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.