Human NHE1 binds full-length calcineurin with high affinity and inhibits substrate dephosphorylation

Calcineurin (Cn) engages with the Na+/H+-exchanger NHE1 via its calcineurin-binding domain (NHE1-CnBD). The manner in which full-length human Cn affects this interaction and its implications has been unclear until now. Researchers utilized fluorescence anisotropy and quantitative size-exclusion chromatography to measure the interaction between full-length human Cn and NHE1-CnBD in various activation states.

Activation of Cn via Ca2+/calmodulin increased the affinity for NHE1-CnBD from a submicromolar range to tens of nanomolar, and stabilized the complex. Notably, NHE1-CnBD displaced the immunosuppressive cyclosporin A/cyclophilin A complex from binding to Cn, and selectively inhibited the dephosphorylation of a phosphopeptide substrate.

However, it enhanced the hydrolysis of p-nitrophenyl phosphate. These results establish NHE1 as a high-affinity, substrate-competitive regulator of calcineurin, inhibiting substrate recognition rather than catalysis. This discovery offers a mechanistic framework for the reciprocal regulation between Cn and NHE1.