Methyl isobutyl amiloride: A new probe to assess the number of NaH antiporters

We measured the binding of [ 3H]-5-(N-methyl-N-isobutyl) amiloride (MIA) to purified rabbit renal brush border membranes. MIA binding was protein, temperature and time dependent with optimal binding at pH 8.0 or above. At low pH MIA binding was inhibited, suggesting competition between H + ions and MIA for the MIA binding site. There was 70–80% specific binding which reached a plateau at 30 min and remained stable thereafter for 150 min. Scatchard analysis revealed one family of binding sites with Bmax of 3.4 ± 0.4 pmoles/mg protein and Kd of 30.5 ± 2.3 nM. MIA inhibited the Vmax of the NaH antiporter (assessed by acridine orange quenching) in a dose dependent fashion with 100% inhibition at MIA concentration of 10 −3 M and this inhibition was greater than that of amiloride. We conclude that MIA, a potent inhibitor of the NaH antiporter, displays a high percentage of specific binding to renal brush border membranes and can be used to assess the number of the NaH antiporters.