AMILORIDE AND HARMALINE ARE POTENT INHIBITORS OF NHAB, A NA+ H+ ANTIPORTER FROM ESCHERICHIA-COLI/

The diuretic drug amiloride is a specific inhibitor of sodium transpor ting proteins in several cell types, Attempts to inhibit this activity in membrane vesicles derived from various bacteria, did not yield cle ar results, Therefore, we tested the effect of amiloride and its deriv atives on the purified Na+/H+ antiporters of E. coli reconstituted in functional form in proteoliposomes. Whereas NhaA is not inhibited by a miloride, both amiloride and harmaline are potent inhibitors of NhaB w ith K-0.5 of 6 and 15 mu M, respectively. The pattern of inhibition by amiloride derivatives is different from that reported for mammalian a ntiporters but similar to that reported for the Na+/H+ antiporter of D . salina [Katz, A., Kleyman, T.R. and Pick, U. (1994) Biochemistry 33, 2389-2393]. Clonidine is a poor inhibitor (K-0.5 = 200 mu M) while ci metidine had no effect on the antiporter up to concentration of 1 mM. These new potent inhibitors provide us with important tools for the st udy of the mechanism of action of NhaB.