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 microM cariporide, 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 microM) and extracellular sodium (248 +/- 63.7 mM). This mutated exchanger displays a Phe162Ser substitution in its fourth transmembrane segment. Using intracellular acidifications in the presence of 3 mM external sodium on the CR5 fibroblasts, we isolated two revertants which exhibited a complete recovery for sodium affinity but were still resistant to cariporide. Sequencing the cDNAs encoding these revertants revealed the presence of two mutations situated at a distant location from Phe162 in the same fourth transmembrane segment (Ile169Ser and Ile170Thr). Interestingly, introducing these two mutations in the wild-type cDNA did not result in a significant increase in affinity for sodium. Furthermore, all the mutants characterized in this study display an unchanged affinity for lithium, another transported cation. These data suggest that the mutation resulting in the low sodium affinity and the 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 geometrical integrity of a highly constrained sodium coordination site.