Enzymatic and thermodynamic analysis of calcineurin inhibition by RCAN1

Calcineurin (CN) is the target of the immunophilin-immunosuppressant complex, cyclophilin/cyclosporin A (CyP/CsA). RCAN1 has recently been shown to be an endogenous regulator of CN activity. We determined the enzymatic and thermodynamic aspects of CN inhibition by RCAN1. The IC50 values of isoforms RCAN1-1L and RCAN1-4 for CN were 2.7 μM and 2.6 μM, respectively. Two deletions in the CN catalytic subunit, one a deletion of Val314 in the Loop7 domain (ΔV314) and the other in the autoinhibitory domain (CNAabc), increased the sensitivity of CN to inhibition by RCAN1-1L. The IC50s of RCAN1-1L and RCAN1-4 for CN in homogenates of mouse brain were 141 nM and 100 nM, respectively. Using isothermal titration calorimetry (ITC), we found that the RCAN1-1L/CN or CyP/CsA/CN interactions were exothermic with a dissociation constant of 0.46 μM or 0.17 μM, respectively. Our ITC results show that the interactions between CN and its two inhibitors were both characterized by a favorable binding enthalpy change. We also confirmed that overexpression of RCAN1-1L could inhibit the transcriptional activation of an NFAT-dependent promoter in response to PMA and ionomycin by inhibiting CN activity in HEK293T cells. Our data should contribute to our understanding of the regulation of CN activity by endogenous inhibitors.