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-propylamiloride (MPA) are potent inhibitors of the growth factor-activatable Na+/H+ exchanger isoform 1 (NHE1). This inhibitor competes with Na+, presumably by interacting with the ion-transport site of the NHE molecule. As an approach to identify this site, we previously reported the use of a specific H(+)-killing selection technique for isolating amiloride-resistant variants of Chinese hamster lung fibroblasts. After long-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 1000-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. Producing this mutant protein from human NHE1 cDNA by site-directed mutagenesis increased the Ki for MPA by 30-fold, as seen in AR300 cells. (ii) An approximately 10-fold amplification of the mutated allele, which contributes to the acquired MPA resistance, accounts for the Vmax increase. Mutating a close residue, Phe-165--> Tyr, increased by 40-fold the Ki for amiloride and reduced Na+ transport rate 3- to 4-fold, indicating that we have identified a critical domain of the NHE molecule that controls amiloride binding and Na+ transport. Interestingly, the epithelial amiloride-resistant NHE isoforms that occurred naturally possess some of the amino acid substitutions described here.
Centre de Biochimie-Centre National de la Recherche Scientifique, Université de Nice-Sophia Antipolis, France.
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L
Counillon
A
A
Franchi
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J
Pouysségur
D000483:Alleles; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors; D015183:Restriction Mapping; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D012964:Sodium