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Voltage-gated sodium channels (VGSCs) are fundamental to the initiation and propagation of action potentials in excitable cells. Ca 2+ /calmodulin (CaM) binds to VGSC type II (Na V 1.2) isoleucine and glutamine (IQ) motif. An autism-associated mutation in Na V 1.2 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 Ca 2+ -regulated binding of CaM to Na V 1.2 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 V 1.2 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 [Ca 2+ ] ≈ free and the lowest at 100 nM [Ca 2+ ]. In the IQ R1902C mutant, Ca 2+ -dependence of CaM binding was nearly lost. Consequently, the binding of CaM to IQ R1902C at 100 and 500 nM [Ca 2+ ] was increased compared to that of IQ wt . Both N- and C lobe of CaM could bind with Na V 1.2 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 V 1.2 IQ motif. This research offers novel insight to the regulation of Na V 1.2 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 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.

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 (NaV1.2) isoleucine and glutamine (IQ) motif. An autism-associated mutation in NaV1.2 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 NaV1.2 IQ (1901Lys-1927Lys, IQwt) and mutant IQ motif (IQR1902C) remains unclear. Here, the binding ability of CaM and CaM's constituent proteins including N- and C lobe to the IQ motif of NaV1.2 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 IQR1902C mutant, Ca2+-dependence of CaM binding was nearly lost. Consequently, the binding of CaM to IQR1902C at 100 and 500 nM [Ca2+] was increased compared to that of IQwt. Both N- and C lobe of CaM could bind with NaV1.2 IQ motif and IQR1902C mutant, with the major effect of C lobe. Furthermore, CaMKII had no impact on the binding between CaM and NaV1.2 IQ motif. This research offers novel insight to the regulation of NaV1.2 IQwt and IQR1902C motif, an autism-associated mutation, by CaM. [ABSTRACT FROM AUTHOR]

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 NaV1.1 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 NaV1.1 IQ domain were detected by pull-down assay. Our data showed that the binding of Ca2+/CaM to the NaV1.1 IQ was concentration-dependent. ApoCaM (Ca2+-free form of calmodulin) bound to NaV1.1 IQ domain preferentially more than Ca2+/CaM. Additionally, the C-lobe of CaM was the predominant domain involved in apoCaM binding to NaV1.1 IQ domain. By contrast, the N-lobe of CaM was predominant in the binding of Ca2+/CaM to NaV1.1 IQ domain. Moreover, CaMKII-mediated phosphorylation increased the binding of Ca2+/CaM to NaV1.1 IQ domain due to one or several phosphorylation sites in T1909, S1918, and T1934 of NaV1.1 IQ domain. This study provides novel mechanisms for the modulation of NaV1.1 by the Ca2+/CaM/CaMKII axis. For the first time, we uncover the effect of Ca2+, lobe-specificity and CaMKII on CaM binding to NaV1.1. [ABSTRACT FROM AUTHOR]