RANDOM MUTAGENESIS REVEALS A NOVEL SITE INVOLVED IN INHIBITOR INTERACTION WITHIN THE 4TH TRANSMEMBRANE SEGMENT OF THE NA+ H+ EXCHANGER-1/

We constructed and expressed human Na+/H+ exchanger (NHE-1 isoform) cD NAs randomly mutagenized within the sequence encoding the transmembran e region of the exchanger. Using acute intracellular acidifications in the presence of the NHE-1 inhibitor amiloride (300 mu M), we selected a clone expressing a NHE-1 protein exhibiting a 3.3-fold increase in K-i for amiloride (10 mu M instead of 3 mu M). Sequencing its cDNA rev ealed one point mutation resulting in a Gly174Ser substitution near th e 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-direct ed Gly174Ala and Gly174Asp substitutions resulted, respectively, in no change and in an approximately 4-fold decrease in the amiloride affin ity. An additional mutation (Leu163Phe) in transmembrane segment four has previously been shown to result in a decreased sensitivity to amil oride and its derivatives. The Leu163Phe/Gly174Ser double mutant posse sses a strongly reduced affinity for various inhibitors (17 mu M for a miloride, 2 mu M for MPA, and 20 mu M 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 hydrog en-bonded, being one helix turn from proline residues, and are therefo re 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.