A POINT MUTATION OF THE NA+ H+ EXCHANGER GENE (NHE1) AND AMPLIFICATION OF THE MUTATED ALLELE CONFER AMILORIDE RESISTANCE UPON CHRONIC ACIDOSIS/

The diuretic drug amiloride and its 5-amino substitute N5-methyl-N5-pr opylamfloride (MPA) are potent inhibitors of the growth factor-activat able Na+/H+ exchanger isoform 1 (NHE1). This inhibitor competes with N a+, presumably by interacting with the ion-transport site of the NHE m olecule. As an approach to identify this site, we previously reported the use of a specific H+-killing selection technique for isolating ami loride-resistant variants of Chinese hamster lung fibroblasts. After l ong-term selection, two variants, AR40 and AR300, 100- and 1000-fold, respectively, resistant to MPA, were isolated. By comparing NHE1 cDNA sequences of parental and two variant cell lines, we show that the 100 0-fold resistance to MPA results from two sequential genetic events. ( i) In one AR40 allele a point mutation, Phe-167 --> Leu, occurs in the middle of the fourth putative transmembrane segment of NHE1. Producin g this mutant protein from human NHE1 cDNA by site-directed mutagenesi s increased the K(i) for MPA by 30-fold, as seen in AR300 cells. (ii) An -10-fold amplification of the mutated allele, which contributes to the acquired MPA resistance, accounts for the V(max) increase. Mutatin g a close residue, Phe-165 --> Tyr, increased by 40-fold the K(i) for amiloride and reduced Na+ transport rate 3- to 4-fold, indicating that we have identified a critical domain of the NHE molecule that control s amiloride binding and Na+ transport. Interestingly, the epithelial a miloride-resistant NHE isoforms that occurred naturally possess some o f the amino acid substitutions described here.