51 / 2021-08-05 20:14:44

Decreased binding of calmodulin to cardiac CaV1.2 channel in the presence of Calmidazolium and Chlorpromazine

Calmodulin; Calmodulin antagonist; Ca2+ concentration; CaV1.2 channnel; Cardiovascular diseases

Calmidazolium (CMZ) and Chlorpromazine (CPZ) are calmodulin (CaM) antagonists that prevent CaM from binding to target proteins by interacting with CaM. CaM/Ca²⁺ regulate many physiological and biochemical process in the body, including regulating the activity and function of ion channels. The complex participate in the process of CDI and CDF feedback mechanism of cardiac Ca_V1.2 channel by binding to the C-terminal of channel and regulate the opening and closing of calcium ion channels and maintain intracellular Ca²⁺ homeostasis. In this study, the change of CMZ and CPZ on the binding of CaM to cardiac Ca_V1.2 channel CT1 protein motif under different Ca²⁺ concentrations and the mechanism of CMZ and CPZ regulating intracellular Ca²⁺ concentration were explored. It provides a certain theoretical ideas for elucidation of the potential molecular mechanism of the pathophysiologic process of related diseases. We examined the binding of CaM to CT1 in the presence of CMZ and CPZ by GST pull-down assay method. The experiments showed that the CaM still have the ability to bind with CT1 at different Ca²⁺concentration (≈free, 100 nM, 10 μM, 2 mM). The binding of CaM to CT1 (in the presence of CMZ and CPZ) were reduced compared with CaM (without CMZ and CPZ) with protein-concentration dependent manner and Ca²⁺-concentration dependent manner. The maximal binding (B_max) estimated of CaM to CT1 without CMZ and CPZ was 0.3937 in 2 mM Ca²⁺. The B_max estimated of CaM to CT1 in the presence of CMZ and CPZ was 0.3850 and 0.1762 in 2 mM Ca²⁺. Meanwhile, the B_max of CaM to CT1 in the presence of CMZ and CPZ were decreased in ≈free, 100 nM, 10 μM Ca²⁺ and the K_d value of CaM to CT1 in the presence of CMZ and CPZ were increased in ≈free, 100 nM, 10 μM Ca²⁺. The date imply that the CMZ and CPZ disrupt the binding of CaM to cardiac Ca_V1.2 channel and reduce the amount of combination with cardiac Ca_V1.2 channel C-terminal protein fragment, which may attenuate Ca_V1.2 inactivation through the loss of Ca²⁺ dependent inactivation (CDI). This study provide a theoretical basis for further study of the regulation of CaM antagonists on myocardial Ca_V1.2 channel function and intracellular Ca²⁺ concentration, and also provided ideas and laid a certain foundation for the development and treatment of new drugs related to cardiovascular diseases.