Phospho-regulation of the K-Cl cotransporter KCC2

钾-氯共转运体KCC2在快速突触抑制中起重要作用。它是神经元中主要的氯离子挤出器，从而在质膜上产生向内定向的氯离子梯度。这种梯度是抑制性神经递质GABA和甘氨酸快速超极化作用的基础。KCC2是必需的，功能障碍与癫痫、神经性疼痛和创伤等神经疾病有关。磷酸化已成为调节KCC2活性的关键机制，并已被提议作为药物治疗的靶点。然而，我们对KCC2在生理和病理生理条件下的磷酸化调节的了解仍然非常有限。通过鉴定天然的磷酸化位点，我们最近发现了一个新的调节磷酸化位点T934/S937。这些氨基酸残基的磷酸化刺激了HEK-293细胞中KCC2的内在转运活性。此外，该位点的磷酸化决定了星孢菌素和NEM敏感但迄今未知的磷酸化位点的作用。该项目通过扩展T934/S937的功能特性来建立在这些结果的基础上。我们将描述它在神经细胞中的作用，并努力识别调节它的分子机制。此外，我们还将进一步分析与星孢菌素和NEM敏感的调节磷酸化位点之间的复杂功能串扰，因为这两个位点相互影响。这些实验将在细胞培养中进行。然而，T934/S937在整个生物体中的作用也是至关重要的。为此，我们将开发必要的工具来表征它们在体内的功能。我们将生产和验证针对磷化位点的抗体。此外，还将产生两个T934/S937突变的小鼠品系。一个小鼠品系将模仿磷酸化位点的去磷酸化状态，另一个将模仿磷酸化位点的磷酸化状态。这两个小鼠品系将由我们以及与其他小组合作描述它们在生理和病理生理过程中的作用。总之，该项目将为KCC2的磷酸调节机制提供重要的见解，这对基础研究和翻译研究都是必不可少的。此外，它还将为破译KCC2磷酸化在体内的作用提供必要的资源。

The K-Cl cotransporter KCC2 plays a seminal role in fast synaptic inhibition. It is the main Cl-extruder in neurons, thereby generating an inwardly directed Cl-gradient across the plasma membrane. This gradient underlies the rapid hyperpolarizing action of the inhibitory neurotransmitters GABA and glycine. KCC2 is essential and dysfunctions are associated with neurological disorders such as epilepsy, neuropathic pain, and trauma. Phosphorylation has emerged as a key mechanism to regulate KCC2 activity and has been proposed as a pharmacotherapeutic target. Yet, our knowledge of phosphorylation-mediated regulation of KCC2 under physiological and pathophysiological conditions is still very limited. By characterizing native phospho-sites, we recently identified the novel regulatory phospho-site T934/S937. Phosphorylation of these amino acid residues stimulates KCC2 intrinsic transport activity in HEK-293 cells. Furthermore, phosphorylation of this site determines the action of a staurosporine and NEM sensitive but hitherto unknown phospho-site. The project builds on these results by extending the functional characterization of T934/S937. We will characterize its role in neuronal cells and strive for the identification of the molecular mechanisms regulating it. In addition, we will further our analysis on the complex functional crosstalk with the staurosporine and NEM sensitive regulatory phospho-site, as both sites influence each other. These experiments will be performed in cell culture. However, it is essential to address the role of T934/S937 in the entire organism as well. To this end, we will generate essential tools to characterize their in vivo function. We will produce and validate antibodies against the phospho-site. Furthermore, two mouse lines with mutations in T934/S937 will be generated. One mouse line will mimic the dephosphorylated state of the phospho-site, the other will mimic the phosphorylated state of the phospho-site. Both mouse lines will be characterized by us as well as in collaboration with other groups for their role in physiological and pathophysiological processes. Altogether, the project will provide important insight in phospho-regulatory mechanisms of KCC2, which is essential for basic and translational research alike. Furthermore, it will supply essential resources to decode the in vivo roles of KCC2 phosphorylation.