Kleyman T R, Cragoe E J, Kraehenbuhl J P
Department of Medicine, Columbia University, New York 10032.
J Biol Chem. 1989 Jul 15;264(20):11995-2000.
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.
在高电阻、转运钠离子的上皮细胞顶端质膜中发现了一种对氨氯吡咪敏感的钠离子通道。我们开发了一种鉴定该通道的方法，该方法基于使用一种新的高亲和力光反应性氨氯吡咪类似物2'-甲氧基-5'-硝基苯甲酰胺（NMBA）以及抗氨氯吡咪抗体来鉴定光标记的多肽。NMBA特异性标记牛肾微粒体中的假定钠离子通道。在用抗氨氯吡咪抗体进行的免疫印迹中检测到一条130 kDa的多肽。NMBA是已建立的两栖类肾上皮细胞系A6中钠离子转运的有效抑制剂，并特异性标记一条130 kDa的多肽。我们利用NMBA光标记和[3H]苯甲酰胺结合来研究钠转运激素调节后假定通道的细胞池。尽管跨上皮钠离子转运相差3.8倍，但盐皮质激素激动剂醛固酮或拮抗剂螺内酯对这个细胞池没有显著影响。
