The Na + /H + exchanger NHE1 is critical for cell vitality as it controls intracellular pH and cell volume. Its functionality is influenced by calcineurin B homologous proteins (CHPs). The human isoform CHP3 is important for transport of NHE1 to the plasma membrane and for its activity. Here, we characterized the binding interaction of human CHP3 with the regulatory domain of NHE1. The exact binding site of CHP3 was previously debated. CHP3 as well as both regions of NHE1 in question were

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... ed and purified. CHP3 specifically formed stable complexes with the CHP-binding region (CBD) of NHE1 (residues 503-545) in size-exclusion chromatography (SEC), but not with the C-terminal region (CTD, residues 633-815). CTD was functional as shown by Ca 2+ -dependent binding of calmodulin in SEC analysis. CHP3 bound with high affinity to CBD with an equilibrium dissociation constant (K D ) of 56 nM determined by microscale thermophoresis. The high affinity was substantiated by isothermal calorimetry analysis (K D = 3 nM), which also revealed that the interaction with CBD is strongly exothermic (ΔG° = −48.6 kJ/mol, ΔH = −75.3 kJ/mol, −TΔS° = 26.7 kJ/mol). The data provide insights in the molecular mechanisms that underlie the regulatory interaction of CHP3 and NHE1 and more general of calcineurin homologous proteins with their target proteins. A constant intracellular pH is critical for cell vitality and is thus tightly controlled in living cells 1 . Important for this fundamental cellular process is the interaction of the sodium/proton exchanger NHE1 with calcineurin B homologous proteins (CHPs) of which three isoforms exist. NHE1 is embedded in the cytoplasmic membrane and transports excess protons in exchange to sodium ions into the extracellular space 2-4 . It is a secondary active transport which is driven by the inward sodium ion gradient generated by Na + /K + ATPase activity. NHE1 is ubiquitous in human cells. The mature protein consists of an N-terminal transmembrane domain that facilitates the ion exchange and a cytoplasmic C-terminal domain which acts as a hub for regulatory protein-protein interactions and posttranslational modifications 5,6 . The astonishing regulatory network reflects the central role of NHE1 to maintain cell vitality upon changes in the metabolic and developmental cell state and in the cellular environment. The integrative control of NHE1 activity is essential for cell proliferation and differentiation. NHE1 malfunction is implicated in a variety of pathophysiological conditions. Enhanced NHE1 activity for instance triggers cell death during episodes of ischemia-reperfusion in cardiac and neural tissues and has been linked to cardiac hypertrophy and heart failure 7 . Inherited loss-of-function mutations of NHE1 result in the Lichtenstein-Knorr syndrome, a recessive neurologic disorder characterized by cerebellar ataxia and severe sensorineural hearing loss 8 . The calcineurin B homologous proteins CHP1, CHP2, and CHP3 are important both for trafficking of NHE1 to the plasma membrane and for influencing NHE1 half-life and transport activity 9 . They belong to the large class of calcium-ion binding EF-hand proteins. The functional importance of their interaction with NHE1 is stressed