The Effect of Ca, Lobe-Specificity, and CaMKII on CaM Binding to Na1.1.

Calmodulin (CaM) is well known as an activator of calcium/calmodulin-dependent protein kinase II (CaMKII). Voltage-gated sodium channels (VGSCs) are basic signaling molecules in excitable cells and are crucial molecular targets for nervous system agents. However, the way in which Ca/CaM/CaMKII cascade modulates Na1.1 IQ (isoleucine and glutamine) domain of VGSCs remains obscure. In this study, the binding of CaM, its mutants at calcium binding sites (CaM, CaM, and CaM), and truncated proteins (N-lobe and C-lobe) to Na1.1 IQ domain were detected by pull-down assay. Our data showed that the binding of Ca/CaM to the Na1.1 IQ was concentration-dependent. ApoCaM (Ca-free form of calmodulin) bound to Na1.1 IQ domain preferentially more than Ca/CaM. Additionally, the C-lobe of CaM was the predominant domain involved in apoCaM binding to Na1.1 IQ domain. By contrast, the N-lobe of CaM was predominant in the binding of Ca/CaM to Na1.1 IQ domain. Moreover, CaMKII-mediated phosphorylation increased the binding of Ca/CaM to Na1.1 IQ domain due to one or several phosphorylation sites in T1909, S1918, and T1934 of Na1.1 IQ domain. This study provides novel mechanisms for the modulation of Na1.1 by the Ca/CaM/CaMKII axis. For the first time, we uncover the effect of Ca, lobe-specificity and CaMKII on CaM binding to Na1.1.