Rui Feng

Voltage-gated sodium channels (VGSCs) are fundamental to the initiation and propagation of action potentials in excitable cells. Ca²⁺/calmodulin (CaM) binds to VGSC type II (Na_V1.2) isoleucine and glutamine (IQ) motif. An autism-associated mutation in Na_V1.2 IQ motif, Arg1902Cys (R1902C), has been reported to affect the combination between CaM and the IQ motif compared to that of the wild type IQ motif. However, the detailed properties for the Ca²⁺-regulated binding of CaM to Na_V1.2 IQ (1901Lys-1927Lys, IQ_wt) and mutant IQ motif (IQ_R1902C) remains unclear. Here, the binding ability of CaM and CaM's constituent proteins including N- and C lobe to the IQ motif of Na_V1.2 and its mutant was investigated by protein pull-down experiments. We discovered that the combination between CaM and the IQ motif was U-shaped with the highest at [Ca²⁺] ≈ free and the lowest at 100 nM [Ca²⁺]. In the IQ_R1902C mutant, Ca²⁺-dependence of CaM binding was nearly lost. Consequently, the binding of CaM to IQ_R1902C at 100 and 500 nM [Ca²⁺] was increased compared to that of IQ_wt. Both N- and C lobe of CaM could bind with Na_V1.2 IQ motif and IQ_R1902C mutant, with the major effect of C lobe. Furthermore, CaMKII had no impact on the binding between CaM and Na_V1.2 IQ motif. This research offers novel insight to the regulation of Na_V1.2 IQ_wt and IQ_R1902C motif, an autism-associated mutation, by CaM.

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 Ca²⁺/CaM/CaMKII cascade modulates Na_V1.1 IQ (isoleucine and glutamine) domain of VGSCs remains obscure. In this study, the binding of CaM, its mutants at calcium binding sites (CaM₁₂, CaM₃₄, and CaM₁₂₃₄), and truncated proteins (N-lobe and C-lobe) to Na_V1.1 IQ domain were detected by pull-down assay. Our data showed that the binding of Ca²⁺/CaM to the Na_V1.1 IQ was concentration-dependent. ApoCaM (Ca²⁺-free form of calmodulin) bound to Na_V1.1 IQ domain preferentially more than Ca²⁺/CaM. Additionally, the C-lobe of CaM was the predominant domain involved in apoCaM binding to Na_V1.1 IQ domain. By contrast, the N-lobe of CaM was predominant in the binding of Ca²⁺/CaM to Na_V1.1 IQ domain. Moreover, CaMKII-mediated phosphorylation increased the binding of Ca²⁺/CaM to Na_V1.1 IQ domain due to one or several phosphorylation sites in T1909, S1918, and T1934 of Na_V1.1 IQ domain. This study provides novel mechanisms for the modulation of Na_V1.1 by the Ca²⁺/CaM/CaMKII axis. For the first time, we uncover the effect of Ca²⁺, lobe-specificity and CaMKII on CaM binding to Na_V1.1.