Authors :
Presenting Author: Huangling Lai, PhD – Guangzhou National Laboratory
Yue Xing, BS – Guangzhou
Ruili Niu, PhD – Guangzhou National Lab
Xuan Guo, PhD – Guangzhou National Lab
Xiaofeng YANG, MD – Guangzhou National Lab
Rationale:
The potassium-chloride cotransporter KCC2 is critical for maintaining GABAergic inhibitory signaling by regulating neuronal chloride homeostasis. Although KCC2 dysfunction is linked to epilepsy, the spatiotemporal mechanisms driving its dysregulation during epileptogenesis remain poorly understood.
This study aimed to identify phosphorylation-dependent mechanisms underlying KCC2 dysfunction in epilepsy and validate their clinical relevance.
Methods:
We integrated human epileptic tissues and an epileptic rat model, using experimental approaches included single-cell transcriptomics, in vivo electrophysiology and patch-clamp technology, phosphoproteomics, biochemistry, and functional validation using neuron-specific phosphomimetic mutations (T906E and T1007E), to investigated the mechanism of KCC2 dysfunction during epileptogenesis. Results:
We demonstrate that T906 phosphorylation dynamically increases prior to seizure initiation, correlating with KCC2 internalization and membrane loss. Single cell transcriptomics revealed neuron-specific KCC2 downregulation, patch-clamp indicated KCC2 dysfunction in pyramidal neuron, phosphoproteomics uncovered T906 as the dominant phosphorylation site upregulated in epileptic rats, contrasting with static T1007 and S940 phosphorylation. Functional validation showed that neuron specific phosphomimetic T906E mutation accelerated seizure onset and exacerbated severity, stronger than T1007E. Crucially, we provide the first evidence of T906 hyperphosphorylation in human epileptic tissues, establishing its clinical relevance.
Conclusions:
T906 phosphorylation acts as an early standalone driver of epileptogenesis by disrupting KCC2 function, with clinical validation in human epilepsy. Targeting T906 offers a novel precision therapeutic strategy to restore inhibitory signaling and mitigate seizure pathogenesis.
Funding: This work was funded by the National Natural Science Foundation of China (Grant No. 550 XFY 82271492, HLL82401701), Major Project of Guangzhou National Laboratory 551 Fund (Grant No. GZNL2024A02001).