Increased NaV1.2 expression and its interaction with CaM contribute to the hyperexcitability induced by prolonged inhibition of CaMKII.

Abstract

Objective: Dysfunction of calcium/calmodulin (CaM)-dependent kinase II (CaMKII) has been involved in hyperexcitability-related disorders including epilepsy. However, the relationship between CaMKII and neuronal excitability remains unclear.

Methods: Neuronal excitability was detected in vivo and in vitro by electroencephalography (EEG), patch clamp and multi-electrode array (MEA), respectively. Next, we assessed the currents of voltage-gated sodium channels (VGSCs) by patch clamp, and  mRNA and protein expressions of VGSCs were determined by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot, respectively. Meanwhile, the association between the nuclear receptor subfamily 4 group A member 2 (NR4A2) and promoters of Scn2a, was determined by chromatin immunoprecipitation (ChIP)-qPCR. In addition, we utilized co-immunoprecipitation (Co-IP), immunofluorescence labeling, and pull-down to determine the interaction between VGSCs and CaM.

Results: Prolonged CaMKII inhibition by KN93, an inhibitor of CaMKII, for 24 h and CaMKII knockdown induced more seizure-like events in Wistar rats, TRM rats and C57BL/6 mice, and led to hyperexcitability in primary hippocampal neurons and human induced-pluripotent stem cell (hiPSC)-derived cortical neurons. In addition, prolonged CaMKII inhibition resulted in elevated persistent sodium current (I_NaP)/transient sodium current (I_NaT) and increased mRNA and protein expressions of Na_V1.2. Meanwhile, prolonged CaMKII inhibition by KN93 decreased NR4A2 expression and contributed to a transcriptional repression role of NR4A2 in Scn2a regulation, leading to increased Na_V1.2 expression. Moreover, an increased interaction between Na_V1.2 and CaM was attributable to enhanced binding of CaM to the isoleucine-glutamine (IQ) domain at the C-terminus of the Na_V1.2 channel, which may also lead to the potentiation in I_NaP/I_NaT and channel activity. Furthermore, a peptide that antagonized CaM binding to Na_V1.2 IQ domain (ACNp) rescued hyperexcitability following prolonged CaMKII inhibition.

Significance: We unveiled that prolonged CaMKII inhibition induced hyperexcitability through increasing the expression of Na_V1.2 and its association with CaM. Thus, our study uncovers a novel signaling mechanism by which CaMKII maintains appropriate neuronal excitability.