Utilization of amiloride analogs for characterization and labeling of the plasma membrane Na+/H+ antiporter from Dunaliella salina

The interactions of amiloride analogs with the Na+/H+ antiporter from plasma membrane of the halotolerant alga Dunaliella salina [Katz et al. (1989) Biochem. Biophys. Acta 983, 9-14] have been investigated. Analogs bearing hydrophobic substitutions at the guanidino moiety of amiloride, such as benzamil, are the most effective inhibitors of Na+ uptake in plasma membrane vesicles, whereas substituents of the 5-amino group are less effective inhibitors than amiloride. This order of specificity is opposite to that found for most Na+/H+ antiporters. The photoaffinity amiloride analog 2'-methoxy-5'-nitrobenzamil (NMBA), a competitive inhibitor with respect to Na+ with Ki = 10 microM, photolabels upon illumination two polypeptides of apparent MW 30 and 50 kDa in purified plasma membrane vesicles. Similar labeling is obtained by immunodetection with antiamiloride antibodies and by incorporation of [125I]NMBA. The specificity of the labeling was ascertained by competition with benzamil. Plasma membrane preparations from high-salt or ammonia-adapted cells, which have higher Na+/H+ antiporter activity [Katz et al. (1992) Plant Physiol. 100, 1224-1229], also show increased incorporation of NMBA into the 30- and 50-kDa polypeptides. It is suggested that: (1) the structure of the Na+ binding site of the D. salina Na+/H+ antiporter differs from that of most Na+/H+ antiporters and (2) the 50- and/or 30-kDa polypeptides are subunits of the plasma membrane antiporter of this alga.