Impact of neuronal chloride transport on treatment of seizures.

描述(申请人提供)：在这笔赠款的最后一个周期，我们建立了阻断向内定向的阳离子-氯化物共转运改变发育中神经元的神经元稳态氯浓度(CI-I)，从而提高了GABA介导的突触抑制和新生儿癫痫发作的效果。这一发现构成了美国和欧洲新的临床试验的基础。我们还发现阳离子-氯转运体在静止的氯-i处处于平衡状态。然而，我们不知道如何将这一发现与利用氯敏感的荧光双波长荧光团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邻居截然不同的唯一的氯-I。我们在这里讨论的问题是：阳离子-氯转运体如何在这么多不同的氯-i中处于平衡状态？认为平衡只取决于氯和阳离子浓度的经典观点不能解释氯-i的变化。神经元阳离子-氯离子转运体主要与阳离子和氯一起运输水，因此这些转运体将等渗(135 MM)阳离子-氯离子溶液移入和移出神经元。因此，神经元的阳离子-氯转运体也运输细胞质的体积，从而改变细胞质的静水压力。这预示着跨膜静水压梯度和渗透压梯度有助于阳离子-氯迁移的自由能，从而有助于平衡氯-i。例如，一个含有大量渗透活性蛋白质的神经元应该比一个蛋白质较少的神经元具有更低的平衡氯-I。我们的主要假设是，神经细胞膜上的压力梯度有助于运输的自由能，从而导致运输处于平衡状态的氯-i。这一假说对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，一个相关的次要假设是，癫痫引起的渗透压变化有利于阳离子、氯-i和水通过共转运体进入神经元，从而使氯-i增加，GABA信号变得兴奋。我们将通过测量稳态和对离子和渗透挑战、癫痫发作和特定转运抑制剂的反应的神经元体积和Cl-i来检验这些假说。我们将使用基因表达Clomeleon的小鼠、急性和器官型切片准备、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。阳离子-氯转运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令人难以置信的精确几何结构，尽管渗透活性蛋白质含量在亚细胞和细胞间动态波动。这些假说还预测，临床上可用的利尿剂和细胞骨架改变的抑制剂也可能在治疗发育中和成熟的神经系统中的癫痫持续状态方面有用。

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.