Second-site revertants of a low-sodium-affinity mutant of the Na+/H+ exchanger reveal the participation of TM4 into a highly constrained sodium-binding site

On the basis of intracellular acidifications in the presence of 30,muM cari poride, we selected a fibroblast cell line termed CR5, expressing a mutated Na+/H+ exchanger NHE-1 with a low affinity for cariporide (87 +/- 11.6 muM ) and extracellular sodium (248 +/- 63.7 mM). This mutated exchanger displa ys a Phe162Ser substitution in its fourth transmembrane segment. Using intr acellular acidifications in the presence of 3 mM external sodium on the CR5 fibroblasts, we isolated two revertants which exhibited a complete recover y for sodium affinity but were still resistant to cariporide. Sequencing th e cDNAs encoding these revertants revealed the presence of two mutations si tuated at a distant location from Phe162 in the same fourth transmembrane s egment (Ile169Ser and Ile170Thr). Interestingly, introducing these two muta tions in the wild-type cDNA did not result in a significant increase in aff inity for sodium. Furthermore, all the mutants characterized in this study display an unchanged affinity for lithium, another transported cation. Thes e data suggest that the mutation resulting in the low sodium affinity and t he two mutations responsible for the reversion of this phenotype affect the binding of sodium itself instead of the conformational changes triggering substrate translocation. Taken together, these results allow us to propose that optimal sodium binding by the Na+/H+ exchangers requires the geometric al integrity of a highly constrained sodium coordination site.