Abstract
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.
Highlights
Voltage-gated sodium channels (VGSCs) are basic signaling molecules in excitable cells and are molecular targets for local anesthetic agents and antiepileptic agents [1,2]
Our data showed the binding of Ca2+/CaM to IQ was in a concentration-dependent and Ca2+-dependent manner, but apoCaM had the highest affinity to NaV1.1 IQ domain
Our study was aimed at clarifying the molecular mechanism underlying the modulation of Ca2+/CaM/calmodulin-dependent protein kinase II (CaMKII) on NaV1.1 channel, which is a key issue in understanding the regulatory mechanism of VGSCs
Summary
Voltage-gated sodium channels (VGSCs) are basic signaling molecules in excitable cells and are molecular targets for local anesthetic agents and antiepileptic agents [1,2]. A sequence within C-terminal of NaV1.1 contains a classical calmodulin (CaM)-binding IQ (isoleucine and glutamine) domain [14,15,16,17], which is involved in Ca2+ signal transduction and alters the activity in response to changes in free Ca2+ concentration ([Ca2+]). It has been found that Ca2+/CaM modulates VGSCs activity [12,23,24], but the molecular mechanism of how Ca2+/CaM binds to NaV1.1 is still unclear. We have previously found that N- and C-lobe of CaM have lobe-specific properties in modulating CaV1.2 channel [26].
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