We constructed and expressed human Na+/H+ exchanger (NHE-1 isoform) cDNAs randomly mutagenized within the sequence encoding the transmembrane region of the exchanger. Using acute intracellular acidifications in the presence of the NHE-1 inhibitor amiloride (300 microM), we selected a clone expressing a NHE-1 protein exhibiting a 3.3-fold increase in K(i) for amiloride (10 microM instead of 3 microM). Sequencing its cDNA revealed one point mutation resulting in a Gly174Ser substitution near the carboxy-terminal end of the putative fourth transmembrane domain of NHE-1. The introduction of this mutation into the wild-type NHE-1 cDNA and its expression reproduced the features of the mutant. Site-directed Gly174Ala and Gly174Asp substitutions resulted, respectively, in no change and in an approximately 4-fold decrease in the amiloride affinity. An additional mutation (Leu163Phe) in transmembrane segment four has previously been shown to result in a decreased sensitivity to amiloride and its derivatives. The Leu163Phe/Gly174Ser double mutant possesses a strongly reduced affinity for various inhibitors (17 microM for amiloride, 2 microM for MPA, and 20 microM for HOE694) and also a decreased affinity (28 mM instead of 14 mM) for sodium. Although distant in the transmembrane segment, Leu163 and Gly174 residues are both not hydrogen-bonded, being one helix turn from proline residues, and are therefore located in highly flexible regions of the protein. This flexibility and the availability of free carbonyls may play an important role in the interaction with the inhibitors and transported cations.