Molecular cloning of NHE1 from winter flounder RBCs: activation by osmotic shrinkage,cAMP, and calyculin A

In this report, wedescribe the cloning, cellular localization, and functionalcharacteristics of Na⁺/H⁺ exchanger 1 (NHE1)from red blood cells of the winter flounder Pseudopleuronectesamericanus (paNHE1). The paNHE1 protein localizes primarily to themarginal band and exhibits a 74% similarity to the trout -NHE, and65% to the human NHE1 (hNHE1). Functionally, paNHE1 sharescharacteristics of both -NHE and hNHE1 in that it is activated bothby manipulations that increase cAMP and by cell shrinkage,respectively. In accordance, the paNHE1 protein exhibits both proteinkinase A consensus sites as in -NHE and a region of high homology tothat required for shrinkage-dependent activation of hNHE1. Aftershrinkage-dependent activation of paNHE1 and resulting activation of aCl/HCO exchanger, their paralleloperation results in net uptake of NaCl and osmotically obliged water.Activation of paNHE1 by cAMP is at least additive to that elicited byosmotic shrinkage, suggesting that these stimuli regulate paNHE1 bydistinct mechanisms. Finally, exposure to the serine/threoninephosphatase inhibitor calyculin A potently activates paNHE1, and thisactivation is also additive to that induced by shrinkage or cAMP.