The Na/H exchanger isoforms NHE1 and NHE3 are regulated differently by various stimuli. Calcium has been recognized as one of the major second messengers in such exchanger regulation. We previously proposed that Ca-induced activation of NHE1 occurs via displacement of its autoinhibitory domain from the H modifier site due to direct binding of Ca/calmodulin. To further validate this hypothesis, the functional role of the cytoplasmic domain was studied in both wild-type and chimeric exchangers, i.e. NHE1, NHE3, NHE1 with the cytoplasmic domain of NHE3(N1N3), and NHE3 with the cytoplasmic domain of NHE1(N3N1). After expression in exchanger-deficient fibroblasts (PS120), early response (<80 s) to external stimuli was assessed as 5-(N-ethyl-N-isopropyl)amiloride-sensitive Na uptake. Among stimuli tested (ionomycin, -thrombin, phorbol ester, hyperosmotic stress, and platelet-derived growth factor) that are all known to activate NHE1, only ionomycin and thrombin induced a significant intracellular Ca mobilization and early activation of Na uptake, implying that Ca is a main regulator of NHE1 in the early phase of the agonist response. However, all the stimuli did not activate NHE3 or N1N3. In contrast, a significant stimulation of Na uptake in response to ionomycin and thrombin was observed in N3N1, accompanied by an alkaline shift of pH sensitivity (0.2 pH units). Deletion of the cytoplasmic calmodulin-binding domain within N3N1 resulted in a constitutive alkaline shift of pHsensitivity and abolished the activation by ionomycin and thrombin. Together, these data reinforce our concept of Ca-induced activation of NHE1. Furthermore, they provide evidence for a functional interaction of the autoinhibitory domain of NHE1 with the H-modifier site of a different isoform, NHE3.

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