SEA0400 is a potent and selective
Na(+)/Ca(2+) exchanger (NCX) inhibitor. We evaluated the inhibitory effects of
SEA0400 on Na(+)(i)-dependent (45)Ca(2+) uptake and whole-cell Na(+)/Ca(2+) exchange currents in NCX-transfected fibroblasts.
SEA0400 preferentially inhibited (45)Ca(2+) uptake by NCX1 compared with inhibitions by NCX2, NCX3, and NCKX2.
SEA0400 also selectively blocked outward exchange currents from NCX1 transfectants. We searched for regions that may form the
SEA0400 receptor in the NCX1 molecule by NCX1/NCX3 chimeric analysis. The results suggest that the first intracellular loop and the fifth transmembrane segment are mostly responsible for the differential
drug responses between NCX1 and NCX3. Further site-directed mutagenesis revealed that multiple mutations at Phe-213 markedly reduced sensitivity to
SEA0400 without affecting that to
KB-R7943. We also found that Gly-833-to-Cys mutation (within the alpha-2 repeat) greatly reduced the inhibition by
SEA0400, but unexpectedly the NCX1 chimera with an alpha-2 repeat from NCKX2 possessed normal
drug sensitivity. In addition, exchangers with mutated
exchanger inhibitory peptide regions, which display either undetectable or accelerated Na(+)-dependent inactivation, had a markedly reduced sensitivity or
hypersensitivity to
SEA0400, respectively. To verify the efficacy of the NCX inhibitor, we examined the renoprotective effect of
SEA0400 in a hypoxic injury model using porcine renal tubular cells.
SEA0400 protected against
hypoxia/reoxygenation-induced cell damage in tubular cells expressing wild-type NCX1 but not in cells expressing SEA0400-insensitive mutants. These results suggest that Phe-213, Gly-833, and residues that eliminate Na(+)-dependent inactivation are critical determinants for the inhibition by
SEA0400, and their mutants are very useful for checking the pharmacological importance of NCX inhibition by
SEA0400.