The Effect of Ca 2+ , Lobe-Specificity, and CaMKII on CaM Binding to Na V 1.1.

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 ²⁺ /CaM/CaMKII cascade modulates Na _V 1.1 IQ (isoleucine and glutamine) domain of VGSCs remains obscure. In this study, the binding of CaM, its mutants at calcium binding sites (CaM ₁₂ , CaM ₃₄ , and CaM ₁₂₃₄ ), and truncated proteins (N-lobe and C-lobe) to Na _V 1.1 IQ domain were detected by pull-down assay. Our data showed that the binding of Ca ²⁺ /CaM to the Na _V 1.1 IQ was concentration-dependent. ApoCaM (Ca ²⁺ -free form of calmodulin) bound to Na _V 1.1 IQ domain preferentially more than Ca ²⁺ /CaM. Additionally, the C-lobe of CaM was the predominant domain involved in apoCaM binding to Na _V 1.1 IQ domain. By contrast, the N-lobe of CaM was predominant in the binding of Ca ²⁺ /CaM to Na _V 1.1 IQ domain. Moreover, CaMKII-mediated phosphorylation increased the binding of Ca ²⁺ /CaM to Na _V 1.1 IQ domain due to one or several phosphorylation sites in T1909, S1918, and T1934 of Na _V 1.1 IQ domain. This study provides novel mechanisms for the modulation of Na _V 1.1 by the Ca ²⁺ /CaM/CaMKII axis. For the first time, we uncover the effect of Ca ²⁺ , lobe-specificity and CaMKII on CaM binding to Na _V 1.1.