Thermodynamics and Kinetics of the Interactions of Calmodulin with Orai

Abstract

Calcium dependent inactivation (CDI) is a key regulator of calcium release‐activated channels. Calmodulin (CaM) has been shown to interact with Orai1, evoking CDI. Though the calmodulin‐binding sequence (CBS) of Orai has been identified, the mechanism is not clear. Here we generated recombinant CaM and characterized its interaction with a peptide corresponding Orai CBS (Orai‐Pep) using isothermal titration calorimetry (ITC) and fluorescence. The peptide binding to CaM increases the extent of hydrophobicity as indicated by the increase of ANS fluorescence. ITC revealed that such a binding is moderate with Ka= 9.5 × 105M−1 and the reaction is exothermic with ΔH = −15.8 kJ/mol. To better define how CaM promotes CDI, we focused on the interaction of CaM and troponin C (TnC) chimeras with Orai‐Pep. 1TnC, in which the 1st EF‐hand of CaM is replaced with the corresponding EF‐hand of TnC, and 2TnC have one‐order lower binding affinity while 3TnC and 4TnC show similar binding strength to that of CaM. This data indicates that the 1st and 2nd EF‐hands of CaM are more important for binding, possibly through their interaction with Trp76 of Orai, in which its accessibility to acrylamide and KI decreases in the complex of 1TnC and 2TnC. Currently we are investigating the kinetics and heat capacity difference of CaM and chimeras upon peptide binding. This work is supported in part by National Science Foundation.