmacology*; D010777:Photochemistry; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013148:Spironolactone / Q000378:metabolism; D014982:Xenopus laevis
D000345:Affinity Labels; D008901:Mineralocorticoids; D015222:Sodium Channels; D013148:Spironolactone; D000450:Aldosterone; D000584:Amiloride