The neuron-specific K/Cl co-transporter 2, KCC2, which is critical for brain development, regulates γ-aminobutyric acid-dependent inhibitory neurotransmission. Consistent with its function, mutations in KCC2 are linked to neurodevelopmental disorders, including epilepsy, schizophrenia, and autism. KCC2 possesses 12 transmembrane spans and forms an intertwined dimer. Based on its complex architecture and function, reduced cell surface expression and/or activity have been reported when select disease-associated mutations are present in the gene encoding the protein, SLC12A5. These data suggest that KCC2 might be inherently unstable, as seen for other complex polytopic ion channels, thus making it susceptible to cellular quality control pathways that degrade misfolded proteins. To test these hypotheses, we examined KCC2 stability and/or maturation in five model systems: yeast, HEK293 cells, primary rat neurons, and rat and human brain synaptosomes. Although studies in yeast revealed that KCC2 is selected for endoplasmic reticulum-associated degradation (ERAD), experiments in HEK293 cells supported a more subtle role for ERAD in maintaining steady-state levels of KCC2. Nevertheless, this system allowed for an analysis of KCC2 glycosylation in the ER and Golgi, which serves as a read-out for transport through the secretory pathway. In turn, KCC2 was remarkably stable in primary rat neurons, suggesting that KCC2 folds efficiently in more native systems. Consistent with these data, the mature glycosylated form of KCC2 was abundant in primary rat neurons as well as in rat and human brain. Together, this work details the first insights into the influence that the cellular and membrane environments have on several fundamental KCC2 properties, acknowledges the advantages and disadvantages of each system, and helps set the stage for future experiments to assess KCC2 in a normal or disease setting.

中文翻译：

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表达系统影响氯化钾协同转运蛋白 KCC2 的稳定性、成熟效率和寡聚特性

神经元特异性 K/Cl 协同转运蛋白 2 KCC2 对大脑发育至关重要，可调节 γ-氨基丁酸依赖性抑制性神经传递。与其功能一致，KCC2 的突变与神经发育障碍有关，包括癫痫、精神分裂症和自闭症。 KCC2 具有 12 个跨膜跨度并形成相互缠绕的二聚体。据报道，根据其复杂的结构和功能，当编码蛋白质 SLC12A5 的基因中存在选定的与疾病相关的突变时，细胞表面表达和/或活性会降低。这些数据表明，KCC2 可能本质上不稳定，就像其他复杂的多胞体离子通道一样，因此使其容易受到降解错误折叠蛋白质的细胞质量控制途径的影响。为了测试这些假设，我们在五个模型系统中检查了 KCC2 的稳定性和/或成熟度：酵母、HEK293 细胞、原代大鼠神经元以及大鼠和人脑突触体。尽管酵母研究表明 KCC2 被选择用于内质网相关降解 (ERAD)，但 HEK293 细胞中的实验支持 ERAD 在维持 KCC2 稳态水平方面发挥更微妙的作用。然而，该系统允许分析 ER 和高尔基体中的 KCC2 糖基化，作为通过分泌途径转运的读数。反过来，KCC2 在原代大鼠神经元中非常稳定，这表明 KCC2 在更天然的系统中有效折叠。与这些数据一致，KCC2 的成熟糖基化形式在原代大鼠神经元以及大鼠和人脑中大量存在。总之，这项工作详细介绍了细胞和膜环境对 KCC2 几个基本特性的影响的初步见解，承认每个系统的优点和缺点，并有助于为未来在正常或疾病环境中评估 KCC2 的实验奠定基础。