Calmodulin Modulates the Ca-dependent Inactivation and Expression Level of Bovine Ca2.2 Expressed in HEK293T Cells

Ca influx through voltage-gated Ca channels (Cas) at the plasma membrane is the major pathway responsible for the elevation of the intracellular Ca concentration ([Ca]), which activates various physiological activities. Calmodulin (CaM) is known to be involved in the Ca-dependent inactivation (CDI) of several types of Cas; however, little is known about how CaM modulates Ca2.2. Here, we expressed Ca2.2 with CaM or CaM mutants with a Ca-binding deficiency in HEK293T cells and measured the currents to characterize the CDI. The results showed that Ca2.2 displayed a fast inactivation with Ca but not Ba as the charge carrier; when Ca2.2 was co-expressed with CaM mutants with a Ca-binding deficiency, the level of inactivation decreased. Using glutathione S-transferase-tagged CaM or CaM mutants as the bait, we found that CaM could interact with the intracellular C-terminal fragment of Ca2.2 in the presence or absence of Ca. However, CaM and its mutants could not interact with this fragment when mutations were generated in the conserved amino acid residues of the CaM-binding site. Ca2.2 with mutations in the CaM-binding site showed a greatly reduced current that could be rescued by CaM (Ca-binding deficiency at the N-lobe) overexpression; in addition, CaM enhanced the total expression level of Ca2.2, but the ratio of Ca2.2 present in the membrane to the total fraction remained unchanged. Together, our data suggest that CaM, with different Ca-binding abilities, modulates not only the inactivation of Ca2.2 but also its expression to regulate Ca-related physiological activities.