Impact of neuronal chloride transport on treatment of seizures.

神经元氯转运对癫痫治疗的影响。

• 批准号: 8789396
• 负责人: Kevin J. Staley
• 金额: $ 37.1万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8789396
• 关键词: Acute; Address; Adult; Anions; Anticonvulsants; Automobile Driving; Cations; Cells; Chloride Ion; Chlorides; Clinical Trials; Cytoskeleton; Data; Diuretics; Dyes; Epilepsy; Equilibrium; Europe; Free Energy; Geometry; Goals; Grant; Health; Hydrostatic Pressure; In Vitro; Intracellular Space; Ions; Isotonic Exercise; Link; Measurement; Measures; Mediating; Membrane; Microscopy; Movement; Mus; Neonatal; Nervous system structure; Neurons; Neurotransmitters; Newborn Infant; Osmolar Concentration; Osmotic Pressure; Phosphorylation; Potassium; Preparation; Property; Proteins; Relaxation; Resistance; Rest; Seizures; Signal Transduction; Slice; Sodium Chloride; Solutions; Status Epilepticus; Sum; Techniques; Testing; Water; base; clomeleon; extracellular; fluorophore; gamma-Aminobutyric Acid; improved; in vivo; inhibitor/antagonist; neuronal transport; pressure; prevent; research study; response; synaptic inhibition; transport inhibitor; two-photon

DESCRIPTION (provided by applicant): During the last cycle of this grant, we established that block of inwardly-directed cation-chloride co-transport alters the neuronal steady-state chloride concentration (Cl-i) in developing neurons, and consequently improves the efficacy of GABA-mediated synaptic inhibition and the control of seizures in the newborn. This finding forms the basis of new clinical trials in the US and Europe. We also found that cation-Cl transporters are at equilibrium at resting Cl-i. However, we don't know how to reconcile this finding with data from experiments utilizing the Cl-sensitive fluorescent dual wavelength fluorophore, Clomeleon, which demonstrate that each neuron has a unique Cl-i that is quite different from its neighbors. The question we address here is: how can cation-Cl transporters be at equilibrium at so many different Cl-i? The classic view that equilibrium depends only on Cl and cation concentrations does not explain the variance in Cl-i. Neuronal cation-chloride transporters obligately move water with cations and Cl, so that these transporters move isotonic (135 mM) cation-Cl solution into and out of neurons. Thus neuronal cation-Cl transporters also transport cytoplasmic volume, which alters the cytoplasmic hydrostatic pressure. This predicts that transmembrane hydrostatic and osmotic pressure gradients contribute to the free energy of cation-Cl transport and thus the equilibrium Cl-i. For example, a neuron with lots of osmotically active protein should have a lower equilibrium Cl-i than a neuron with less protein. Our primary hypothesis is that the pressure gradient across the neuronal membrane contributes to the free energy of transport and thus the Cl-i at which transport is at equilibrium. This hypothesis has important implications for prolonged seizures, which induce changes in the neuronal cytoskeleton that increase the volume of neurons, thereby lowering the osmotic pressure. Thus a linked secondary hypothesis is that seizure-induced changes in osmotic pressure favor movement of cations, Cl-i and water into neurons via cotransporters so that Cl-i increases and GABA signaling becomes excitatory. We will test these hypotheses by measuring neuronal volume and Cl-i at steady state and in response to ionic and osmotic challenges, seizures, and specific transport inhibitors. We will use mice that genetically express Clomeleon, acute & organotypic slice preparations, in vitro and in vivo multiphoton microscopy, pH-sensitive dyes, electrophysiological recordings, and transporter phosphorylation studies. Sensitivity of cation-Cl transport to local pressure gradients would allow neurons to maintain the incredibly precise geometries needed for stable cable properties and connectivity despite dynamic subcellular and intercellular fluctuations in osmotically active protein content. The hypotheses also predict that clinically available diuretics and inhibitors of cytoskeletal changes might also be useful in the treatment of status epilepticus in both developing and mature nervous systems.

描述（由申请人提供）：在本次资助的最后一个周期中，我们确定阻断向内定向的阳离子-氯离子共转运会改变发育神经元中的神经元稳态氯离子浓度（Cl-i），从而提高 GABA 介导的突触抑制和新生儿癫痫发作控制的功效。这一发现构成了美国和欧洲新临床试验的基础。我们还发现，阳离子-Cl 转运蛋白在静息 Cl-i 时处于平衡状态。然而，我们不知道如何使这一发现与使用 Cl 敏感荧光双波长荧光团 Clomeleon 的实验数据相一致，该实验表明每个神经元都有一个与其相邻神经元完全不同的独特 Cl-i。我们在这里解决的问题是：阳离子-Cl 转运蛋白如何在如此多的不同 Cl-i 下保持平衡？平衡仅取决于 Cl 和阳离子浓度的经典观点并不能解释 Cl-i 的变化。神经元阳离子-氯离子转运蛋白必然将水与阳离子和 Cl 一起移动，因此这些转运蛋白将等渗 (135 mM) 阳离子-Cl 溶液移入和移出神经元。因此，神经元阳离子-Cl 转运蛋白还转运细胞质体积，从而改变细胞质静水压。这预测跨膜静水压和渗透压梯度有助于阳离子-Cl 传输的自由能，从而有助于平衡 Cl-i。例如，具有大量渗透活性蛋白质的神经元应该比具有较少蛋白质的神经元具有更低的平衡 Cl-i。我们的主要假设是，跨神经元膜的压力梯度有助于传输自由能，从而有助于传输处于平衡状态的 Cl-i。这一假设对于长期癫痫发作具有重要意义，癫痫发作会引起神经元细胞骨架的变化，从而增加神经元的体积，从而降低渗透压。因此，一个相关的第二个假设是，癫痫发作引起的渗透压变化有利于阳离子、Cl-i 和水通过协同转运蛋白进入神经元，从而使 Cl-i 增加，GABA 信号传导变得兴奋。我们将通过测量稳态下的神经元体积和 Cl-i 以及对离子和渗透压挑战、癫痫发作和特定转运抑制剂的反应来测试这些假设。我们将使用基因表达 Clomeleon 的小鼠、急性和器官切片制剂、体外和体内多光子显微镜、pH 敏感染料、电生理记录和转运蛋白磷酸化研究。尽管渗透活性蛋白质含量存在动态的亚细胞和细胞间波动，但阳离子-Cl传输对局部压力梯度的敏感性将使神经元能够保持稳定的电缆特性和连接所需的极其精确的几何形状。这些假设还预测，临床上可用的利尿剂和细胞骨架变化抑制剂也可能有助于治疗发育中和成熟神经系统的癫痫持续状态。