Regulation of chloride homeostasis and inhibitory synapses by palmitoylation

描述（由申请人提供）：兴奋性和抑制性神经传递之间的精确平衡对于健康的大脑功能至关重要。在成熟的哺乳动物大脑中， 抑制性信号传导主要通过神经递质γ-氨基丁酸（GABA）与配体门控氯（Cl-）通道（GABAA受体）结合来实现。相反，在未成熟的大脑中，GABA能神经传递是兴奋性的，以促进发育所必需的营养活动。GABA介导的神经传递的强度和极性由细胞内氯离子浓度决定。两个氯离子协同转运体NKCC 1和KCC 2的相对活性的变化负责GABA能信号从兴奋性到抑制性的发育转换。重要的是，由功能失调的NKCC 1和/或KCC 2活性引起的细胞氯稳态的破坏导致神经元低兴奋性或高兴奋性，其涉及癫痫、慢性缺血和神经性疼痛的发病机制。缺乏对负责调节NKCC 1和KCC 2活性的机制的理解，这对于开发针对由Cl-稳态改变引起的疾病的新治疗策略至关重要。虽然协同转运蛋白活性的长期差异可能是由基因表达的变化介导的，但越来越多的证据表明，通过翻译后修饰和膜运输进行急性调节。蛋白质棕榈酰化是一种可逆的翻译后修饰，已知其调节神经元蛋白质运输和功能的各个方面。这项拟议的研究扩展了初步发现，即NKCC 1和KCC 2都是棕榈酰化的，以检验棕榈酰化通过以下方式调节协同转运蛋白活性的假设： 调节它们的蛋白质表达、成熟、寡聚化、磷酸化、运输和/或氯转运。阻止协同转运蛋白棕榈酰化的抑制剂和突变方法将应用于各种生物化学和细胞生物学测定以测试效果 棕榈酰化对协同转运蛋白生物学的影响。为了确定协同转运蛋白活性的发育变化是否与棕榈酰化的变化相关，我们实验室开发的用于定量评估蛋白质棕榈酰化的酰基-生物素交换（ABE）方法将用于表征脑发育不同阶段内源性NKCC 1和KCC 2的棕榈酰化。最后，ABE方法结合敲低和显性阴性方法将用于鉴定负责介导协同转运蛋白棕榈酰化的酶。总之，这项工作将探索一种潜在的新机制，用于调节NKCC 1和KCC 2的活性，并可以提供洞察各种病理，从改变Cl-稳态，以及确定新的治疗策略。

DESCRIPTION (provided by applicant): The precise balance between excitatory and inhibitory neurotransmission is critical for healthy brain function. In the mature mammalian brain, inhibitory signaling is predominantly accomplished through the neurotransmitter ¿-aminobutyric acid (GABA) binding to ligand-gated chloride (Cl-) channels (GABAA receptors). In contrast, in the immature brain GABAergic neurotransmission is excitatory in order to promote trophic activities necessary for development. The strength and polarity of GABA-mediated neurotransmission is determined by the intracellular chloride concentration. Changes in the relative activities of two chloride cotransporters, NKCC1 and KCC2, are responsible for the developmental switch in GABAergic signaling from excitatory to inhibitory. Importantly, disruptions in cellular chloride homeostasis resulting from dysfunctional NKCC1 and/or KCC2 activity result in neuronal hypo- or hyperexcitability that is implicated in the pathogenesis of epilepsy, chronic ischemia, and neuropathic pain. An understanding of the mechanisms responsible for regulating NKCC1 and KCC2 activity is lacking and is critical to the development of novel therapeutic strategies that target diseases resulting from altered Cl- homeostasis. While long-term differences in cotransporter activity are likely mediated by changes in gene expression, there is increasing evidence for acute regulation through post-translational modification and membrane trafficking. Protein palmitoylation is a reversible post-translational modification known to regulate various aspects of neuronal protein trafficking and function. The proposed research expands upon the preliminary finding that both NKCC1 and KCC2 are palmitoylated to test the hypothesis that palmitoylation regulates cotransporter activity either by modulating their protein expression, maturation, oligomerization, phosphorylation, trafficking, and/or chloride transport. Inhibitor and mutational approaches to prevent cotransporter palmitoylation will be applied in various biochemical and cell biological assays to test the effect of palmitoylation on cotransporter biology. To determine if developmental changes in cotransporter activity correlate with changes in palmitoylation, the acyl- biotin exchange (ABE) method, which was developed by our laboratory to quantitatively assess protein palmitoylation, will be used to characterize the palmitoylation of endogenous NKCC1 and KCC2 at various stages of brain development. Lastly, the ABE method in conjunction with knockdown and dominant negative approaches will be used to identify the enzymes responsible for mediating cotransporter palmitoylation. In summary, this work will explore a potentially novel mechanism for the regulation of NKCC1 and KCC2 activity and could provide insight into various pathologies that result from altered Cl- homeostasis as well as identify novel therapeutic strategies.