Characterization of the rat Na+/H+ exchanger isoform NHE4 and localization in rat hippocampus

The kinetics of the Na+/H+ exchanger (NHE) isoform NHE4 were studied by measuring Na-22(+) fluxes in stably transfected NHE-deficient fibroblasts. Unlike NHE1, NHE2, and NHE3, activation of this isoform is dependent on hyperosmolarity-induced cell shrinkage. It is virtually inactive at isosmolarity and most active at 490 mosM. When induced by cell shrinkage, NHE4 exhibits a sigmoidal response to increasing extracellular Na+ concentrations, suggesting allosteric or cooperative binding kinetics. In comparison, NHE1 and -3 exhibit hyperbolic velocity vs. extracellular Na+ concentration responses at both iso- and hyperosmolar conditions. Unlike NHE1 and NHE4, hyperosmolarity-induced cell shrinkage inhibits NHE3 activity in transfected fibroblasts, reducing maximum velocity by 40%, with no effect on binding affinity to extracellular Na+. NHE4 is relatively insensitive to inhibition by amiloride analogues in the order 5-(N,N-dimethyl)amiloride > 5-(N,N-hexamethylene)amiloride > amiloride > 5-(N-ethyl-N-isopropyl)amiloride. Time-dependent inhibition of activity by cytochalasin D suggests a relationship between the actin cytoskeleton and regulation by cell shrinkage. By in situ hybridization of fixed tissues, NHE4 mRNA was found to be highly expressed in the cavi amnoni fields of rat hippocampus. The kinetics of this exchanger, when considered with its unusual tissue distribution in renal inner medullary collecting tubules and hippocampus, are consistent with NHE4 having a specialized role in cell functions.