Calcium affects CHP1 and CHP2 conformation and their interaction with sodium/proton exchanger 1

Calcineurin B homologous proteins (CHPs) belong to the EF-hand Ca(2+) -binding protein (EFCaBP) family. They have multiple important functions including the regulation of the Na(+) /H(+) exchanger 1 (NHE1). The human isoforms CHP1 and CHP2 share high sequence similarity, but have distinct expression profiles with CHP2 levels for instance increased in malignant cells. These CHPs bind Ca(2+) with high affinity. Biochemical data indicated that Ca(2+) can regulate their functions. Experimental evidence for Ca(2+) -modulated structural changes was lacking. With a newly established fluorescent probe hydrophobicity (FPH) assay, we detected Ca(2+) -induced conformational changes in both CHPs. These changes are in line with an opening of their hydrophobic pocket that binds the CHP-binding region (CBD) of NHE1. Whereas the pocket is closed in the absence of Ca(2+) in CHP2, it is still accessible for the dye in CHP1. Both CHPs interacted with CBD in the presence and absence of Ca(2+) . Isothermal titration calorimetry (ITC) analysis revealed high binding affinity for both CHPs to CBD with equilibrium dissociation constants (KD s) in the nanomolar range. The KD for CHP1:CBD was not affected by Ca(2+) , whereas Ca(2+) -depletion increased the KD 7-fold for CHP2:CBD showing a decreased affinity. The data indicate an isoform specific regulatory interaction of CHP1 and CHP2 with NHE1.