Uvarov Pavel, Fudo Satoshi, Karakus Cem, Golubtsov Andrey, Rotondo Federico, Sukhanova Tatiana, Soni Shetal, Di Scala Coralie, Kajander Tommi, Rivera Claudio, Ludwig Anastasia
Neuroscience Center, HiLIFE, University of Helsinki, Helsinki, Finland.
Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, Finland.
Front Mol Neurosci. 2025 Jan 15;17:1505722. doi: 10.3389/fnmol.2024.1505722. eCollection 2024.
The neuron-specific K-Cl cotransporter KCC2 maintains low intracellular chloride levels, which are crucial for fast GABAergic and glycinergic neurotransmission. KCC2 also plays a pivotal role in the development of excitatory glutamatergic neurotransmission by promoting dendritic spine maturation. The cytoplasmic C-terminal domain (KCC2-CTD) plays a critical regulatory role in the molecular mechanisms controlling the cotransporter activity through dimerization, phosphorylation, and protein interaction.
To identify novel CTD regulatory motifs, we used the Mu transposon-based mutagenesis system to generate a library of KCC2 mutants with 5 amino acid insertions randomly distributed within the KCC2-CTD. We determined the insertion positions in 288 mutants by restriction analysis and selected clones with a single insertion site outside known KCC2 regulatory motifs. We analyzed the subcellular distribution of KCC2-CTD mutants in cultured cortical neurons using immunocytochemistry and selected ten mutants with ectopic expression patterns for detailed characterization.
A fluorescent Cl-transport assay in HEK293 cells revealed mutants with both reduced and enhanced Cl-extrusion activity, which overall correlated with their glycosylation patterns. Live-cell immunostaining analysis of plasma membrane expression of KCC2-CTD mutants in cultured cortical neurons corroborated the glycosylation data. Furthermore, the somatodendritic chloride gradient in neurons transfected with the KCC2-CTD mutants correlated with their Cl-extrusion activity in HEK293 cells. Gain- and loss-of-function mutant positions were analyzed using available KCC2 cryo-EM structures.
Two groups of mutants were identified based on 3D structural analysis. The first group, located near the interface of transmembrane and cytoplasmic domains, may affect interactions with the N-terminal inhibitory peptide regulating KCC2 activity. The second group, situated on the external surface of the cytoplasmic domain, may disrupt interactions with regulatory proteins. Analyzing CTD mutations that modulate KCC2 activity enhances our understanding of its function and is essential for developing novel anti-seizure therapies.
神经元特异性钾氯协同转运体KCC2维持细胞内低氯水平，这对快速的γ-氨基丁酸能和甘氨酸能神经传递至关重要。KCC2还通过促进树突棘成熟在兴奋性谷氨酸能神经传递的发育中起关键作用。细胞质C末端结构域（KCC2-CTD）通过二聚化、磷酸化和蛋白质相互作用在控制协同转运体活性的分子机制中发挥关键调节作用。
为了鉴定新的CTD调节基序，我们使用基于Mu转座子的诱变系统生成了一个KCC2突变体文库，其中5个氨基酸插入随机分布在KCC2-CTD内。我们通过限制性分析确定了288个突变体中的插入位置，并选择了在已知KCC2调节基序之外具有单个插入位点的克隆。我们使用免疫细胞化学分析了KCC2-CTD突变体在培养的皮质神经元中的亚细胞分布，并选择了十个具有异位表达模式的突变体进行详细表征。
在HEK293细胞中进行的荧光氯转运测定显示，突变体的氯外排活性既有降低的也有增强的，这总体上与其糖基化模式相关。对培养的皮质神经元中KCC2-CTD突变体的质膜表达进行的活细胞免疫染色分析证实了糖基化数据。此外，用KCC2-CTD突变体转染的神经元中的树突棘-体细胞氯梯度与其在HEK293细胞中的氯外排活性相关。使用可用的KCC2冷冻电镜结构分析了功能获得和功能丧失突变体的位置。
基于三维结构分析鉴定出两组突变体。第一组位于跨膜结构域和细胞质结构域的界面附近，可能影响与调节KCC2活性的N末端抑制肽的相互作用。第二组位于细胞质结构域的外表面，可能破坏与调节蛋白的相互作用。分析调节KCC2活性的CTD突变可增强我们对其功能的理解，这对于开发新型抗癫痫疗法至关重要。
